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Manganese Centers in Oxalate Decarboxylase

Permanent Link: http://ufdc.ufl.edu/UFE0021119/00001

Material Information

Title: Manganese Centers in Oxalate Decarboxylase
Physical Description: 1 online resource (117 p.)
Language: english
Creator: Moomaw, Ellen Winger
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: epr, metalloenzyme, oxalate
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO2 and formate by a mechanism that remains poorly understood. The bacterial form of the enzyme, present in Bacillus subtilis, is composed of two cupin domains, each of which contains a Mn(II) ion coordinated by four conserved residues. My work reports an in vivo strategy for obtaining recombinant, wild type OxDC in which manganese is substituted by cobalt, together with the first conditions for in vitro reconstitution of the apoenzyme with Mn(II). My work also examines the question of whether both Mn-binding sites in Bacillus subtilis OxDC can independently catalyze the decarboxylation reaction by expressing and characterizing a series of OxDC mutants in which metal binding is perturbed. A linear relationship between Mn occupancy and catalytic activity is demonstrated. Electron Paramagnetic Resonance (EPR) measurements reveal that the apparent line broadening observed for the Mn signals of wild type OxDC arises from dipolar coupling between neighboring Mn ions. These results are consistent with the proposal that there is only a single catalytic site in the enzyme. The similarity between the two Mn(II) sites has precluded previous attempts to distinguish them spectroscopically and complicated efforts to understand the catalytic mechanism. My research utilizes a multifrequency EPR approach to distinguish the two Mn ions on the basis of their differing fine structure parameters, and observed that acetate and formate bind to Mn(II) in only one of the two sites. The EPR evidence is consistent with the hypothesis that this Mn-binding site is located in the N-terminal domain, in agreement with predictions based on a recent X-ray structure of the enzyme.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Ellen Winger Moomaw.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Richards, Nigel G.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021119:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021119/00001

Material Information

Title: Manganese Centers in Oxalate Decarboxylase
Physical Description: 1 online resource (117 p.)
Language: english
Creator: Moomaw, Ellen Winger
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: epr, metalloenzyme, oxalate
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO2 and formate by a mechanism that remains poorly understood. The bacterial form of the enzyme, present in Bacillus subtilis, is composed of two cupin domains, each of which contains a Mn(II) ion coordinated by four conserved residues. My work reports an in vivo strategy for obtaining recombinant, wild type OxDC in which manganese is substituted by cobalt, together with the first conditions for in vitro reconstitution of the apoenzyme with Mn(II). My work also examines the question of whether both Mn-binding sites in Bacillus subtilis OxDC can independently catalyze the decarboxylation reaction by expressing and characterizing a series of OxDC mutants in which metal binding is perturbed. A linear relationship between Mn occupancy and catalytic activity is demonstrated. Electron Paramagnetic Resonance (EPR) measurements reveal that the apparent line broadening observed for the Mn signals of wild type OxDC arises from dipolar coupling between neighboring Mn ions. These results are consistent with the proposal that there is only a single catalytic site in the enzyme. The similarity between the two Mn(II) sites has precluded previous attempts to distinguish them spectroscopically and complicated efforts to understand the catalytic mechanism. My research utilizes a multifrequency EPR approach to distinguish the two Mn ions on the basis of their differing fine structure parameters, and observed that acetate and formate bind to Mn(II) in only one of the two sites. The EPR evidence is consistent with the hypothesis that this Mn-binding site is located in the N-terminal domain, in agreement with predictions based on a recent X-ray structure of the enzyme.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Ellen Winger Moomaw.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Richards, Nigel G.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021119:00001


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8f8eeebcccceb4f1961f31a5faa8432c703de4c5










MANGANESE CENTERS IN OXALATE DECARBOXYLASE


By

ELLEN WINGER MOOMAW














A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007

































O 2007 Ellen Winger Moomaw


































To my parents and to my sisters









ACKNOWLEDGMENTS

There are many people that I would like to thank for their help and support throughout my

doctoral research at the University of Florida. I am particularly indebted to my advisor Dr. Nigel

G. J. Richards and EPR collaborator Dr. Alexander Angerhofer for making this work a reality as

well as for their guidance. I thank Dr. George Christou, Dr. Thomas J. Lyons, and Dr. Daniel L.

Purich for their input and support as members of my doctoral dissertation committee. This study

was supported by grants from the National Institutes of Health (DK61193 and DK61666) and by

the University of Florida Chemistry Department.

I am grateful to my coworkers and friends in the Richards research group, especially Dr.

Patricia Moussatche, for the molecular biological training and collaboration that she provided. I

also thank Kai Li, Ewa Wroclawska, Dr. Drazenka Svedruzic, Dr. Christopher Chang, Cory

Toyota, and Dr. Jemy Gutierrez for technical assistance, training, and helpful discussions.

I owe a debt of gratitude to my EPR collaborators in addition to Dr. Alexander Angerhofer,

Dr. Andrew Ozarowski and Dr. Jurek Kryztek of the National High Magnetic Field Laboratory,

Dr. Ines Garcia-Rubio of the ETH Zurich, and Dr. Ralph T. Weber of the Bruker Biospin Corp.

I appreciate the technical support of Vij ay Antharam and Omj oy Ganesh for the CD

studies and of Witcha Imaram for the EPR spin-tapping experiments.

I thank Dr. Ben Smith, Graduate Coordinator, and Lori Clark in the Graduate Student

Office for their help and advice.












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ............ ..... ._ ...............8...


LIST OF FIGURES .............. ...............9.....


AB S TRAC T ............._. .......... ..............._ 12...


CHAPTER


1 INTRODUCTION ................. ...............14.......... ......


Oxalic Acid in Biological Systems............... ...............14
Oxalate Degrading Enzymes .............. ...............15....
Oxalate Decarboxylase ................ ...............17.................
Biological Distribution .............. .... ..... ......... .... .... ......... ......... ... .......1
Oxalate Decarboxylase Belongs to the Cupin Superfamily (DSBH) of Proteins ...........18
Structural Features ................. ...............20.................
M echanistic Information .............. ..... .............. ..........2

Oxygen Dependence and the Formation of Hydrogen Peroxide ................ ..........._.._. .25
Research Objectives............... ...............2

2 CHARACTERIZATION OF THE MN-DEPENDENCE OF OXALATE
DECARB OXYLASE ACTIVITY ................. .......__ .........27_.....


Introducti on ............ ..... ._ ...............27....
Results and Discussion .............. .... .. .. ..... ....................2

Optimization of Expression of Recombinant Wild Type OxDC. ........._._... ................27
Effect of Addition of Other Metals in the Growth Medium .................. ... .. ........... ......28

Preparation of the OxDC "Apoenzyme" and Reconstitution of the Wild Type, Mn-
Containing Enzyme............... ...............29.
Gepasi Simulations ................. ...............3.. 1..............
Experimental Section............... ...............34
M materials .............. .... .... .... ...... ........ .... ...... .. .. .............3
Expression and Purification of Recombinant, Wild Type OxDC. ............._ ...............34
Expression and Purification of Go-Sub stituted, Wild Type OxDC ................. ...............3 5
Preparation of the OxDC "Apoenzyme" and Reconstitution of the Wild Type, Mn-
Containing Enzyme............... ...............36.
Metal Content Determination ............ ...... .._ ...............36...
Steady-State Kinetic Assays............... ...............37.
Gepasi Simulations ............_ ...... .._ ...............38...

3 SPECTROSCOPIC CHARACTERIZATION OF THE TWO MANGANESE
CENTERS ............_ ...... .._ ...............42...












Introducti on ................ .... ......... ...... ... .............4
Electron Paramagnetic Resonance Spectroscopy ................. ............... ......... ...42
Electronic Configuration of Mn(II) .............. ... ............... ...............43. ....
Electron Paramagnetic Resonance Properties of Mn(II) ................ .......................43
Oxalate Decarboxylase EPR ................. ...............46........... ....
Results and Discussion .............. ...............47....
X-band EPR ................... ....... ...... .. .......... ........ ........4
Field Dependence of the EPR Signal in Storage Buffer. ......____ ..... ... .._............48
Spectral Simulations and Magnetic Parameters .............. ... ..... ...............49
Field Dependence of the EPR Signal in Acetate Buffer, pH 5.2 ................. .................5 1
Experimental Section............... .... ... .. .. ........5
Oxalate Decarboxylase Sample Preparation .............. ...............53....
Electron Paramagnetic Resonance Spectroscopy ................. ............... ......... ...54

4 SPECTROSCOPIC CHANGES OF THE MANGANESE CENTERS IN THE
PRESENCE OF SUBSTRATE .............. ...............55....


Introducti on ................. ...............55.................
Results and Discussion .............. ...............56....
X-band (9.5 GHz) ................... ....... ....__ .. ...... ....... .. ..........5
Chemical Oxidation of OxDC Observed at X-band............... ........ ............ 5
X-band Spin-Trapping of an Oxygen Species Formed During Oxalate
Decarboxylase Turnover............... ...............60
Q-band ( 35 GHz) ................. ...............63.............
W -band (94 GHz) ................. ...............63....... .....
324 GH z............... ...............64..
690 GH z............... ...............64..
Experimental Section............... ...............65

5 SITE-DIRECTED MUTAGENESIS STUDIES TO PROBE WHICH MANGANESE-
BINDING SITE(S) IS INVOLVED IN CATALYSIS ................. ......__ .........___..67

Introducti on ............ ..... ._ ...............67....
Results and Discussion ................. ...... ...... ......................6

Design and Steady-State Characterization of OxDC Mutants with Domain-Specific
Modified Mn Affinity ........................ ...............6
Size-Exclusion Chromatography (SEC) ................. ...............70........... ....
Circular Dichroism Measurements ................. ...............71................
Electron Paramagnetic Resonance Properties .............. ...............73....
Relaxation Enhancement Measurements ................. .. ............. ........_.._.........7

Implications for the Location of the Catalytic Site(s) in OxDC ................. ................ .78
Motivation for the Preparation of Single Domain OxDC Mutants ............... .................79
N-Terminal OxDC Single Domain Mutant (OxDC-N1) Does Not Catalyze the
Decarboxylation Reaction.................. ......... .. .......................8
EPR Characterization of Reconstituted N-terminal OxDC Single Domain Mutant
(OxDC-N 1) .............. ...............8 1....











C-Terminal OxDC Single Domain Mutant (OxDC-C) Does Not Catalyze the
Decarboxylation Reaction.................. .. .......... ..... .................8
Combining the N- and C-Terminal Single Domain Mutants Did Not Result in
Decarboxylase Activity ................. ...............82.................
Experim ental Section............... .............. ........... .......8
Expression and Purification of Site-Specific OxDC Mutants. ................... ...............8
Oxalate Oxidase Assays ................. ...... .............8
Size-Exclusion Chromatography Measurements .............. ...............84....
Circular Dichroism Studies .............. ...............85...
Electron Paramagnetic Resonance Spectroscopy ................. ............... ......... ...85
Expression of OxDC Single Domain Mutants ................ ...............86...
Purification of Single Domain OxDC Mutant OxDC-N1 .............. .....................8
Purification of Single Domain OxDC Mutant OxDC-C .............. ....................8

6 CONCLUSIONS AND FUTURE WORK .....___.....__.___ .......____ ............8

APPENDIX

A KINETIC PARAMETERS USED INT GEPASI SIMULATIONS .............. .....................9

B SIMULATIONS OF EPR SPECTRA AT DIFFERENT FIELD/FREQUENCY
COMBINATIONS OF OXALATE DECARBOXYLASE INT STORAGE BUFFER...........99

C HIGH FIELD SPECTRA AND SIMULATIONS OF WT OXDC AND THE E280Q
M UTANT .............. ...............105....

LIST OF REFERENCES ................. ...............109................

BIOGRAPHICAL SKETCH ................ ............. ............ 117...










LIST OF TABLES


Table page

2-1 Effect of MnCl2 and CoCl2 in the growth medium on metal incorporation and
specific activity of recombinant, wild type OxDC. ........... ...............29......

2-2 Metal content of "apoenzyme" and enzyme reconstituted with Mn. ............ .................30

3-1 Magnetic parameters of OxDC species I and II. ............ ...............50.....

5-1 Mn incorporation and steady-state kinetic parameters for metal-binding OxDC
m utants ........... ..... .._ ...............68...

5-2 Metal content of Mn-binding OxDC mutants. .....__.....___ .......... ............6

5-3 Estimates of size for the oligomeric forms of recombinant wild type OxDC and
OxDC Mn-binding mutants obtained using size exclusion chromatography. ...................71

5-4 Metal content of single domain mutant preparations............... ..............8

5-5 Primers used in the construction of Mn-binding mutants ......___ ........ ..............83

5-6 Primers used in the preparation of OxDC single domain mutants............... ................8










LIST OF FIGURES


FiMr page

1-1 Enzymes that catalyze oxalate degradation. .......... ...............16......

1-2 Sequence alignments of OxDCs from Bacilhts subtilis, Bacilhts clausii, Collybia
vehttipes, and Aspergilhts oryzae showing the positions of the two conserved motifs
(motif 1 in blue and motif 2 in red) in the two domains. ........... ......................1

1-3 Ribbon structures of the Bacilhts subtilis OxDC monomer, trimer, and hexamer. .........21

1-4 Residues defining the Mn-binding sites in the N-terminal (1UW8) and C-terminal
(1J5 8) domains of OxDC ........._. ...... .... ...............22.

1-5 Proposed catalytic mechanism for oxalate decarboxylase based on heavy-atom
isotope effect measurements. ............. ...............24.....

2-1 The dependence of OxDC specific activity on the extent of Mn incorporation. .............31

2-2 Numerical simulations of the dependence of catalytic activity on the extent of Mn
i ncorp orati on ................. ...............32................

3-1 Absorption of microwave irradiation by an unpaired electron in a magnetic Hield. .......42

3-2 Electron spin energy levels and hyperfine splitting for Mn(II) in spherical symmetry.
.............................44.

3-3 Overlay of the N-terminal (shown in magenta) and C-terminal (shown in green)
manganese-coordinating ligands (PDB code: 1UW8) ........... ......................4

3-4 X-band cw-EPR spectra of wild type OxDC in storage buffer and in acetate buffer........48

3-5 Field dependence of the EPR spectra of OxDC in storage buffer (20 mM
Hexamethylenetetramine HC1, pH 6.0) with 0.5 M NaC1. ............. .....................4

3-6 W-Band (94 GHz) EPR spectra of OxDC. ............. ...............50.....

3-7 Field dependence of the EPR spectra of OxDC in acetate buffer (50 mM, pH 5.2)
with 0.5 M NaC 1.. ............ ...............51.....

4-1 Spectral changes of the g 2 signal at X-band upon addition of acetate and oxalate
to OxD C. ............. ...............56.....

4-2 Spectral changes of the g 2 signal at X-band upon addition of oxalate to OxDC in
storage buffer at pH 5.2. ............. ...............57.....











4-3 Spectral changes of the g 2 signal at X-band upon addition of oxalate to OxDC in
storage buffer at pH 5.2 under anaerobic conditions followed by the reintroduction of
air. ............. ...............58.....

4-4 Mn(II) signal intensity and carbon-based radical formation as a function of the
concentration of sodium (meta) periodate. ............. ...............60.....

4-5 EPR spectra of the spin-trapped radical formed during OxDC turnover. ........................61

4-6 EPR spectrum of a short-lived DMPO-oxygen species .................... ............... 6

4-7 Spectral changes of the Mn(II) signal at Q-band upon addition of acetate and oxalate
to OxDC. ............. ...............62.....

4-8 Spectral changes of the Mn(II) signal at W-band upon addition of acetate and oxalate
to OxD C. ............. ...............63.....

4-9 Spectral changes of the Mn(II) signal at 324 GHz upon addition of acetate and
oxalate to OxDC. ............. ...............64.....

4-10 Spectral changes of the Mn(II) signal at 690 GHz upon addition of acetate and
oxalate to OxDC. ............. ...............65.....

5-1 CD spectra of recombinant wild type OxDC and the Mn-binding OxDC mutants. ..........72

5-2 EPR spectra of the Mn(II) signals in wild type OxDC (red) and the E280Q OxDC
mutant (blue) at 10 K. .............. ...............74....

5-3 Inversion-recovery experiments on wild type OxDC (12.3 mg/mL) and the E280Q
OxDC mutant (16.8 mg/mL)................ ...............76

5-4 Data for the Hahn-echo decay experiment on wild-type OxDC and the E280Q
m utant. ........ ...............77.......

5-5 Topology diagram of OxDC. ........... ...............80......

5-6 Effect of buffer and oxalate on the g 2 X-band Mn(II) signal of reconstituted N-
terminal OxDC mutant (OxDC-N1) .............. ...............81....

B-1 X-band spectrum of OxDC in SB pH6.0 at T = 10 K. ................. ....__. ...............99

B-2 V-band spectrum of OxDC in SB pH6.0 at T = 20 K. ................. ....__. ...............100

B-3 W-band EPR spectrum of OxDC in SB pH6.0 at T = 50 K............... ....................101

B-4 Sub-mm EPR spectrum of OxDC in SB pH6.0 at T = 20 K and 222 GHz. .................... 102

B-5 Sub-mm EPR spectrum of OxDC in SB pH6.0 at T = 20 K and 324 GHz. .................... 103










B-6 Sub-mm EPR spectrum of OxDC in SB pH6.0 at T = 20 K and 412.8 GHz. ................104

C-1 High field EPR of wild-type OxDC enzyme at 3 86. 116 GHz and 10 K. ......................106

C-2 High field EPR of E280Q OxDC mutant at 3 31.2 GHz and 20 K. ........._.._... ...............107

C-3 High field EPR of E280Q OxDC mutant at 3 82.826 GHz and 10 K. ........._.._... .............108









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

STUDIES OF THE MANGANESE CENTERS INT OXALATE DECARBOXYLASE

By

Ellen Winger Moomaw

August 2007

Chair: Nigel G. J. Richards
Major: Chemistry

Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO2 and format

by a mechanism that remains poorly understood. The bacterial form of the enzyme, present in

Bacillus subtilis, is composed of two cupin domains, each of which contains a Mn(II) ion

coordinated by four conserved residues. My work reports an in vivo strategy for obtaining

recombinant, wild type OxDC in which manganese is substituted by cobalt, together with the

first conditions for in vitro reconstitution of the apoenzyme with Mn(II). My work also

examines the question of whether both Mn-binding sites in Bacillus subtilis OxDC can

independently catalyze the decarboxylation reaction by expressing and characterizing a series of

OxDC mutants in which metal binding is perturbed. A linear relationship between Mn

occupancy and catalytic activity is demonstrated. Electron Paramagnetic Resonance (EPR)

measurements reveal that the apparent line broadening observed for the Mn signals of wild type

OxDC arises from dipolar coupling between neighboring Mn ions. These results are consistent

with the proposal that there is only a single catalytic site in the enzyme.

The similarity between the two Mn(II) sites has precluded previous attempts to distinguish

them spectroscopically and complicated efforts to understand the catalytic mechanism. My

research utilizes a multifrequency EPR approach to distinguish the two Mn ions on the basis of









their differing fine structure parameters, and observed that acetate and format bind to Mn(II) in

only one of the two sites. The EPR evidence is consistent with the hypothesis that this Mn-

binding site is located in the N-terminal domain, in agreement with predictions based on a recent

X-ray structure of the enzyme.









CHAPTER 1
INTTRODUCTION

Oxalic Acid in Biological Systems

Oxalate producing plants, which include numerous crop plants, accumulate oxalate in the

range of 3% to 80% (w/w) of their dry weight either as the free acid, as sodium or potassium

oxalate, or as an insoluble salt, most commonly calcium oxalate (1-4). A number of possible

pathways for the biosynthesis of oxalic acid in plants have been described (5). These pathways

include the oxidation of glycolate and glyoxylate (byproducts of photorespiration) by glycolate

oxidase (6, 7) and the activity of isocitrate lyase on isocitrate (8, 9). Possible functions of

soluble oxalate and calcium oxalate crystals in plants include protection against insects and

foraging animals, ion regulation, and detoxification of heavy metals (2, 4, 10).

Oxalic acid and its calcium salt accumulate in many fungi but knowledge of its

biosynthetic pathway remains fragmentary. Oxaloacetase, which cleaves oxaloactetate to yield

acetate and oxalate, is present in many fungal species and has been proposed as one biosynthetic

route (5, 11, 12). Phytopathogenic fungi use oxalic acid at the site of infection to lower the pH

which enhances the activity of lytic enzymes such as polygalacturonase and cellulase (13, 14).

Utilization of oxalate by wood rotting fungi to degrade lignin and cellulose has been the subj ect

of much research (15-18).

Bacteria capable of using oxalate as a sole carbon and energy source (oxalotrophic) play

essential roles in soil fertility and retention and/or recycling of elements necessary for plant

growth and are important modulators in the biological carbon cycle (19, 20). Oxalotrophic

bacteria inhabiting the gastrointestinal tracts of mammals provide the only known route for the

catabolism of dietary oxalate in these organisms.









At high concentrations, oxalate causes death in humans and animals as a result of its

corrosive properties. At lower concentrations, oxalate leads to a variety of pathological

disorders, including hyperoxaluria, pyridoxine deficiency, cardiomyopathy, cardiac conductance

disorders, calcium oxalate stones and renal failure (20-24). The administration of oxalate

degrading bacteria (Oxalobacter formigenes) has been proposed as a treatment for hyperoxaluria

(25).

Several oxalate degrading enzymes have either actual or potential commercial significance

with applications in medicine, agriculture, and industry. Oxalate oxidase and oxalate

decarboxylase are used in clinical assays of oxalate in blood and urine (26, 27). Transgenic

plants have been engineered to express oxalate degrading enzymes as a means of protection

against pathogens and to reduce the amount of oxalate present (28, 29). Structural, mechanistic,

and biochemical information is needed in order to further the application of oxalate degrading

enzymes in medicine, agriculture, and industry (28, 30, 31).

Oxalate Degrading Enzymes

Three maj or classes of enzymes have evolved to degrade oxalate. Plants employ oxalate

oxidase (32), fungi (33) and soil bacteria (34) utilize oxalate decarboxylase, and bacteria exploit

oxalyl-CoA decarboxylase (35) (Figure 1.1A). Oxalate oxidases (OxOx), also known as

germins, catalyze the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that

is coupled with the formation of hydrogen peroxide (32, 36, 37). Hydrogen peroxide formation

is believed to serve as a defense mechanism against infection by pathogens (38, 39) and to

contribute to cell wall crosslinking (40). X-ray crystallographic structure determination revealed

that OxOx crystallizes as a hexamer (41) and electron paramagnetic resonance (EPR) studies

demonstrated the presence of Mn(II) in the resting enzyme (36). The Mn ion is coordinated by

four conserved residues (three His and a Glu) (42) and each monomer possesses the P-barrel









topology characteristic of the cupin superfamily of enzymes (28, 43-46). EPR spectroscopic

changes of the Mn signal upon the addition of oxalate supports the hypothesis that the Mn ion is

the site of catalysis (47). UV-visible spectroscopy, spin trapping studies, and structural studies

have lead to proposed mechanisms that involve the binding of oxalate directly to Mn(II), the

formation of Mn(III), and a radical intermediate species (36, 37, 48).


A O 2, H+
HO O =2CO2 + H1202





O O



ThDP CO H So
O O

Figure 1-1 Enzymes that catalyze oxalate degradation. (A) Oxalate oxidases, found in plants.
(B) Oxalate decarboxylases, present in fungi and in some bacteria (C) Oxalyl-CoA
decarboxylases, thiamin-dependent enzymes present in bacteria.

Oxalyl-CoA decarboxylase (OXC) catalyzes the cleavage of oxalyl-CoA to formyl CoA

and carbon dioxide (37, 49, 50) (Figure 1-1C). In Oxalobacter formigenes OXC is coupled to

formyl-CoA transferase, which catalyzes an acyl transfer from formyl-CoA to oxalate to yield

format and oxalyl-CoA (51). The overall coupled reactions convert oxalate to format and

carbon dioxide with the consumption of a proton. Insight into this system was provided by the

isolation and characterization of a membrane bound formate-oxalate antiporter (52-54), which

imports oxalate into the cell and exports format, creating a proton gradient across the membrane

which is then used to drive ATP synthesis (55, 56).









Oxalate Decarboxylase


Biological Distribution

Oxalate decarboxylase (OxDC) catalyzes the difficult carbon-carbon bond cleavage of

oxalate to yield carbon dioxide and format (57) (Figure 1-1B). This enzyme was first reported

in the basidiomycete fungi Collybia (Flammulina) velutipes and Coriolus hersutus more than 50

years ago (33, 58). OxDC has since been reported in a number of fungal species including the

following: Sclerotinia sclerotiorum (59) Coriolus (Traumetes) versicolor (18), Mythrothecium

verrucaria (60), Aspergillus niger (61, 62), Agaricus bisporus (63) and Postia placenta (64).

Fungal OxDCs can be both an intracellular enzyme (18) or excreted from the hyphal cells (18,

64, 65). Expression of OxDC in fungal cultures is induced by decreasing the pH and/or adding

carboxylic acids such as oxalate, glycolate, and citrate (18, 59-61, 66, 67). It has been proposed

that differences in the regulation of OxDC expression may imply differences in the function of

the enzyme in a specific organism (34, 68). Acid-induced OxDCs might be involved in proton

consumption whereas those induced by oxalate may protect the organism from the harmful

metabolic effects of oxalate.

The first prokaryotic OxDC was reported in 2000 when Bacillus subtilis, a soil bacterium,

was shown to express a cytosolic OxDC. Bacterial OxDC is induced by acid but not by oxalate

which may suggest that its metabolic function is not related to its oxalate degrading activity (34,

69). This enzyme, encoded by the yvrK gene (renamed oxdC) has been overexpressed in E. coli

and further characterized (57, 70-74). Comparison of the OxDC fungal sequences with the B.

subtilis genome (75), suggested that two other genes, yoaN (renamed oxdD) and yxaG may also

code for enzymes with OxDC activity (70). The yoaN gene product OxdD was shown to have

low levels of OxDC activity (70) and the yxaG gene product has been reported to be a novel Fe-

containing quercetin 2,3-dioxygenase (76-78).









Oxalate Decarboxylase Belongs to the Cupin Superfamily (DSBH) of Proteins

The cupin superfamily of proteins have been well recognized as possessing remarkable

functional diversity with representatives found in Archaea, Eubacteria, and Eukaryota (28, 43-

46). The identification of the cupin superfamily was originally based on the recognition that the

wheat protein germin shared a nine amino acid sequence with another protein, spherulin,

produced by the slime mold Physarum polycephahmn during starvation (44). This sequence

similarity was also observed in a number of seed storage proteins called germin-like proteins

(GLPs). Knowledge of the three dimensional structures of these proteins led to the collective

name cupin on the basis of their P-barrel shape (' cupa' means "small barrel" in Latin) (46).

Characteristic features of proteins with this fold include high thermal stability and resistance to

proteases. These features are consistent with a high degree of subunit contacts, hydrophobic

interactions, and short loops. The cupin domain was originally described as two conserved

motifs, each composed of two P-strands separated by a less conserved region composed of

another two P-strands separated by a loop of varying length (28, 45). Motif 1 was originally

designated as G(X)SHXH(X)3,4E(X)6G (shown in blue in Figure 1-2) and Motif 2 as

G(X)SPXG(X)2H(X)3H(X)3N (shown in red in Figure 1-2). With more sequences analyzed, it

has become clear that the primary sequence of the two motifs is not as highly conserved as

previously thought (42, 43).

The cupin superfamily of proteins exemplifies a general trend emerging from comparative

genomics: classes of proteins are being expanded beyond the presence of a set of conserved

residues which had previously been the cornerstone of their identification. In 2003,

Anantharaman et al. described the use of information from recently reported X-ray










B subtilis 1 - - - - - - - M
B. clausii 1 - - - - - - K G N K L
C. velutipes 1 MFNNFQRL~LTVI LLSGFTAGVPIAS TTTGTGTATGT STAAEP SATVPFAS TDPNPVLWNE
A. oryzae 1 - - - - -M A SA IF PV GV HK


B. subtilis 3 KQND IPQP IRGD -KGATVKI PRNI ERDRQNPDMLVPPE TD HGTVSNMKFS FSDTHNRL~EK
B. clausii 12 GNPNI PQP IRADGAGGVDRGPRNLMRDLQNPNI LVPPE TDRGLI PNLRFS FSDAHMIQLNH
C. velutipes 61 TSDPALVKPERNQLGATI QGPDNLPI DLQNPDLLAPPTTD HGFVGNAKWPFS FS KQRL~QT
A. oryzae 25 SGFKDGQP ISDNGKGAPLLGGTNKAL~DLQNPDNLGQPS TDNGFVPNLKWS FSD SKTRL~FP


B. subtilis 62 GGYAREVTVRELPI SENLASVNMRL~KPGAI RELHWHKEAEWAYMIYGSARVT IVDEKGRS
B. clausii 72 GGWSRE I TRDLPIATTLAGVNMS LTPGG;VRELKWHKQAEWSYMLLGHARI TAVDQNGRN
C. velutipes 121 GGHABRQQNEVVLPLATNLAC TNMRL~EAGAI RELHWHKNAEWAYVLKGS TQ ISAVDNEGRN
A. oryzae 85 ---VREQVIQDLPQSHDI SGAQQHLKKGAIRELHWHRVAEWGFLYSGSLLLSG;VDENGQ
W ** :: .: *.:******: WW**.: ** @**:*:

B. subtilis 122 FI DDVGEGDLWYFP SGLPH SIQAL~EEG- --AEFLLVFDDGSF SEN- STFQLTDWIAHTPK
B. clausii 132 FIADVGPGDLWYFPPGIPH SIQGLDDG- -CEFLLVFDDGMF SDL- STLS LSDWMAHTPK
C. velutipes 181 YI STVGPGDLWYFPPGI PHSLQATADDPEGSEFI LVFDSGAFNDD- GTFLLTDWLS HVPM
A. oryzae 142 TTEKL~EEGDIWYFPKGVAHNVQGLDDE- --NEYLLVFDDGDFEKVGTTFMVDDWI THTPR
*:** *:. A *. W**WWWW W W *: : **:: W

B. subtilis 178 EVIAANFGVT-KEE ISNLPGKEKY IFENQLPGSLKDD IVEGPNGEVPYPFTYRL~LEQEPI
B. clausii 188 DVLSANFGVP-ESVFATIPTEQVYIYQDEVPGPLQSQQINSYAPTKELPL
C. velutipes 240 EVILKNFRAKNPAANS H IPAQQLY IFPSEPPADNQPDPVS -PQGTVPLPY SFN S SVEPT
A. oryzae 199 D IIAKNFGVD -ASVFDKVPEKFPY ILNGTVSDEANNTPQGTLTGNS SYVYHTYKHP SEPV
:::* ** .* : *

B. subtilis 237 E SEGGKVY IAD STNFKVS KT IASALVTVEPGAMRELHWHPNTHEWQYY I SGKARM~TVFAS
B. clausii 247 VTPGGSVRIVD SRNPVS KT IAAALVEVEPGAMREMHWHPNNDEWQYYLTGQARMTVFTG
C. velutipes 299 QY SGGTAKIAD STTFNI SVAIAVAEVTVEPGAL~RELHWHPTEDEWTFF I SGNARVT IFAA
A. oryzae 258 PGSGGTFRKI D SKNPVS QT IAAALVELEPKGLRELHWHPNAEEWLYFHKGNARATV~LG
**. ** *W :** *W :* .*****WWW .** ::.:*@ @*:

B. subtilis 297 DGHARTFNYQAGDVGYVPFAMGHYVENI GD -EPLVFLE IFKDDHYADVSLNQWLAMLPET
B. clausii 307 NGVARTFDYRAGDVGYVPFATGHYIQNTGN-ESVWF~LEMFKDEVLNLATE
C. velutipes 359 QSVAS TDYQGGD IAYVPASMGHYVENIGN- TTLTYLEVFNTDRFADVSL SQWLAL~TPPS
A. oryzae 319 DSKARTFDFTAGDTAVF~PDNSGHYIENTSETEKLVWIEIYKSRVDSLAQWLAL~TPAD
S*: ** WW .* ***: .: :*:: ** *** ** ***@@ WW*

B. subtilis 356 FVQAHLDLGKDFTDVLSKEKHPVVKKKCSK
B. clausii 366 LVQHNIHVDSKFTNKLRKEKWPVVKYPTI-
C. velutipes 418 VVQAHLNLDDETIAELKQFATKATVVGPVN
A. oryzae 378 VVATTLKVDIEVVKQIKKEKQVLVKGK- --





Figure 1-2 Sequence alignments of OxDCs from Bacilhts subtilis, Bacilhts clausii, Collybia
vehttipes, and Aspergilhts oryzae showing the positions of the two conserved motifs
(motif 1 in blue and motif 2 in red) in the two domains. Alignment was made using
the Clustal W method (79, 80). Asterisks indicate identical residues, colons(:)
indicate conservative substitutions, and periods (.) indicate semi-conservative
substitutions. The Mn-binding residues of the Bacilhts subtilis OxDC are underlined.


crystallographic structures and sequences to gain a perspective on the major principles that


appear to have shaped the emergence of diverse enzymatic activities within structurally similar









and evolutionarily related scaffolds (81). The absence or presence of various metals such as Ni,

Fe, Zn, Mn, or Cu contribute to the functional diversity of the cupin superfamily (43, 78). The

term cupin has been expanded into the Structural Classification of Proteins (SCOP)

(http:.//scop.berkeley .edu/data/scop.b .html) database as the double-stranded P-helix (DSBH)

multicatalytic fold. The terms cupin and DSBH are now used synonymously (82-84). Since

OxDC has two of these characteristic DSBH domains it is further classified as a bicupin. It has

been suggested that OxDC evolved from OxOx by gene duplication and selection (28, 37, 45).

This suggestion is consistent with current models of enzyme evolution (85, 86).

Anantharaman et al. (81) propose that ancestral forms of the DSBH can be evolutionarily

reconstructed as simple, small-molecule-binding domains that perhaps bound sugars and cyclic

nucleotides (45, 81, 87) and that it is from these sugar-binding domains that sugar-modifying

domains such as isomerases and epimerases arose. They further propose (81) that a set of

conserved histidine residues employed in sugar-binding in the ancestral non-enzymatic domain

evolved into the metal coordinating histidine residues observed in germin (88) and oxalate

decarboxylase (72) and that another lineage of DSBH domains acquired a new set of conserved

residues with the ability to bind 2-oxoglutarate which gave rise to the iron-2-OG-dependent

dioxygenases.

Structural Features

High resolution X-ray crystal structures of~acillus subtilis OxDC (72, 73) have confirmed

that the OxDC monomer is composed of two P-barrel domains, each of which contains a metal-

binding site (Figure 1-3A). These metal ions are 26 angstroms apart from each other in the

monomer. Evidence from inductively-coupled plasma mass spectrometry (ICP-MS) (89) and









EPR spectroscopy initially suggested that OxDC activity was Mn-dependent (70). This


hypothesis is consistent with crystallographic observations (72, 73).


(A)


(c)


Figure 1-3 Ribbon structures of the Bacillus subtilis OxDC monomer, trimer, and hexamer. (A)
The N- and C-terminal cupin domains of the OxDC monomer are colored green and
purple, respectively, and the N-terminal segment that contributes to the secondary
structure of the C-terminal domain is colored red. The yellow spheres show the
locations of the two Mn centers. (B) Structure of an OxDC trimer in which the
monomers are colored red, green and purple. The locations of Mn ions are shown by
the purple (N-terminal domain) and yellow spheres (C-terminal domain). (C)
Structure of the OxDC hexamer in which one monomer is colored red to emphasize
the role of a-helical regions in mediating monomer/monomer interactions. These
structures were visualized using the CAChe Worksystem Pro V6.5 software package
(Fujitsu America Inc., Beaverton, OR).










The quaternary structure of OxDC is hexameric (Figure 1-3C) composed of two trimeric

layers (Figure 1-3B) packed face to face that have 32 (D3) pOint symmetry. The OxDC trimer

resembles the OxOx hexamer (37). The OxDC hexamer has a diameter of approximately 90

angstroms and a thickness of 85 angstroms. A large solvent channel (15 angstroms wide) runs

through the hexamer along the 3-fold axis (72). The trimeric layers of the hexamer are

stabilized by ot-helical protrusions of adjacent monomers (Figure 1-3C).


A QPhe-155 G u-101d Phe-134 B .Phe-335

,J Val-313
~cChe-160 'Hi -97 G~y u-33 T

J / 140O Tyr-340 His-275
His-319


Glu-162 ~3 :His-95
His-273


Arg- ) Arg-27 '


Figure 1-4 Residues defining the Mn-binding sites in A) the N-terminal (1UW8) domain of
OxDC and B) the C-terminal (1J58) domain of OxDC. Residue numbering is for the
enzyme encoded by the OxdC gene in Bacillus subtilis. For clarity, hydrogen atoms
bound to carbon atoms are omitted. Atom coloring: C, black; H, white; N, blue; O,
red; Mn, silver. These structures were visualized using the CAChe Worksystem Pro
V6.5 software package (Fujitsu America Inc., Beaverton, OR).

Both of the Mn-binding sites in the OxDC monomer resemble the Mn-binding site of

OxOx in that each Mn ion is coordinated by the side chains of four conserved residues (Figure 1-

4) in a distorted octahedral environment. The manganese-binding residues in the N-terminal

domain are His95, His 97, His 140, and Glul01 and in the C-terminal domain are His273,

His275, His3 19, and Glu280. In one of the available X-ray crystallographic structures ("open"

conformation), the N-terminal Mn-binding site contains one water molecule and one format









molecule while the C-terminal Mn-binding site contains two water molecules (72). In another

available X-ray crystallographic structure ("closed" conformation), however, the N-terminal

Mn-binding site contains two water molecules and the C-terminal Mn-binding site contains a

single water molecule in a penta-coordinated form (73).

Not only do oxalate oxidase and oxalate decarboxylase possess remarkably similar Mn-

binding sites, the metal-binding cavity is also intriguingly similar in that it is lined primarily by

hydrophobic residues. Given these similarities and common substrate, it appears that only subtle

changes are necessary to promote different biochemical activities. It has been proposed that the

absence of a proton donor in the active site of OxOx prevents it from catalyzing the

decarboxylation of oxalate (72). The putative proton donors) in OxDC have been proposed to

be Glul62 in the N-terminal domain (73) and/or Glu333 in the C-terminal domain (72).

Mechanistic Information

Oxalate decarboxylase requires molecular oxygen for catalytic turnover (57, 58, 61, 70)

even though the production of format and CO2 frOm oxalate involves no net oxidation or

reduction. Furthermore, all of the OxDCs that have been characterized possess optimum activity

at acidic pH and exhibit a high substrate specificity for oxalate (37, 57, 58, 70). Efforts to

elucidate the catalytic mechanism through the use of heavy atom isotope effects (57), electron

paramagnetic resonance spectroscopy (71), density functional theory calculations (90),

homology modeling with oxalate oxidase sequences (91), structural information and site-directed

mutagenesis studies (72, 73) have led to a number of mechanistic proposals.

Heavy-atom (13C and IsO) kinetic isotope effect (KIE) measurements (92) were used to

probe the structure of the transition state for the decarboxylation step for the recombinant, wild

type OxDC from B. subtilis (57). Since V/K KIEs were measured in these competition

experiments, no information can be obtained for the steps that occur after carbon-carbon bond









cleavage (93). The pH dependence of the enzyme catalyzed reaction suggests that

monoprotonated oxalate is the actual substrate for OxDC (57) and that the substrate likely binds

directly to the Mn in the enzyme-substrate complex (37).







H O O PCET Hi M "s
Hisl~ O His' I lu OT
His' FluHis NH,
His NH,
U~~ I-.NK N'r g
HN N ~Arg H

-CO,

O O
O' -H GuO'O~ O Gl
His,, basexO- Hisi e 0 *-
His' I Flu O His' I Flu O~
His NHf His NH,+
H N 1 'Ar H N N^'Ar
H H


Figure 1-5 Proposed catalytic mechanism for oxalate decarboxylase based on heavy-atom
isotope effect measurements.

Heavy-atom KIEs measured at pH 4.2 and 5.7 are consistent with a two step model in

which a reversible step precedes carbon-carbon bond cleavage and decarboxylation (Figure 1-5)

(37, 57). In this proposal, a reversible proton-coupled electron transfer (94, 95) yields a Mn-

bound oxalate radical anion, which then decarboxylates to form CO2 and a format radical anion.

Protonation of the Mn-bound format radical anion produces format which is then liberated

from the enzyme. In this proposal, active site glutamate residue(s) serve as a general acid/base

catalyst and active site arginine residue(s) act to polarize the oxalate carbonyl bond (57). The

oxidation state in the above mechanism is purely hypothetical and remains to be demonstrated by










experimental methods. Other proposals have invoked that the oxidation state of manganese

alternates during catalysis, between Mn(II) and Mn(III) (70, 74) or Mn(III) and Mn(IV) (72).

Only Mn(II) has been detected experimentally by either standard perpendicular-mode or parallel-

mode EPR spectroscopy of the resting enzyme (70, 71, 74) or during turnover (71).

Oxygen Dependence and the Formation of Hydrogen Peroxide

The first report of an oxalate decarboxylase observed that decarboxylation did not proceed

under strictly anaerobic conditions and that the introduction of air into the manometric apparatus

restored the activity to the original level (33). Subsequently reported OxDCs have shown a

similar oxygen dependence (37, 57, 58, 60-62, 70) but vary with respect to the level of activity

restored upon the reintroduction of oxygen. The role of dioxygen in the catalytic cycle is

unknown and cannot be replaced by other oxidizing agents such as H202, paraquinOne, 2-methyl-

1,4-naphthoquinone, flavin adenine dinucleotide, flavin mononucleotide, and cytochrome c (58).

The most extensive characterization of the oxygen dependence of OxDC was carried out

on the enzyme from Aspergillus niger using manometric techniques (62). In this study, the

influence of the partial pressure of 02 On the enzyme was observed by replacing the air in the

Warburg apparatus by mixtures of 02 and N2. In the absence of o-phenylenediamine (oPDA),

maximal activity was obtained at 0.04 atm of Oz, whereas, in the presence of oPDA maximal

activity was obtained at 0.2 atm. Pressures greater than optimal accelerated the irreversible

inactivation of the enzyme even in the presence of oPDA. Inactivation occurs, however, only

during catalytic turnover since bubbling 02 through the enzyme solution prior to the addition of

substrate did not affect product formation. This suggests that if 02 binds directly to the metal, it

does so after oxalate binding. Oxalate oxidase activity has been reported for both fungal and

bacterial oxalate decarboxylases. The rate of oxalate oxidation relative to decarboxylation is 1.5-









3.0% for the A. niger enzyme (62) and 0.2% for the recombinant, wild type B. subtilis enzyme

(70).

Research Objectives

The overarching goals of this research are motivated by the fact that the metal centers in

oxalate decarboxylase and oxalate oxidase are evolutionarily related (28) even though the

chemical transformations catalyzed by the enzymes are different (32, 33). This research seeks to

employ the techniques of bioinorganic chemistry, molecular spectroscopy, enzyme kinetics, and

protein engineering to characterize oxalate decarboxylase. Increased knowledge of this enzyme

may impact our general understanding of metalloenzyme evolution and the role of the protein

environment in modulating reactivity (86, 96).

The specific obj ectives of the presented work were: 1) to optimize the expression and

purification procedures to obtain OxDC with high manganese occupancy; 2) to characterize the

manganese dependence of the enzyme; 3) to distinguish the manganese-binding sites

spectroscopically; and, 4) to determine which manganese center is the site(s) of catalysis.









CHAPTER 2
CHARACTERIZATION OF THE MN-DEPENDENCE OF OXALATE DECARBOXYLASE
ACTIVITY

Introduction

Three pieces of indirect evidence support the idea that OxDC activity is Mn-dependent.

First, Mn(II) is present in the resting form of recombinant, wild type Bacilhts subtilis OxDC

when the enzyme is expressed in Escherichia coli (70, 71). Second, the successful expression of

correctly folded OxDC with reasonable catalytic activity specifically requires the presence of

Mn(II) in the growth medium (70). Third, the X-ray scattering factors for Mn which were used in

the refinement of the high-resolution structures of recombinant Bacilhts subtilis OxDC were

fully consistent with this metal being bound within both DSBH domains (72, 73). On the other

hand, the native form of OxDC from Bacilhts subtilis could not be purified in sufficient

quantities for accurate metal analysis (34), and other enzymes in the bicupin family appear to be

able to employ a variety of metals in catalysis (76, 78, 97). In order to characterize the Mn-

dependence of recombinant B. subtilis OxDC, an in vivo strategy was employed for obtaining

recombinant, wild type OxDC in which Mn is substituted by Co, and in vitro conditions for re-

constituting the recombinant enzyme with Mn were developed.

Results and Discussion

Optimization of Expression of Recombinant Wild Type OxDC

Expression conditions for obtaining OxDC were optimized so that pure samples of the

enzyme could be routinely obtained with a metal content of 1.6-1.9 Mn/monomer rather than the

0.86-1.14 Mn/monomer reported in prior studies of the enzyme (57, 70). At this level of Mn

incorporation, recombinant OxDC exhibits a specific activity of 40-65 U/mg as measured in an

endpoint assay employing format dehydrogenase (FDH) (98). To obtain reproducibly high

levels of Mn incorporation, protein expression was induced at a lower optical (0.6 at 600 nm)









density than previously reported (57), and cells were grown at a post-induction temperature of

30oC so as to promote the transport of manganese ions into the bacterial cells (99, 100).

Consistent with previous reports, we observed the oxalate dependence of the decarboxylase

activity followed Michaelis-Menton kinetics, with our Km value being lower than previously

reported (8.4 vs. 15 mM) (70).

Effect of Addition of Other Metals in the Growth Medium

Having established conditions for obtaining recombinant OxDC with high specific activity,

we next investigated whether including other salts in the growth medium might yield enzyme in

which manganese had been replaced by other transition metals. Introducing FeCl2, FeCl3, Of

CoCl2 into to the growth medium in place of MnCl2 yielded samples of the recombinant, wild

type enzyme with varying levels of Mn incorporation. All of these variants behaved similarly to

the wild type, Mn containing OxDC on purification but exhibited activities that correlated best

with their Mn content leading to the conclusion that metals such as Co or Fe do not support

catalysis.

When CoCl2 (2 mM) was used as a supplement, ICP-MS analysis showed that samples of

recombinant OxDC contained 0.80 Co/monomer and 0.05 Mn/monomer (Table 2-1). Given the

very low specific activity of the Co-substituted OxDC was similar to that expected solely on the

amount of Mn present in the enzyme sample, we assumed that enzyme-bound Co(II) did not

catalyze the decarboxylation reaction and therefore investigated the effects of expressing the

enzyme with mixtures of the chloride salts of both Co(II) and Mn(II) in the growth medium

(Table 2-1). We anticipated that different concentration ratios of the exogenous salts would yield

OxDC samples substituted with different levels of Mn, and this proved to be the case although

no obvious correlation was observed between the Mn:Co ratio and the extent to which Mn or Co

was incorporated into the recombinant enzyme.










Table 2-1 Effect of MnCl2 and CoCl2 in the growth medium on metal incorporation and specific
activity of recombinant, wild type OxDC. Metal content is expressed as the number
of metal ions/OxDC monomer. n. d. indicates that the value was not determined. These
samples contained <0.01 atoms/monomer Mg.

OxDC MnCl2 C0012 Mn CO Zn Fe Cu Specific Activity
Preparation (mM) (mM) (U/mg)

1 5 0 1.87 n.d. 0.51 0.07 0.01 61.2
2 5 0 1.87 n.d. 0.13 0.19 < 0.01 50.1
3 5 0 1.63 n.d. 0.08 < 0.01 < 0.01 40.9
4 0 2 0.05 0.80 0.14 < 0.01 < 0.01 2.2
5 0.25 2 0.27 1.03 0.22 0.18 < 0.01 8.0
6 1 1 0.12 1.32 0.26 0.13 < 0.01 4.5
7 5 0.25 0.56 0.68 0.22 0.13 < 0.01 13.5
8 5 0.05 0.47 0.09 0.19 0.06 < 0.01 19.0

When a 20: 1 Mn:Co ratio was employed the two metals were incorporated into the enzyme

in approximately equal amounts. Increasing the amount of MnCl2 relative to CoCl2 in the growth

medium, however, did not yield wild type OxDC containing more Mn than Co. This finding

likely reflects the tight regulation of Mn metabolism that is observed in bacteria such as

Escherichia coli (100-103). Although a positive correlation between Mn content and

decarboxylation rate was evident on assaying the activity of the Mn/Co-substituted enzymes, we

could not definitely conclude that decarboxylase activity was linearly correlated with Mn content

on the basis of these in vivo experiments because no expression conditions could be identified

that gave OxDC samples containing 0.7-1.5 Mn/monomer.

Preparation of the OxDC "Apoenzyme" and Reconstitution of the Wild Type, Mn-
Containing Enzyme

In order to correlate specific activity with metal content in the 0.7 1.5 Mn/monomer

range, we examined alternate strategies to obtain samples of the OxDC apoenzyme (104-109),

then prepare enzyme samples reconstituted with manganese. Following procedures that had

been reported for removing the metals from other metalloenzymes, we tried a variety of

chelating agents with and without chaotropic agents. In contrast to the bicupin quercetin 2,3-










dioxygenase (76, 78, 97), it proved remarkably difficult to remove the metal ion from wild type

OxDC. Many literature conditions either did not remove the metal or led to irreversible protein

denaturation. The removal of Mn from recombinant OxDC and its subsequent reconstitution with

Mn was eventually accomplished, however, following a protocol based on that used to obtain the

apoenzyme of Mn-dependent superoxide dismutase (Mn-SOD) (110-112).

Table 2-2 Metal content of "apoenzyme" and enzyme reconstituted with Mn. Metal content is
expressed as the number of metal ions/OxDC monomer. n. d. indicates that the value
was not determined.

Enzyme Preparation Mn Co Zn Fe Cu Mg Specific Activity


WT OxDC 1.87 n.d. 0.51 0.07 0.01 0.01 61.2 U/mg
"Apoenzyme" 0.01 n.d. 2.0 0.01 0.01 0.01 0 U/mg
Reconstituted OxDC 1 0.90 n.d. 0.79 0.14 0.01 0.01 25.1 U/mg
Reconstituted OxDC 2 0.64 0.01 1.20 0.01 0.02 n.d. 14.4 U/mg
Reconstituted OxDC 3 0.84 0.01 0.60 0.01 0.01 n.d. 19.5 U/mg


This multi-step procedure (see Experimental Methods section) involved partially unfolding

the protein in 3.5 M guanidinium hydrochloride (GuHC1) with ethylenediaminetetraacetic acid

(EDTA) present. Refolding samples into a buffered solution without added metals resulted in a

manganese-free "apoenzyme" (2 Zn/monomer) in which Zn(II) had replaced Mn(II) in wild type

OxDC (Table 2-2). Alternate metal ions could also be introduced into the "apoenzyme" by

refolding samples into a buffered solution containing salts such as MnCl2. These conditions

were used to prepare samples of recombinant OxDC containing 0.64, 0.84, and 0.90

Mn/monomer (the remaining metal sites being occupied by Zn) for kinetic characterization. The

specific activities of these samples were determined (Table 2-2). Combining these data with the

data obtained adding CoCl2 to the growth medium resulted in a plot of manganese content vs.

specific activity which suggests a linear correlation between decarboxylation rate and Mn

incorporation (Figure 2-1)











70 ,

60 --


O 30



50 *l *





0 0.5 1 1.5 2
M n/m onom er

Figure 2-1 The dependence of OxDC specific activity on the extent of Mn incorporation. The
line shows the specific activity that would be expected assuming a linear correlation
with Mn content.

Gepasi Simulations

The observation of a linear dependence of OxDC specific activity on Mn incorporation

places an important constraint on kinetic models for the number of active sites in OxDC that may

mediate catalysis. The fact that OxDC is a bicupin capable of binding up to 2 Mn/monomer

raises questions concerning the number and location of the catalytic sites that mediate C-C bond

cleavage (72, 73). Previous efforts to address these questions by steady state kinetic

characterization of OxDC mutants in which residues implicated in proton transfer (Glu-162 and

Glu-333) were site-specifically modified have given ambiguous results (72, 73). The steady-

state behavior of seven kinetic models was, therefore, simulated using the GEPASI simulation

package (113, 114) to evaluate the effects of varying active site number and Mn-binding site

affinity on the observed Mn-dependence of catalytic activity.






























n n.5 I L5s
kI1*lhnll mmi n per ain


OnDC Gepai Simulation*














11 LSI 1.)
rEnid hostal Mn per unit


OxDC Gecpasi Simulations
Case 7 innl) full Ce~r.Olc me anne, conPert hani~ng













Lt$ I L5
real Exrull MIn pr ulrn


OxDC Gepasi Simlulation~s
Case 4 (site I iund2 have eqaluo acurity,


OxDC Gepasi Simulations
Cas h Irate I acturE. cemperatiS a inding)














total lusurl Min pe use


Figure 2-2 Numerical simulations of the dependence of catalytic activity on the extent of Mn
incorporation. Full details of the kinetic parameters used in the simulations are

provided in Appendix A. All panels show the amount of product formed after a

reaction time of 5 s. Note that site 1 and site 2 cannot be associated with a specific N-

or C- terminal Mn-binding site.









To date only three models have been identified that are consistent with experimental

observations (Case la, Case 4a,b,and c, and Case 6a)(Figure 2-2). In Case la one site (site 1) has

catalytic activity that is independent of metal occupancy of the second, inactive Mn-binding site

(site 2) and these two sites possess the same affinity for Mn. In Case 4 a linear relationship is

observed independent of affinity for Mn as both sites have equal levels of catalytic activity.

Case 6 assumes that although catalytic activity is only associated with a single site (site 1), the

affinity of Mn for site 2 is increased 100-fold when Mn occupies the active site (site 1). Kinetic

models that seek to simulate an earlier proposal (8) in which the N-terminal site is responsible

for the maj ority (if not all) of activity, with the C-terminal site being primarily important in

maintaining enzyme structure, only gave a non-linear relationship between activity and Mn

incorporation (Cases 3, 5 and 7).

A model in which both sites have equal activities (Case 4) reproduces the observed linear

relationship between activity and Mn content. On the other hand, for models where it is assumed

that only a single site (site 1) can mediate catalysis, it is difficult to obtain a linear plot. For

example, a model (Case 3) in which the catalytic activity of site 1 is turned on when Mn(II)

occupies the second binding site (site 2) yields a non-linear relationship between activity and Mn

incorporation. Similarly, permitting differences in the affinity of the two sites for Mn while

requiring that catalytic activity be localized within a single site (site 1), irrespective of the

occupation of the second site (site 2), results in non-linear behavior (Cases lb and 10, Cases l'b

and l'c). Linear behavior is anticipated for this model only if the two sites initially have an equal

affinity for Mn. It is possible, however, to obtain a linear dependence using a kinetic model

(Case 6a) in which the affinities of the two binding sites are initially identical but Mn occupancy

of site 1 results in an enhanced affinity of site 2 for the metal. We note that such a model









corresponds to a recent suggestion that structural interactions between the cupin domains may be

important for yielding enzyme with full catalytic activity (73).

Experimental Section

Materials

Unless otherwise stated, all chemicals and reagents were purchased from Sigma-Aldrich

(St. Louis, MO), and were of the highest available purity. Protein concentrations were

determined using a modified Bradford assay (Pierce, Rockford, IL) for which standard curves

were constructed with bovine serum albumin (14). All DNA primers were obtained from

Integrated DNA Technologies, Inc. (Coralville, IA), and DNA sequencing was performed by the

core facility in the Interdisciplinary Center for Biotechnology Research (ICBR) at the University

of Florida. The metal content of wild type OxDC, and all site-directed OxDC mutants, was

quantified at the University of Wisconsin Soil and Plant Analysis Laboratory on the basis of

ICP-MS measurements (89).

Expression and Purification of Recombinant, Wild Type OxDC.

Recombinant wild-type Bacillus subtilis OxDC was expressed and purified using a

modified literature procedure (57). Thus, Luria-Bertani broth (50 mL) containing 50 Cpg/mL

kanamycin (LBK) was inoculated with oxdC:pET-9a/BL21 (DE3) and incubated overnight at 37

oC. An aliquot (4 mL) of this stationary phase culture was then used to inoculate Luria-Bertani

broth (5 x 400 mL) and the resulting cultures were incubated at 30 oC until reaching an OD600

value of 0.6. At this time, the bacteria were heat-shocked at 42 oC for 10 minutes before the

addition of isopropyl thiogalactoside and MnCl2 to Einal concentrations of 1 and 5 mM,

respectively. The induced cells were then grown at 30 oC with shaking to ensure maximal

aeration for 4 h. The cells were harvested by centrifugation (6000 rpm, 20 min, 4 oC), and the

pellets re-suspended in 50 mM imidazole-C1, pH 7.0, (100 mL) before sonication. The lysate was









clarified by centrifugation (10,000 rpm, 20 min, 4 oC) and stored overnight at 4 oC. The lysis

pellets were re-suspended in 50 mM imidazole-C1, pH 7.0, containing IM sodium chloride, 10

CLM MnGl2, 0.1% Triton X-100, and 10 mM 2-mercaptoethanol (total volume 100 mL), and the

resulting mixture stirred overnight at 4 oC. After centrifugation (10,000 rpm, 20 min, 4 oC), the

solubilized extract was combined with the lysate and diluted 7-fold before being applied to a

DEAE-Sepharose Fast Flow column (2.5 x 25 cm) equilibrated with 50 mM imidazole-HC1, pH

7.0 (buffer A). Elution was performed using a 500 mL linear gradient from buffer A to buffer A

containing 1 M NaC1. Fractions containing OxDC were pooled, and solid (NH4)2SO4 added to a

Einal concentration of 1.7 M. The resulting solution was applied to a phenyl-Sepharose Hi-

Performance column (2.5 x 18 cm) (GE Healthcare, Piscataway, NJ) equilibrated with 50 mM

imidazole-C1, pH 7.0, containing 1.7 M (NH4)2SO4 (buffer B). Bound proteins were eluted using

a 500 mL linear gradient from buffer B to buffer A, and fractions containing purified OxDC

were pooled, and concentrated by ultrafiltration in an Amicon stirred cell (Millipore, Billerica,

MA) to a Einal volume of 10 mL before being exhaustively dialyzed against 20 mM

hexamethylenetetramine hydrochloride, pH 6.0, containing 0.5 M NaC1. The dialyzed enzyme

was then concentrated to approximately 9 mg/mL, and stored in aliquots at -80 oC.

Expression and Purification of Co-Substituted, Wild Type OxDC

The recombinant Co-containing, wild-type enzyme was obtained following the standard

protocol for expressing the Mn-substituted enzyme, except that CoGl2 (Fisher Scientific,

Pittsburgh, PA) or various CoGl2/MnGl2 mixtures were added to the cell culture in place of

MnGl2, after the heat shock step but prior to induction of OxDC expression. After cell lysis, and

extraction of the recombinant protein from the crude lysate as described above for recombinant

wild-type OxDC, the Co-containing enzyme was purified by DEAE column chromatography.

Fractions containing OxDC were pooled and dialyzed for 4 h against 50 mM imidazole-HCI









buffer, pH 7.0 (2 L). The resulting sample was then applied to a Q-Sepharose Hi-Performance

column (2.5 x 18 cm) column equilibrated with buffer A, and eluted using a 500 mL linear

gradient from buffer A to buffer A containing 1 M NaC1. Fractions containing OxDC were

pooled and exhaustively dialyzed against 20 mM hexamethylenetetramine hydrochloride, pH

6.0, containing 0.5 M NaC1. The purified, Co-substituted enzyme was concentrated and stored as

described for recombinant, wild type OxDC.

Preparation of the OxDC "Apoenzyme" and Reconstitution of the Wild Type, Mn-
Containing Enzyme

Recombinant Mn-containing, wild type OxDC was dialyzed against 3.5 M guanidinium

hydrochloride (GuHC1), 20 mM Tris-HC1, and 10 mM EDTA, pH 3.1, for 8 h at 4 oC. A second

round of dialysis against 2.5 M GuHC1, 20 mM Tris-HC1, and 10 mM EDTA, pH 7.0, was

performed, and excess EDTA removed in a third dialysis against 2.5 M GuHCI containing 20

mM Tris-HC1, pH 7.0. Both of the latter steps were carried out for 8 h at 4 oC. At this stage, the

protein could be re-folded by dialysis against 20 mM hexamethylenetetramine-HC1, pH 6.0,

containing 0.5 M NaC1, over 8 h at 4 oC, to yield a Mn-deficient form of OxDC ("apoenzyme")

that exhibited no catalytic activity when incubated with oxalate. Alternatively, a round of

dialysis over 8 h, at 4 oC, against 20 mM Tris-HC1, pH 7.0, containing 10 mM MnGl2 COuld be

used to re-introduce Mn(II) into the enzyme before exhaustive exchange into 20 mM

hexamethylenetetramine-HC1, pH 6.0, containing 0.5 M NaGl 4 oC. The latter re-folding step

gave samples of reconstituted OxDC containing Mn.

Metal Content Determination

Two methods were used to prepare samples and blanks for determination of metal content

by ICPMS (University of Wisconsin Soil and Plant Analysis Lab). In the first method,

approximately 0.2 mM enzyme samples (200 CIL of ~10 mg/mL) were made 1 mM









ethylenediaminetetracetic acid (EDTA) and incubated on ice for 15 minutes. Samples were then

desalted on a G-25 pasteur pipet column equilibrated with dH20. The desalting column had been

previously treated with EDTA. 200 CIL of storage buffer was put through an identical procedure

for use as a blank. In the second method, divalent cations were removed from 20 mM

hexamethylenetetramine hydrochloride, pH 6.0 containing 0.5 M sodium chloride by passing

through a 1.5 x 16 cm column containing Chelex 100 (Bio-Rad) in the Na+ form. Purified

protein samples were exchanged into the resulting buffer by washing 2.5 mg samples three times

with 10-fold volumes of the "scrubbed" buffer in Centricon or Centriprep 30 (Amicon)

concentrators (104). The final filtrates were recovered and used as blanks, which routinely

possessed insignificant metal content. Samples were sent to the University of Wisconsin Soil

and Plant Analysis Laboratory for the determination of metal content by ICPMS. Both methods

yielded similar results, results reported here are from the second method.

Steady-State Kinetic Assays

Assay mixtures consisted of 50 mM NaOAc, pH 4.2, 0.2% Triton X-100, 0.5 mM o-

phenylenediamine, 1-50 mM potassium oxalate, and either the wild type OxDC (1-4 CLM) or

OxDC mutant (80-120 CLM) (100 CLL total volume). Reactions were initiated by the addition of

substrate, incubated at ambient temperature (21-23o C), and quenched by the addition of 1 N

NaOH (10 CLL). The amount of format product was determined by an end-point assay (98)

consisting of 50 mM potassium phosphate, pH 7.8, 0.09 mM NAD and 0.4-1.0 U/mg of

format dehydrogenase (1 mL final volume). The absorbance at 340 nm was measured after

overnight incubation at 37oC, and format was quantitated by comparison to a standard curve

generated by spiking pre-quenched OxDC assay mixtures with known amounts of sodium

format. Measurements were made at specific substrate and enzyme concentrations in duplicate,










and data were analyzed to obtain the specific activity by standard computer-based methods

(115). The initial rate of format production is expressed in millimoles per liter per minute.

Gepasi Simulations

The rates used for the reactions for the various simulations (Figure 2-2) have been cut and

pasted from the Gepasi output files into Appendix A. The equations used for the simulations are

given in Figure 2-3. To ensure that the binding equilibria for the Mn ions are well established

before catalysis takes place, the kl values for the forward reaction in equations [R1-4] were

chosen to be fairly large, i.e., close to the diffusion limit: k1(R1-R4) = 1 x 109 (SM)- k2(R1-R4)

= 5(s)l


kg (R1)
kz(R1)
kg (R12)
k2(R2)
kg (R3)
k2(R3)
kz (R4)
kZ(R4)
ksl(R5)
k2z(R5)
kl(H61
kgl(RT)
k2(R7)
ky (RS)

ky (RS)
kfl(R10)
---------+


Mn +- apo

Mn +- apo

Mn + EMnA

Mun + IE~lns

]E S

IES

EMnA +- S

EMnB + S

EMnAS
EMnBS


EhInx

Eh3nB

E

]E

ES

]E + P

EhlnxS

EMusS

EMnA P
EMnB + P


Figure 2-3 Equations used for Gepasi simulations to describe the amount of product formed after
5 s as a function of Mn incorporation.

The rate constants for dissociation were small to ensure good binding. 5 s-l represents a

KD Of 5 nM. For low affinity 5,000 s^l was chosen, representing 5 CIM affinity with unchanged









kl. As a result this ended up giving full sites for a complement of Mn ions present. The binding

of substrate to enzyme is given by:

kl(R5,R7,R8) = 1 x10s (s M)1

k2(R5,R7,R8) = 8.4 x 105 (s)l

kz(R5, R7, R8) + kl(R6, R9, R10)
These values were chosen to give a Km 8.4 mM
ki(R5, R7, R8)

and keet = kl(R6,R9,R10) = 53 (s)l

In order to simulate inactive sites, the keet value was reduced to 5 x 10-6 S-1. For "less

active" sites the keatvalue was changed to values between 17 and 35 s^l. For "half active" sites

the keetvalue was chosen to be 26.5 s^l. In case 3 where site 2 was mechanistically required all

k1(R9,R10) = 5 x 10-6 S-1 and only kl(R6) = 53 s^l. This resulted in non-linear behavior in all

instances due to the fact that only the full enzyme is capable of catalysis.

The linear relationships in Case 4 are due to the fact that each bound Mn participates

equally in catalysis. This case is thus insensitive to the affinity of the two sites. To simulate

cooperative binding (Case 6) positive feedback was assumed for the binding of the second site.

Only site 1 is equally active in both the single Mn as well as the full enzyme. To simulate

cooperativity of binding the second site was assumed to have a factor of 100 higher affinity once

the first site was filled. To see what happens when only the full enzyme was active, case 7 was

created which is otherwise equal to case 6. It shows essentially the same result as case 3 where

only the full enzyme was capable of catalysis. In all cases, the starting conditions involved 1

mM apoenzyme concentration, up to 2 mM Mn concentration, and a saturating substrate

concentration of 10 M.

Case 1 (site 1 active, site 2 unimportant): In this case only site 1 is active and the presence or

absence of site 2 is simply irrelevant. However, as the results show it is not quite so irrelevant if









it is allowed to bind substrate (and thus sequester the enzyme in that state). Thus the "dip" seen

in case Ic is artifactual since in reality one would expect the enzyme to also allow substrate to

bind in the other site.

Case 1': To avoid the problem with the "dip" in case 1, a lower catalytic rate was assumed for

the singly bound Mn, i.e., 26.5 s^l for the single Mn case vs. 53 s^l for the full enzyme. As it

turns out the difference in activities between the singly and doubly occupied enzyme starts to

"curve" the former straight line predicted for equal affinity of the Mn sites. This is expected

since the model introduces some form of cooperativity into the catalytic mechanism.

Case 2 (site 1 most active, site 2 less active): is another modification of case 1, in which the rates

were input as 53s-l for the full enzyme, 35 s^l for site 1, and 8 s^l for site 2.

Case 3 (site l active, and site 2 required structurally or mechanistically): The quadratic behavior

of the case with equal affinity is confirmed and makes sense because the number of fully loaded

enzymes is quadratic with the concentration of bound Mn.

Case 4 (site 1 and site 2 have equal activityi): Case 4 doesn't show any difference in its kinetics

except a small difference in total bound Mn for the different data points, just as expected.

Case 5 (site 2 required for f dll activity of the enzyme): This case is similar to case 3 but relaxes

the necessity of the mechanistic site 2 a bit by allowing for "half activity" of site I when site 2 in

not occupied.

Case 6 (site 1 is active, binding is cooperative 0I ithr the affinity for the first Mlln being different for

the two sites): Case 6 represents cooperative binding of the Mn sites. It is assumed that the

subsequent Mn binds with a 100 fold higher affinity than the first one. However, the cases 6b

and 6c differ, just like in all other cases treated before, by 3 orders of magnitude in their initial

affinity for sites 1 and 2. In other words, when the first affinity is 5 nM the second affinity is









now 50 pM, and when the first affinity is 5 CIM the second is now 50 nM. It should be noted that

the activity of site I was assumed to be 53 sl for both the fully loaded enzyme as well as for the

singly loaded one at site 1.

Case 7: Case 7 is similar to case 6, except that it assumes only the full enzyme containing both

Mn to be active.










CHAPTER 3
SPECTROSCOPIC CHARACTERIZATION OF THE TWO MANGANESE CENTERS

Introduction

Electron Paramagnetic Resonance Spectroscopy

Electron paramagnetic resonance is a spectroscopic technique for detecting species

containing unpaired electrons, generally organic radicals or transition metal ions with partially

filled d orbitals. Since an unpaired electron possesses circular motion about its axis (spin angular

momentum) and a charge, it has a magnetic moment. An external magnetic field interacts with

the magnetic moment of the unpaired electron (Zeeman interaction) and can result in two

possible orientations, parallel to the magnetic field or antiparallel to it. The two orientations

(states) possess different energies and the difference in energy (Zeeman energy) increases with

increases in the magnetic field.






Energy d~zhr~~Ts~BBA ine is generated inwhnhte









Magnetic Fiel (B)



Figure 3-1 Absorption of microwave irradiation by an unpaired electron in a magnetic field.
Figure adapted from http ://www.bruker-biospin. com/cw.html.









Application of radiation at an appropriate frequency results in a transition between the two

states (resonance condition, AE = hu = gpuBo). In EPR spectroscopy, the sample is irradiated

with a fixed frequency microwave energy and the magnetic field is gradually increased. A line is

generated in the EPR spectrum when the Zeeman energy matches the photon energy (Figure 3-1)

(116).

Electronic Configuration of Mn(II)

The shape of the magnetization of Mn(II) in any given coordination environment dictates

its EPR properties. This shape is determined by the distribution of unpaired electrons around the

Mn nucleus and is related to the types of ligands present and how they are geometrically

arranged around the ion. As a free ion in the gaseous phase, Mn2+ pOSSCSSCS flVe 3d electrons in

a spherical distribution around the nucleus (an S-state ion). Ligand interactions with the d

electrons in the condensed phases break both the degeneracy of the 3d orbitals and their spherical

symmetry which influences the EPR properties of Mn2+. The splitting that results from a

noncubic environment is known as zero-field splitting (zfs) or fine structure splitting (117).

Electron Paramagnetic Resonance Properties of Mn(II)

The characteristic six line EPR spectrum of Mn(II) arises from the interaction hyperfinee

coupling) between the unpaired electrons (S = 5/2) and the 55Mn nucleus (I = 5/2). While this

interaction decreases the sensitivity of the EPR measurement by a factor of six, it provides a tool

for distinguishing between octahedral and tetrahedral coordination states (118). Interactions

between electron spin (2M) and nuclear spin (2M) result in 36 states and 30 allowed transitions

where A 2 = + 1 and A 2M1= 0 (five EPR transitions, each split by hyperfine fine coupling to a

sextet) (Figure 3-2) (117).










Ms M,
5/2/ /2
3/2 ~-1/2
12

3/2

1/2/
5/2

-1/25/

-5/2
-5/2




zero-field high-fieldhypefiine5/
Ms levels levels splitting




Figure 3-2 Electron spin energy levels and hyperfine splitting for Mn(II) in spherical symmetry.
One sextet of the five-fold allowed transitions (A M = 1, a 2M1= 0) is indicated by
arrows. Figure adapted from (117).

The schematic representation in Figure 3-2 depicts the electronic Zeeman and nuclear


hyperfine interactions of a hypothetical case in which there is a five-fold degeneracy of the A M

= & 1 fine structure transitions. This situation would yield a spectrum of six well resolved EPR


signals and is close to what is observed for hexaaquo manganese in solution. For hexaaquo

manganese there is, however, some inhomogenous broadening of the EPR lines that is the result

of an inexact superpositioning of the five A 2 = & 1 fine structure transitions (11 7, 119). When


Mn(II) is bound in a symmetry lower than cubic, the asymmetry of the ligand field (or crystal

field) removes the degeneracy of the electronic spin levels in the absence of an applied magnetic


field. This means that the approximate degeneracy of the five A 2 = & 1 fine structure


transitions is lifted resulting in a zero field splitting. The three Kramers doublets (5/2, 13/2,


1/2) possess different energies in the absence of an external magnetic field.










Four magnetic parameters are essential to define a paramagnetic species, these are g, A, E,

and D. The gyromagnetic ratio of an electron, ge, is the ratio of its magnetic dipole moment to

its angular momentum. A free electron has a g value of 2.0023 193043 86 (which is ge, the

electronic g-factor). When an unpaired electron is in an atom, it is affected by not only the

external magnetic Hield, Bo, but also by any local magnetic Hields. The effective Hield, Beef, felt by

an electron is described by

Begf= AE = hu = gguBo (1-o)

where o allows for the effects of the local fields. The resonance condition is, therefore,

AE = hu = gegBBo(1-o)

The quantity ge(1-o) is called the g-factor, given by the symbol g, so

AE = hu = gguBo

The g-factor (or g-value) is determined in an EPR experiment by measuring the Hield, Bo,

and the frequency, u, at which resonance occurs. If g is different than ge, the ratio of the

electron's magnetic moment to its angular momentum has changed from the free electron value.

Since the electron's magnetic moment (the Bohr magneton, 4U) is constant, it must have gained

or lost angular momentum (116).

As illustrated in Figure 3-2, the hyperfine interaction is the interaction between the

magnetic moment of an electron with the magnetic moment of the nucleus. The electron-nuclear

interaction, depends on the proj sections of both electron and nuclear spins:

Eelectron-nuclear = A 2ME2

where A is the hyperfine coupling constant. A depends not only on the g-values for the

electron and the nucleus but also on the distance between them and their orientation with respect

to the external field.









The Eine structure parameters D and E, reflect the deviation of the ligand Hield from

spherical and axial symmetry, respectively. D- and E- strain reflect the inhomogeniety of these

values and depend on the metal-ligand distances and bond angles (117).

Oxalate Decarboxylase EPR

Since the two Mn(II) ions in the resting monomer are in very similar coordination (Figures

1-4 and 3-3) environments, spectroscopic efforts to establish whether catalysis takes place in

only one or both of the two metal sites have been significantly complicated. In previous X-band

studies, it has been shown that addition of small molecules like format and oxalate have small

but reproducible effects on the Mn(II) EPR spectra indicating the possibility of using EPR as a

sensitive probe of the ligand environment (74). Distinguishing which metal signals) was

perturbed, however, is difficult in X-band because the signals are very broad.3.




















Figure 3-3 Overlay of the N-terminal (shown in magenta) and C-terminal (shown in green)
manganese-coordinating ligands (PDB code: 1UW8) The manganese ion is shown
in blue. This image was prepared using the CAChe Worksystem Pro V6.5 software
package (Fujitsu America Inc., Beaverton, OR).









A multifrequency EPR approach has been employed to address the question of whether it

is possible to distinguish the two Mn ions in OxDC spectroscopically. Specifically this set of

experiments was designed to (i) distinguish the two Mn(II) sites and to (ii) determine their

respective magnetic parameters. The rationale of using this approach is based on the fact that

Mn(II) linewidths generally become narrower at higher fields (and thus higher frequencies)

allowing for better spectral resolution of small differences in g and A. Effects associated with

differing fine structure parameters, however, are more prominent at low and intermediate fields

(frequencies).

Results and Discussion

To avoid complications arising from the binding of ligands other than water to the two free

ligand positions on each Mn, initial experiments used OxDC dissolved in 20 mM

hexamethylenetetramine (HMTA) HCI buffer at pH6.0, 0.5 M NaCl (storage buffer). HMTA is

not expected to bind to Mn(II) because it is positively charged and too bulky to fit into the Mn-

binding pockets in the protein. OxDC has maximum activity at a pH value of around 4.0 and it

is common practice in the literature to use various types of negatively charged buffer molecules

to control pH for spectroscopic and kinetic analysis (57, 72-74). It was of interest, therefore, to

investigate the effect of acetate buffer at pH 5.2 on the EPR spectra. At this pH value the enzyme

possesses substantial activity but is also highly soluble.

X-band EPR

The X-band EPR spectrum of OxDC (Figure 3-4) at low temperature is distributed over a

wide field range with a clearly discernable but weak group of lines at half-field indicating

substantial fine-structure in the Mn(II) S=5/2 ions (71, 74) .










OxDC WT in different buffers
X-band cw-EPR at 7K

I-- in stor-age bulffer (SB)I
I- in SB + acetate pH5.21










1000 2000 3000 4000
Magnetic Field [G;I


Figure 3-4 X-band cw-EPR spectra of wild type OxDC in storage buffer and in acetate buffer.

Figure 3-4 shows that there may be a mixture of Mn(II) species with similar g-(2.001) and

A-(250 MHz) values but with different zero-field splitting constants D. When D is small

compared to v, the EPR signals are mostly around g 2. However, for large D, the signal is

spread over a broad field range with only parts staying near g 2.

Field Dependence of the EPR Signal in Storage Buffer

Figure 3-5 shows the g = 2 region of the EPR spectra of OxDC in HMTA buffer pH 6.0 at

frequencies ranging from X-band (corresponding to 0.34 T) to the sub-mm band (15 T). In

Figure 3-5 the effect of increasing field on the central +'/ '/ sextet of lines is clearly visible.

They are substantially broadened at low frequencies due to higher order contributions of the

zero-field splitting (zfs) (117), but broadening is reduced as the sample is moved toward its high-

field limit. No broadening or field-dependent (g) or -independent (A) splitting of the central lines

was observed up to 15 T where the linewidth is at its narrowest. This indicates that g and A are in

fact very similar for the C- and N-terminal Mn(II) ions.









30 a' I ^nan (Y.YY UnzJ
V-band (49.2 GHz)
-- W-band (94.0 GHII
25 -~ mm-band (222.4 GHz)
sub-mm (324 GHz)
Y- sub-mm (412.8 GHz)
2~ 20--







10-





-0 -20 0 20 40
ABo [mT]

Figure 3-5 Field dependence of the EPR spectra of OxDC in storage buffer (20 mM
Hexamethylenetetramine HC1, pH 6.0) with 0.5 M NaC1. To facilitate comparison of
the +/ t+-+-1/ transitions spectra are shifted along the BO-axis. Field positions in T at
the zero-points: 0.3340 (X-band, 9.4873 GHz), 1.7490 (V-band, 49.200 GHz), 3.3545
(W-band, 94.0214 GHz), 7.9427 (222.40 GHz), 11.567 (324.00 GHz), and 14.730
(412.80 GHz). All spectra were taken at temperatures between 5 and 20 K. Reprinted
from (120) with permission.

Spectral Simulations and Magnetic Parameters

Spectral simulations were performed with the "easyspin" toolbox for Matlab (121) by Ines

Garcia-Rubio and are shown along with the experimental settings in Appendix B. The main

sextet lines could be simulated considering a Mn(II) center with zfs parameters D = 1200 MHz

and E = 283 MHz (site I in table 3-1). However, this did not account for the weaker shoulders on

the high- and low-field sides of the sextet lines and a second Mn(II) species was considered (site

II in Table 3-1). Figure 3-6A shows the simulation together with the experimental W-band

spectrum of OxDC in HMTA buffer. Note that both sites are present in the same proportion and

site II has a considerably higher D-value (2700 MHz). This explains why its signal intensity is

spread out over a broad field range and is seen only in the form of relatively weak shoulders on

the narrow and intense lines of the site I signals, even at high frequencies. For this reason the









multi-frequency approach was crucial to detect, identify, and characterize the signals from the


Mn(II) ion in site II.


Figure 3-6 W-Band (94 GHz) EPR spectra of OxDC. A) HMTA buffer pH6.0. B) Acetate buffer
pH5.2. C) HMTA buffer pH6.0 and 50 mM format. The right panel shows the
simulation of site I (blue) and site II (green) with the magnetic parameters given in
Table 3-1. The left panel shows the experimental spectra (black) and the sum of the
simulations of the two sites in the same proportion. Reprinted from (120) with
permission.

Table 3-1 Magnetic parameters of OxDC species I and II. Modified from (120) with permission.

g: A /MHz] D [MHz] E/D D- and E-Strain
SB, site I 2.00087 25312 1200 & 50 0.23 & 0.02 24% 24%
SB, site II 2.00094 25013 2700 & 50 0.25 & 0.02 20% 20%
AB, site I 2.00086 25212 1200 & 50 0.23 & 0.02 40% 40%
AB, site II 2.00086 25013 2150 & 50 0.05 & 0.02 33% 60%
SB + format, I 2.00087 25312 1200 & 50 0.23 & 0.02 24% 24%
SB + format, II 2.00086 25013 2150 & 50 0.05 & 0.02 33% 60%


The best simulations required substantial D- and E-strain (20-24%) which is not

uncommon for transition metal ions in proteins and in particular for Mn(II) (117). The E/D ratio

was found to be approximately 25%, indicating considerable rhombicity of the distorted

octahedral coordination environment of both Mn(II) ions.










Field Dependence of the EPR Signal in Acetate Buffer, pH 5.2

The spectra recorded at various frequencies from X-band to 420 GHz are shown in Figure

3-7. At high fields the wings that are characteristic for the fine structure are strongly suppressed

compared to the main 6-line transitions. Therefore, the change in D upon acetate binding is less

visible. The differences between this and the previous set of spectra are small and are most

obvious in the intermediate to high frequency ranges (W-band and up). They mainly involve the

shoulders associated with site II.


-- X-band (9.49 GHz)
I I I I V-band (49.2 GHz)
30t W-band (94.0 GHz)
mm-band (222.4 GHz)
sub-mm (324 GHz)
25 sub-mm (412.8 GHz)


15;



S0-







-4O -30 -20 -10 0 10 20 30 40
ABo [mT]


Figure 3-7 Field dependence of the EPR spectra of OxDC in acetate buffer (50 mM, pH 5.2) with
0.5 M NaC1. The spectra were shifted along the Bo-axis. Field positions in T at the
zero-points: 0.3339 (X-band, 9.4853 GHz), 1.7470 (V-band, 49.200 GHz), 3.353 (W-
band, 94.0206 GHz), 7.9415 (222.40 GHz), 11.563 (324.00 GHz), and 14.7253
(412.80 GHz). Temperatures were set between 5 K and 20 K. Reprinted from (120)
with permission.

Figure 3-6B shows the experimental W-band signal of OxDC in acetate buffer pH5.2

with its simulation. The changes in the features of site II are mainly due to a decrease of D and E

from 2700 to 2150 MHz, and 675 to 108 MHz, respectively as well as a small decrease in g (see









Table 3-1). The magnetic parameters of site I were unchanged except for an increase in zfs

parameter strain which was also seen for site II. Note that the rather dramatic change in E

indicates a more axial ligand field environment for site II in the presence of acetate.

The zfs parameters of Mn(II) have been demonstrated to be sensitive to electrostatic

charges in their vicinity (122). The replacement of one or two water molecules in the

coordination sphere of Mn by acetate is certainly expected to change the electrostatic potential

around the Mn-center and could lead to the observed changes in D and E. The observation that

only site II changes upon exposure to acetate buffer suggests that only site II is solvent-

accessible. When format is added to OxDC in HMTA buffer the spectral changes observed for

site II are very similar to those found for acetate (see Figure 3-2C). This is not surprising given

that format and acetate are alike in the polar parts of their structure and are expected to show the

same coordination geometries with the metal ion.

The simplest interpretation of these results points to a correlation between the two

magnetic parameter sets and the two Mn-binding sites in the protein. The differences in the fine

structure are due to subtle differences in the charge distribution in the N- and C-terminal binding

sites while the almost identical g and A is due to similar octahedral coordination in both sites.

The fact that both species are present in approximately the same concentration in all preparations

investigated so far supports this interpretation.

The observation of changes in the fine structure parameters of only site II upon addition of

acetate buffer or format is intriguing and suggests that small molecule binding mainly takes

place at site II and not site I under our experimental conditions. Just et al. (73) observed a

channel leading from the N-terminal Mn binding site to the solvent which may be accessible by

the hinge-motion of a flexible loop region while they report no obvious solvent channel available









for the C-terminal site. Moreover, format was found coordinated to the N-terminal Mn(II) in the

X-ray structure by Anand et al. (72). Therefore, it seems reasonable to identify site II with the

N-terminal Mn-binding site, and site I with the C-terminal site.

The open and closed conformations (72, 73) of OxDC show the C-terminal Mn-binding

site in hexa- and penta-coordinated forms, respectively. It is worth noting that D-values for

penta-coordinated Mn(II) centers in MnSOD have been reported as one order of magnitude

higher than what we found for site I (123). Therefore, our site I magnetic parameters are

compatible with the hexa-coordinated Mn(II) ion in the C-terminal Mn site that is observed in

the X-ray structure of OxDC published by Anand et al. (72).

A multi-frequency EPR approach has allowed us to spectroscopically distinguish two

Mn(II) species that are present in equal proportions in the resting state of the enzyme oxalate

decarboxylase in HMTA storage buffer. The main difference between these two species is the

value of the fine structure parameters with DI = 1200 MHz, DuI = 2700 MHz, and E/D = 0.25.

When the enzyme is placed in acetate buffer pH5.2 or when format is added, DuI is reduced to

2150 MHz and EII/DII = 0.05 while DI and EI remain the same indicating that only one Mn(II) is

solvent accessible. Based on published crystal structure data, we conclude site I is the C-terminal

Mn site while site II is the solvent-exposed N-terminal site and, therefore, the site of small

molecule (acetate and format) binding.

Experimental Section

Oxalate Decarboxylase Sample Preparation

Several different batches of OxDC enzyme preparations were used. They were prepared

according to the procedures listed in Chapter 2. Final concentrations ranged from 7.7 to 12.3

mg/mL. Samples in HMTA pH6.0 were used without further modifications. Samples in 50mM

acetate buffer (AB) pH5.2 were prepared from stock by addition of a concentrated acetate buffer









solution (500 mM, pH5.2). Typically, 90 CIL of sample were mixed with 10 CIL of concentrated

AB buffer. The volumes were scaled in the same ratio for experiments requiring smaller (W-

band) or larger quantities (sub-mm bands). The same procedure was used for the addition of

format to the HMTA buffered samples. 10 CLL of 500 mM format solution was added to 90 CLL

of HMTA pH6.0 buffered sample to arrive at a final concentration of 50 mM format.

Electron Paramagnetic Resonance Spectroscopy

X-band spectra (9.5 GHz) were recorded on an Elexsys E580 spectrometer (Bruker

Biospin Corp.) and the OxDC samples were placed in 3 x 4 mm2 (IDxOD) homemade clear

fused quartz tubes and frozen in liquid nitrogen before insertion into the Oxford ESR900 cryostat

which had been pre-cooled to~-10 K.

W-band (94 GHz) spectra were recorded on an Elexsys E680 spectrometer (Bruker Biospin

Corp.) and the samples were placed into 0.7 x 0.79 mm2 (IDxOD) clear fused quartz capillaries.

The samples were then frozen in liquid nitrogen prior to insertion into the precooled Bruker

ER4118CF-W cryostat.

V-band and sub-mm bands (50, 200-420 GHz) spectra were recorded with a home built

instrument using a 15/17 T superconducting magnet as described by Hassan et al. (124).

Samples were placed into 7.2 x 8.2 mm2 (IDxOD) home-made Teflon cups. The cups have a

depth of 9.5 mm and were supplied with a Teflon stopper. Typically, 200 CIL of sample was

inserted into the cup which was then closed with the stopper to protect the sample from

contamination. The sample was pre-frozen in liquid nitrogen, the field standard (P-doped Si

sample) was then placed on top of the stopper before it was inserted into the sample holder. The

sample holder was also pre-cooled to liquid nitrogen temperatures before it was inserted into the

pre-cooled Oxford Spectrostat CF DY LT cryostat.

Experimental settings and simulations are givens in Appendix









CHAPTER 4
SPECTROSCOPIC CHANGES OF THE MANGANESE CENTERS IN THE PRESENCE OF
SUBSTRATE

Introduction

Previous EPR spectroscopic characterization of the Mn centers in oxalate decarboxylase by

workers in this laboratory identified a tyrosyl radical formed during oxalate turnover (71).

Formation of this species requires OxDC, oxalate, and oxygen. The time course of radical

formation and decay compared to the overall rate of enzyme turnover suggested that radical

formation may be related to catalysis but is not on the catalytic pathway. Furthermore, no

spectroscopic signature for Mn(III) or Mn(IV) was observed in samples frozen during catalytic

turnover (71). X-band EPR spectral perturbations of the Mn centers have been observed upon

oxalate addition indicating that EPR spectroscopic characterization of the manganese centers in

the presence of substrate may yield insights into the mechanistic role that they play during

catalysis (74). Since, as noted in Chapter 3, the effects of differing fine structure parameters are

more prominent at low and intermediate fields and the fact that linewidths generally become

narrower at higher fields allowing for better spectral resolution of small differences in g and A,

multifrequency EPR characterization is a rational approach for characterizing the manganese

centers in the presence of substrate. The scientific aim of the experiments described in this

chapter is to follow the decarboxylation reaction of OxDC by monitoring both the Mn as well as

the formation of any intermediate radicals as the reaction progresses. It is anticipated that

experiments of this type will be essential to establishing the redox state of the active Mn site

before, during, and after catalysis.










Results and Discussion


X-band (9.5 GHz)

Figure 4-1 shows spectral changes at X-band of the g 2 signal upon addition of acetate

and oxalate to OxDC. The spectrum in storage buffer (HMTA, pH 6.0, 0.5 M NaC1) is shown in

black. The sample is made 50 mM sodium acetate, pH 5.2 in order to decrease the pH to a level

where OxDC is active yet still soluble enough to maintain the high protein concentration

required (~10 mg/mL) to obtain high quality spectra.






-- in storage buffer (SB)
in SB + acetate pH5.2
---- in AB pH5.2 + ~ablell


2500 3000 3500 4000
Magnetic Field IGI


Figure 4-1 Spectral changes of the g = 2 signal at X-band upon addition of acetate and oxalate to
OxDC.

The intensity of the signal increases upon acetate addition (shown in red). The spectral

intensity decreases to its original value when oxalate is added to 50 mM in acetate buffer pH 5.2

(shown in green) and flash frozen in liquid nitrogen. To detect the spectrum of the tyrosyl

radical, the sample is thawed and allowed to react for 2 min, then freeze-quenched and re-

inserted into the EPR cryostat (shown in blue). Formation of the tyrosyl radical is accompanied

by a further decrease in spectral intensity.










To further explore the changes in spectral intensity during radical formation buffers other

than acetate were used to lower the pH of the storage buffer. Buffers used were sodium citrate,

pH 5.2, PIPES [piperazine-1 ,4-bis(2-ethanesulfonic acid)], pH 5.2, as well as the storage buffer

adjusted to pH 5.2 (Figure 4-2). All tested buffers gave essentially the same spectra indicating

that the effect of the change of the intensity during and after radical formation was not buffer

dependent.


in SB pH5.2
-- + oxalate
2 -.. + oxalate after 3h +dtint
-~~~- + oxalate after 3h +dtint






-2 -







3000 3500 4000jl
Magnletic Field [G]


Figure 4-2 Spectral changes of the g 2 signal at X-band upon addition of oxalate to OxDC in
storage buffer at pH 5.2.

Figure 4-2 shows the spectral changes of the g 2 signal at X-band upon addition oxalate

to OxDC in storage buffer at pH 5.2. Upon making the sample 50 mM oxalate and flash freezing

2 minutes after mixing, the tyrosyl radical is observed (shown in green) as well as minor spectral

changes and a reduction in the spectral intensity. After spectral acquisition the sample was

thawed and stored on ice for 3 hours before being measured again (shown in blue). At this point

the Mn(II) signal as well as the radical signal was almost gone. Upon making the sample 5 mM










dithionite almost all of the signal intensity was restored although spectral changes were

observed, primarily in the wings of the sextet.






-- in SB pH5.2
degassed
-- degassed + oxalate
J;~ de gassed + oxalate + O,(g)
1~ I- degassed + oxalate + more O,(g)










3000 3500 4000
Magnetic Field [G]


Figure 4-3 Spectral changes of the g 2 signal at X-band upon addition of oxalate to OxDC in
storage buffer at pH 5.2 under anaerobic conditions followed by the reintroduction of
air.

With respect to the half-field X-band signal (see Figure 3-1), oxalate almost completely

destroys its multiple pattern (data not shown) and leaves only a broad signal at 1560 G.

Addition of dithionite does not rescue the low-field multiple spectrum. The disappearance of

the Mn(II) intensity and its almost complete restoration with dithionite is strong but indirect

evidence for the formation of Mn(III) or Mn(IV). When, however, X-band spectra were taken of

these identical series of experiments using a parallel mode cavity (data not shown), no

spectroscopic signature of Mn(III) was observed. The parallel polarization experiments,

however, do not preclude the possibility of the formation of long-lived, high-valent manganese

species. It is conceivable that any high-valent manganese species might possess such large zero-









Hield splitting parameters that the anticipated signal in parallel mode would be unobservable at

X-band or simply broadened beyond detection.

Figure 4-3 shows spectral changes of the g = 2 signal at X-band upon addition of oxalate to

OxDC in storage buffer at pH 5.2 under anaerobic conditions followed by the reintroduction of

air. Very little spectral changes are observed when OxDC in storage buffer at pH 5.2 (shown in

red) is made anaerobic by gently bubbling the EPR tube containing the sample with N2(g) for 4

minutes inside a glove box (shown in pink). Addition of degassed oxalate decreases the Mn(II)

signal dramatically (shown in brown) as in the case when oxygen is present (Figure 4-2), but no

radical signal is observed. When the sample was thawed and air was allowed to enter the EPR

tube, the Mn(II) signal intensity decreased further and surprisingly no radical was formed (Figure

4-2, shown in turquoise). Finally, when air was bubbled through the sample the Mn(II) signal

only decreased slightly (shown in purple) and there was still no radical formation. This sample

was then assayed for decarboxylase activity and remarkably showed an increase in specific

activity from 51 U/mg to 88 U/mg. It was later confirmed that samples which have been made

anaerobic then had oxygen reintroduced possessed an increase in specific activity of at least

50%.

Chemical Oxidation of OxDC Observed at X-band

The addition of potassium ferricyanide to 50 mM (or hydrogen peroxide to 3%) to OxDC

did not have any appreciable effect on the Mn(II) signal (data not shown). The intensity of the

Mn(II) signals could be decreased, however, by the addition of the strong oxidizing agents

potassium hexachloroiridate (data not shown) and sodium (meta) periodate (Figure 4-4). In these

experiments, a series of additions were made to the enzyme in storage buffer. At each

concentration of oxidant an X-band spectrum was taken at 7 K. The decrease in signal intensity

was small up to 2 mM of either of the oxidants used. Both of the oxidants showed a marked










decrease in signal intensity at about 4 mM with a concomitant appearance of a carbon-based

radical. The linewidth of the radical signal is consistent with the previously described tyrosyl

radical (71). The Mn(II) could be brought back with the addition of 5 mM dithionite (Figure 4-

4). While it is difficult to interpret these intriguing observations with respect to the redox forms

of the enzyme before, during, and after catalysis, these results indicate that molecules are able to

enter into the Mn-binding sites facilitating future efforts to measure the reduction potentials of

the two sites.



3 I [I I I No Oxidant
0.5 nM NalO,

1.0 tnM NalO,
2- 4
4-1.0 inM NalO,



w 42








2500 3000 3500 4000
Magnetic Field [G]



Figure 4-4 Mn(II) signal intensity and carbon-based radical formation as a function of the
concentration of sodium (meta) periodate.

X-band Spin-Trapping of an Oxygen Species Formed During Oxalate Decarboxylase
Turnover

In the spin-trapping technique, a diamagnetic spin-trap (EPR silent) compound reacts with

reactive short-lived free radicals to form a more persistent spin adduct. From the EPR spectrum

of the spin adduct, the structure of the reactive free radical can be deduced indirectly. The spin

trapping experiments described here showed that this technique can be used to detect radical











species formed during OxDC turnover. These experiments employed the most commonly used

nitrone spin trap 5,5-Dimethyl-1 -pyrroline N-oxide (DMPO). The spectral shape of the EPR


signal of the trapped radical shown in the time course (Figure 4-5) suggests that it may be a


hydroxyl radical (125, 126), but this should be confirmed by analyzing the trapped product by

mass spectroscopy.



3.3 m~g'rmL. CdC
100 mrlVI KAc, pH 4.2
5 uL [DPCIV|
initiated with 75 mlVI oxcalate
5000
4mn
4000 -7mn
-15mn
19mn
3000-

S2000-

S1000



-1000

-2000 ** I
3490 3500 3510 3520 3530 3540
field


Figure 4-5 EPR spectra of the spin-trapped radical formed during OxDC turnover. The
following blanks showed no significant signal: buffer + DMPO, buffer + DMPO +
oxalate, and OxDC + buffer + DMPO.

One problem with the use of DMPO as a spin-trapping agent is that a DMPO-superoxide

adduct has a half-life on the order of 1-2 minutes (125, 126) before decaying to the DMPO-


hydroxide adduct. Since the hypothetical mechanism shown in Chapter 1 (Figure 1-5) proposes

the formation of a manganese-bound superoxide radical, it was of interest to look at an earlier


time point than those shown in Figure 4-5. A 2.5 minute time point is shown in Figure 4-6 and


shows the appearance of a different signal that then decays to that shown in Figure 4-5 raising


the possibility that it represents a DMPO-superoxide adduct. This should be explored further











using a spin trap with a longer-lived superoxide adduct species such as 2-ethoxycarbonyl-2-


methyl-3,4-dihydro-2H-pyrrole-1i-oxide (EMPO) or 5-diethoxyphosphoryl-5-methyl-1 -pyrroline

N-oxide (DEPMPO) (12 7).


75 mM oxalate
10 dB att, 60 dB R/G
first scan ~2.5 min


4000

3000

2000

1000

0

-1000

-2000

-3000


3500 3520
field


Figure 4-6 EPR spectrum

Q-band (3Hz)


of a short-lived DMPO-oxygen species.


r1300 12000 12500
Magnetic Field [G]

Figure 4-7 Spectral changes of the Mn(II) signal at Q-band upon addition of acetate and oxalate
to OxDC.










Q-band EPR (Figure 4-7) was able to reveal the low- and high Hield- wings that belong to

the higher spin manifolds (transitions outside the central +'/ '/ sextet of lines) at least for

one of the Mn(II) species. The sample in storage buffer showed a true half Hield signal (data not

shown) which becomes weaker when the sample is made pH 5.2 with the addition of acetate

buffer.

W-band (94 GHz)

-- in storage buffer (SB)
in AB (acetate buffer, pH 5.2)
-- in AB + oxalate









I

33000 33200 33400 33600 33800 34000 34 00
Magnetic Field [G]


Figure 4-8 Spectral changes of the Mn(II) signal at W-band upon addition of acetate and oxalate
to OxDC.

Just as in X-band, the wild-type enzyme shows an effect of acetate binding on its six line

Mn(II) signal in W-band g 2 region (Figure 4-8). At this higher frequency, where there are no

contributions from zero Hield splitting, there is no half field signal and all the signal is found at

the g 2 region. The spectra taken in storage buffer (shown in black) display a clear spliiting in

their negative troughs which is absent in those taken in acetate buffer, pH 5.2. There is very

little difference in the spectra taken in acetate buffer with (shown in green) and without oxalate,

which is in strong contrast with what is observed at X-band. No radical is observed at W-band.










324 GHz

Figure 4-9 shows the very clean 324 GHz spectrum of OxDC in storage buffer (black). In

this spectrum only a single species is visible (note the two lines of the phosphorus doped silicon

Hield standard in the 5th line and between the 5th and 6th lines). Addition of acetate (red) leads to

a broadening of the sextet lines especially in the higher Hield portions with a clear splitting of the

weaker component. The lines narrow again with the addition of substrate (green).


-- SB pH6.0
-- AB pH5.2
-- AB pH5.2 + oxarlale















I 1.54 1 1.56 I 1.58 1 1.6
Maglner"s Field [T]



Figure 4-9 Spectral changes of the Mn(II) signal at 324 GHz upon addition of acetate and oxalate
to OxDC.

690 GHz

The spectra shown in Figure 4-10 were acquired at the highest field available, 690 GHz.

The larger linewidth of all the spectra displayed may be due to the fact that the Keck magnet

(Bitter type magnet) is less homogeneous or this may be the first indication of an effect of g-

anisotropy. The two Mn(II) species are indistinguishable from each other in the spectrum taken

in storage buffer (shown in black). Addition of acetate buffer broadens the six lines and begins

to split the lower Hield lines (shown in red). The spectrum taken after the addition of substrate










(shown in green) splits approximately 50 % of its signal intensity off into additional sextet

signals. The observed spectrum is consistent with the hypothesis that only one Mn-binding site

is available for substrate binding. This experiment clearly demonstrates that substrate binding to

Mn(II) can be followed spectroscopically by very high field EPR.


-- SB pH6.0
AB pH5.2
AB pHS.2 + oxalate













24.66 24.68 24.7 24 72
Magnetic Field [T]

Figure 4-10 Spectral changes of the Mn(II) signal at 690 GHz upon addition of acetate and
oxalate to OxDC.

Experimental Section

OxDC was purified as described in Chapter 2.

Sample preparation and EPR spectroscopy was as described in Chapter 3.

X-band spectra: microwave frequency 9.48731 GHz, microwave power 0.64 mW, modulation

frequency 100 k
conversion time 41 ms, 1 sweep, 1.465 G/data point.

Q-band spectra: microwave frequency 34.05197 GHz, microwave power 17 CLW, modulation

frequency 100 k
conversion time 41 ms, 1 sweep, 1.953 G/data point.










W-band spectra: Microwave frequency 94.02141 GHz, microwave power 0.6 CIW, modulation

frequency 100 k
conversion time 82 ms, 1 sweep, 1.172 G/data point.

324 GHz spectra: Microwave frequency 324.00 GHz, modulation frequency 41.8 kHz,

modulation amplitude 0.5 G, lock-in sensitivity 50 CIV, time constant 100 ms, sweep speed 5.01

G/s, 1 sweep, 0.367 G/data point.









CHAPTER 5
SITE-DIRECTED MUTAGENESIS STUDIES TO PROBE WHICH MANGANESE-BINDING
SITE(S) IS INVOLVED INT CATALYSIS

Introduction

The fact that the OxDC contains Mn in both the N- and C-terminal cupin domains raises

the question of whether catalysis takes place in only one or both of the two Mn-binding sites.

Multifrequency EPR studies (Chapter 3) suggest that only one Mn-binding site binds acetate and

format. Two structural observations can be cited as support for the hypothesis that the active

site of the enzyme is located in the N-terminal domain. First, this domain appears to contain a

channel along which oxalate can diffuse from solution, which can exist in an "open" or "closed"

form as a result of the conformational rearrangement of residues 161-165 (73). Second, format

has been observed to coordinate the N-terminal Mn ion in one of the OxDC crystal structures

(72). Site-directed mutagenesis studies of conserved arginine (Arg-92 and Arg-270) and

glutamate (Glu-162 and Glu-333) residues in the two Mn-binding sites (Figure 1-4) have,

however, provided conflicting evidence for which of the two domains might mediate catalysis

(72, 73). Interpretation of these studies is complicated by the presence of polyhistidine

purification tags in the recombinant OxDC mutant enzymes, and/or a lack of quantitative

information on their Mn content (37, 73). Resolving the location of the active site(s) in OxDC is

an important problem because if only a single site mediates the OxDC-catalyzed reaction,

legitimate issues are raised concerning the function (if any) of the second Mn-binding domain,

and the extent to which local protein structure in each domain results in the differential reactivity

of the two metal centers.









Results and Discussion

Design and Steady-State Characterization of OxDC Mutants with Domain-Specific
Modified Mn Affinity

Given the possibility that both Mn sites catalyze the decarboxylation reaction, a series of

OxDC mutants were constructed designed to disrupt the Mn-binding capability of a given cupin

domain by modifying the side chains of either Glu-101 and Glu-280, which coordinate the metal

in the N- and C-terminal domains, respectively (Figure 1-4). Prior studies of Flamnmulina OxDC

had shown that mutation of Mn-binding histidine residues in either cupin domain yielded only

inactive enzyme (128). A series of site-specific OxDC mutants was, therefore, constructed in

which Mn-binding glutamate residues in each of the two domains (Figure 1-4) were replaced by

alanine, aspartate and glutamine residues (Table 5-1). It was anticipated that the affinity of the

binding site containing the mutated residue would be severely reduced so as to yield enzyme in

which Mn was incorporated preferentially into the other domain. If catalysis was mediated

independently by both Mn-binding sites, we anticipated these OxDC metal-binding mutants

would exhibit activities reduced by approximately 50% from that of wild type enzyme (assuming

metal incorporation proceeded to give 1 Mn/monomer).

Table 5-1 Mn incorporation and steady-state kinetic parameters for metal-binding OxDC mutants

Enzyme Spe. Mn Km (mM) kct(-) kat/Km (M-'s- )
Act. content

WT OxDC 61.2 U/mg 1.87 8.4 & 0.7 53 A 1.5 6309

E101A 0.05 U/mg 0.18 2.9 & 0.3 0.046 & 0.002 16
E101D 0.79 U/mg 0.09 3.4 & 0.1 0.49 & 0.01 144
E101Q 0.63 U/mg 0.11 4.0 + 0.2 0.62 & 0.01 155

E280A 0.03 U/mg 0.67 3.0 + 0.2 0.019 & 0.001 6
E280D 0.69 U/mg 0.64 5.4 & 0.4 0. 14 & 0.01 26
E280Q 0.15 U/mg 0.73 10.1 & 0.6 0.62 & 0.01 61

E101Q/E280Q 0.01 U/mg 0.07 2.9 & 0.3 0.012 & 0.0003 4










Contrary to this expectation, the steady-state kinetic parameters for the series of OxDC

mutants showed that catalytic turnover was significantly lower than expected on the basis of Mn

incorporation even though the oxalate Km values were not greatly perturbed (Table 5-1). In the

case of the E280Q OxDC mutant, the Mn content was 39% of that present in fully active, wild

type OxDC yet the specific activity of the same mutant was only about 1% of the wild type

activity. In other words, all Mn-binding mutants fell significantly below the line constructed in

Figure 2-1 (the dependence of OxDC specific activity on the extent of Mn incorporation). In

addition, none of the mutant enzymes were found to exhibit oxalate oxidase activity, at least as

assayed with a dye oxidation method to monitor oxalate-dependent hydrogen peroxide formation

(129).

Table 5-2 Metal content of Mn-binding OxDC mutants

Enzyme Preparation Mn Co Zn Fe Cu Mg Specific Activity b


WT OxDC 1.87 n.d." 0.51 0.07 0.01 0.01 100%
E101A 0.18 < 0.01 0.17 < 0.01 < 0.01 n.d." < 0.1%
E101D 0.11 < 0.01 0.08 < 0.01 < 0.01 n.d." 1.0%
E101Q 0.09 < 0.01 0.05 0.30 < 0.01 n.d." 1.3%
E280A 0.67 < 0.01 0.12 < 0.01 < 0.01 n.d." < 0.1%
E280D 0.64 < 0.01 0.07 0.04 < 0.01 n.d." 0.3%
E280Q 0.73 n.d." 0.11 < 0.01 < 0.01 < 0.01 1.1%
E1 0 1Q/E2 80Q 0.07 < 0.01 0.12 0.04 < 0.01 n.d." < 0.1%

a Number of metal ions/OxDC monomer. b Value is relative to that of wild type OxDC. Value was not
determined.

In designing these experiments, it was assumed that the absence of Mn in the domain

lacking a key glutamate side chain would not dramatically affect the three-dimensional fold of

the P-barrel domain structure. This assumption seems reasonable given the existence of stable,

metal-free cupin domains that lack metal-binding residues (130, 131). Unexpectedly, these

experiments showed that Mn incorporation at the C-terminal binding site appears to require the

presence of Mn in the N-terminal domain. Thus, replacement of Glu-101 by alanine, aspartate or









glutamine gave OxDC mutants containing approximately 0.1-0.2 Mn/monomer (Table 5-1).

That this was not merely an effect associated with expressing the OxDC mutant in Escherichia

coli was demonstrated by the failure of efforts to introduce Mn into the E101Q OxDC mutant

using our well-defined in vitro conditions for metal substitution and re-folding. OxDC

molecules containing Mn bound only in the C-terminal domain may be absent in solution, and

hence the activity of samples of recombinant OxDC containing less than 2 Mn/monomer is

associated with enzyme species containing Mn in both domains and/or one Mn in the N-terminal

binding site.

Size-Exclusion Chromatography (SEC)

Because modification of the metal-binding glutamates gave OxDC mutants with catalytic

activities far below those anticipated from their Mn content (assuming two independent catalytic

sites) it was of interest to investigate whether changes to the metal binding glutamate residues

might have caused large perturbations in enzyme structure. X-ray crystal structures show that

Bacillus subtilis OxDC adopts a quaternary structure consisting of a hexamer in which two

trimers are packed face to face so that the complex possesses 32 (D3) pOint symmetry (72)

(Figure 1-3). Because this crystallographic observation is consistent with PAGE studies of native

OxDC (34), we employed size-exclusion chromatography to investigate the quaternary structures

adopted by the series of OxDC mutants (Table 5-3).

Although recombinant, wild type OxDC seemed to elute as a hexamer under our

conditions, approximately 85% of the purified protein sample was present as oligomers of higher

apparent mass, corresponding to complexes formed from approximately 12-18 monomers.

Perhaps more importantly, however, replacement of either of the Mn-binding glutamate residues

did not yield oligomeric forms of the OxDC mutants that were significantly different to those










adopted by the wild type enzyme, although small populations of dimers (based on their elution

properties) were observed for several of the mutants.

Table 5-3 Estimates of size for the oligomeric forms of recombinant wild type OxDC and OxDC
Mn-binding mutants obtained using size exclusion chromatography. aValues shown
are those given for the molecular weight standards. bEstimates obtained from size
exclusion chromatography. Asterix indicates the predominant species observed under
elution conditions. "Double mutant in which Glu-101 and Glu-280 are both replaced
by glutamine residues.

Actual MW Estimated MW Number of OxDC
(kDa)a (kDa)b monomers

Carbonic Anhydrase 29 27
Albumin 66 93
Alcohol Dehydrogenase 150 103
Amylase 200 259
Apoferritin 443 433
Thyroglobulin 669 579

Wt OxDC -596*, 310 14,7
E101A 589*, 208, 80 13, 5, 2
E101D 666 15
E101Q 500* 196 12, 5
E280A -603*, 272, 222, 105 14, 6, 5, 3
E280D -602 14
E280Q -617*, 216 14, 5
E 10 1Q/E2 80Qc 572*, 189 13, 4


Circular Dichroism Measurements

Circular dichroism (CD) measurements were employed to evaluate the extent of secondary

structural changes resulting from site-specific replacement of the metal-binding glutamate

residues (Figure 5-1). The utility of circular dichroism in the analysis of proteins is derived from

the fact that the polypeptide backbone is optically active in the far ultraviolet (170-250 nm) and

that different secondary structures produce characteristic spectra (132). Since CD measurements

give estimates of the fraction of residues in ot- helical, P- sheet, P-turn, and unordered

conformations, the effects of mutations, denaturants, and temperature can be studied and the










kinetics of protein folding and unfolding can be investigated much more efficiently than by high

resolution methods such as X-ray crystallography. Technical considerations of CD data

collection include sample preparation in a buffer which does not absorb in the region of interest

and balancing the factors of sample concentration, background signal, and cuvette pathlength.

As expected on the basis of the X-ray crystal structures, the CD spectrum of wild type

OxDC showed features consistent with the largely P-strand character of the cupin domains

together with minima at 222 and 205 nm that are presumably associated with the a-helices that

mediate monomer/monomer contacts (Figure 5-1). The three OxDC mutants containing

perturbations in the C-terminal Mn-binding site (E280A, E280D and E280Q) exhibited very

similar CD spectra, but which differed considerably from that of wild type OxDC.


8000

6000

S 4000 E11

E 2000


S-2000-

S-4000 n.

-6000-

-8000
200 210 220 230 240 250
Wavelength (nm)


Figure 5-1 CD spectra of recombinant wild type OxDC and the Mn-binding OxDC mutants.

Because these three proteins still bind relatively large amounts of Mn/monomer, the

simplest interpretation of the CD spectra is that they reflect the P-strand character of the N-

terminal (and possibly the C-terminal) cupin domain(s). Thus, the observed changes likely reflect

changes in the amount of a-helix, especially in light of the decreased molar ellipticities observed









at wavelengths between 190 nm and 200 nm. For OxDC mutants in which Glu-101 was replaced

by other residues (E101A, E101D and E101Q) the picture was complicated by the finding that

only E101A and E101D exhibited similar CD spectra. Although there are differences in the

absolute molar ellipticities observed in the CD spectra for E101A, E101D and the three C-

terminal OxDC mutants, the overall shape of the curves for the five enzymes were similar,

suggesting a resemblance of their overall secondary structures. In the case of the E101Q OxDC

mutant, however, the observed spectrum resembled that of the apo-form of Thermococcus

litoralis phosphoglucose isomerase, a cupin for which activity is thought to be Fe-dependent

(133). In many respects, the CD spectrum of the E101Q OxDC mutant is consistent with a higher

proportion of a-helix, and similar levels of P-strand, secondary structure, relative to wild type

OxDC.

Electron Paramagnetic Resonance Properties

To gain additional insight into the structure of the Mn-binding pocket in the OxDC

mutants, we compared the EPR properties (see Chapter 3 for an introduction to the technique) of

the Mn(II) center in the E280Q OxDC mutant with those of the wild type enzyme (Figure 5-2).

In spectra taken for aerobic solutions of the enzyme at 10 K, the six-line Mn signals for the two

proteins showed a strong resemblance, suggesting that the Mn(II) ions in both proteins were

coordinated in similar environments, and the isotropic g-factor (gso = 2.00087) was identical for

the two preparations within experimental limits (+0.00001). Differences were observed,

however, in the signal linewidths (Figure 5-2). The linewidth for the E280Q mutant was 0.85

mT and that for the wild type enzyme was 1.1 mT (see simulations in Appendix C).

The spectra also revealed very little g- and A-strain, because the peak-to-peak amplitude

was approximately the same within each six-line pattern, and did not show any trace of

transitions between the higher electron spin manifolds. The latter is due to relatively large fine









structure values of the order of about 1000 MHz (Table 3-1). Similarly large fine structure

parameters have been observed for the Mn(II) centers in the superoxide dismutases from

Rhodobacter capsulatus and Escherichia coli (134). Taken together, these spectral features

suggest that the Mn ions in both samples are located in a very homogeneous ligand environment,

supporting the proposal that the cupin structure of the N-terminal domain of wild type OxDC is

retained in the Mn-binding mutant enzyme (E280Q).


High Field EPR of WT and E280Q @ 10 K.


Magnetic Field [mT]


Figure 5-2 EPR spectra of the Mn(II) signals in wild type OxDC (red) and the E280Q OxDC
mutant (blue) at 10 K. For the wild type enzyme, these experiments were performed
at 3 86. 116 GHz, with a modulation amplitude and modulation frequency of 2 G and
40 k at 0. 1 mT/s. In the case of the E280Q OxDC mutant, EPR experiments were
performed at 382.826 GHz, with a modulation amplitude and modulation frequency
of 2 G and 43 k 13.628-13.698 T at 0. 1 mT/s. For ease of comparison, the spectrum for the E280Q
OxDC mutant (blue) is offset by 108.8 mT along the magnetic field axis. The
additional sharp lines visible at 13796 and 13800 mT arise from the P-doped silicon
field standard employed to calibrate the magnetic field sweep during acquisition of
the spectrum for wild type OxDC (red).









Relaxation Enhancement Measurements

The significant difference in the spectral linewidth observed for the Mn signal in the two

samples, however, suggested that a line broadening mechanism was operative in wild type

OxDC that was absent in the E280Q OxDC mutant, perhaps because only one Mn-binding site

was occupied in this protein. To answer this question, we measured the inversion recovery

kinetics of the Mn signals for wild type OxDC and the E280QOxDC mutant of OxDC (Figure 5-

3). Relaxation refers to the recovery from a non-equilibrium state to an equilibrium state. The

characteristic time is called the relaxation time and is strongly dependent on the electronic

structure of the paramagnetic center and on its interactions with its environment. The spin-lattice

relaxation time, T1, is the time constant for equilibration of the populations of the two electron

spin Zeeman energy levels. The transverse relaxation time (or spin-spin relaxation time), T2, iS

due to the variation in resonant fields that result from other spins in the vicinity. T1 is usually

much longer than T2 (135).

These relaxation studies were performed using echo-detected EPR spectra for the samples,

which were acquired at 3700 G and 3675 G for the wild type and mutant enzyme, respectively.

For both samples, the observed T1 decay was bi-exponential, with values of 9.25 and 55.4 Cpsec

being obtained for the Mn signal in wild type OxDC. In the case of the E280Q OxDC mutant,

however, the corresponding T1 values were 16.3 and 92.5 Cpsec, meaning that relaxation was

enhanced in the sample of wild type OxDC by a factor of approximately 1.7.A similar

enhancement was observed for Tm (T2) in the two samples (Figure 5-4).















1- Mutant fi280Q I I I
c 1 ~Simulation Mlutant


io T (WT) = (9.25, 55.4)ps






-30-

0 100 200 300 400
Puls Separation Time T [ps]


Figure 5-3. Inversion-recovery experiments on wild type OxDC (12.3 mg/mL) and the E280Q
OxDC mutant (16.8 mg/mL). Samples were dissolved in 20 mM
hexamethylenetetramine-HC1, pH 6.0, containing 0.5 M NaCl (100 pIL total volume),
and both spectra were taken at 5 K with a microwave frequency of 9.70703 GHz. The
fields for the wild type OxDC and the E280Q OxDC mutant samples were 370 mT
and 367.5 mT, respectively. The pulse sequence (see inset) employed n/2 and 2n pulses
of 16 ns and 32 ns, respectively, and a 2-phase CYCLOPS sequence was used to
separate the echo from unwanted spurious echoes. The data in the figure is the sum of
5 individual pulse trains per CYCLOPS phase, separated by a repetition time of 5.1
ms. Microwave attenuation was set to 11 dB. The echo amplitude from the Hahn
readout sequence was integrated and plotted as a function of the pulse separation time
T, with a pulse separation z for the Hahn readout sequence of 140 ns. Both traces
were simulated with bi-exponential recovery kinetics.

The observed changes in both T1 and T2 for the Mn signals in the two spectra demonstrated

that the relaxation kinetics for metal centers in the wild-type enzyme were enhanced when

compared to those of the single Mn in the E280Q OxDC mutant (Figures 5-3 and 5-4). The

inter-monomer Mn-Mn distance of approximately 21 A+ observed in the wild type OxDC

hexamer is close enough for a dipolar interaction between the two metal ions to be observable in

the EPR spectrum.










H~ahn-Echo Decay
In Storage Buffer, T = 5.2K



Simulation WT
Mutant E280Q
Simulation Mutant






100 -, T2(WT)= 183 as





0 500 1000 1500 2000
Pulse separation time T (plsec)


Figure 5-4 Data for the Hahn-echo decay experiment on wild-type OxDC and the E280Q mutant.
Samples and fields were as for Figure 3-3. The experiment (inset) employed the
usual n/2-z-n: Hahn echo pulse sequence with 16 and 32 ns pulses, respectively. A 2-
phase CYCLOPS sequence was used to separate the echo from artifacts, and echo
modulations were observed on the decay traces. Simulations were performed using a
mono-exponential model taking points after the decay modulations.

Similar relaxation enhancements over distances of 15-30 A+ have been seen in other

proteins, including the bacterial photosynthetic reaction center (52) and metmyoglobin (53).

Assuming that E280Q assembles in the same quaternary structure, its closest Mn-Mn distance

would be almost twice as great (39-40 A+) as in the wild type hexamer, reducing the effect of the

magnetic moment of one Mn ion on the relaxation dynamics of its nearest neighbor. Hence, it

seems likely that the difference in the high-field EPR linewidths in the two spectra is due to

paramagnetic relaxation broadening.









Implications for the Location of the Catalytic Site(s) in OxDC

As discussed in Chapter 2, the observation of a linear relationship between Mn occupancy

and activity is consistent with three kinetic models, the simplest of which postulates that both

Mn-binding sites are catalytically active. If the sites are also independent, then we anticipated the

absence of Mn in one domain would yield a mutant enzyme exhibiting 50% of wild type activity

(assuming insignificant structural changes and full metal incorporation). This proved not to be

the case, however, with OxDC mutants (even those containing up to 0.73 Mn/monomer)

exhibiting much lower activity than expected based on their Mn content (Figure 2-1). In fact, the

level of activity observed for the E280Q OxDC mutant was significantly reduced in light of its

Mn occupancy even though EPR experiments showed that Mn coordination by residues in the N-

terminal domain was unaffected. Size-exclusion chromatography also supports the assumption

that the E280Q OxDC mutant is correctly folded, and hence this result implies that (i) either the

C-terminal Mn site mediates catalysis, or (ii) the N-terminal site catalyzes decarboxylation if,

and only if, metal is bound in the C-terminal site. Unfortunately for the first of these two

hypotheses, the E101D and E101Q OxDC mutants (in which Mn binding to the N-terminal

domain is disrupted) exhibit catalytic activity that is lower than that observed for wild type

OxDC containing an equivalent amount of bound Mn. This observation is therefore consistent

with decarboxylation being mediated by the N-terminal Mn site, unless activity in the C-terminal

site is dependent on the presence of metal in the N-terminal domain. Kinetic simulations in

which the activity of one active site is dependent on metal occupancy of the other non-catalytic

site, however, do not predict a linear relationship between bound Mn and catalytic activity, with

one exception (case 6) in which Mn binding in one site causes a significant increase in Mn

affinity of the second site while assuming that both sites have equal affinities prior to metal

binding (Figure 2-2). Although these data rule out the hypothesis that both Mn binding sites can









independently degrade oxalate, this mutagenesis strategy does not permit us to define the

location of the Mn(II) site that mediates catalysis.

Motivation for the Preparation of Single Domain OxDC Mutants

As noted in Chapter 1, it has been suggested that the two domains of oxalate decarboxylase

arose from a gene duplication event (28, 37, 43-46). Two disparate sets of enzymological studies

influenced the preparation of single domain mutants of OxDC. Gerlt and Babbitt (136) raised

the possibility that (P/a)s-barrel fold proteins (unrelated to OxDC) may be derived from mixing

and matching of (P/a)4-half barrels as well as other (P/a)s-barrels by divergent evolution.

Crystallographic studies (137) indicated that imidazole glycerol phosphate synthase (HisF) from

Thermatoga maritima is a (P/a)s-barrel composed of two superimposable domains (HisF-N and

HisF-C). To examine the possibility that HisF evolved from duplication and fusion from and

ancestral half barrel, the N- and C-terminal (P/a)4-half barrels of HisF (His-N and HisF-C) were

produced in Escherichia coli purified and characterized (138). Separately, HisF-N and HisF-C

are folded proteins, but are catalytically inactive. However, coexpression in vivo or joint

refolding in vitro, resulted in these two domains assembling into a stoichiometric and

catalytically active HisF-NC complex (138).

Another example of the expression of single domains in the literature was that of E. coli

catalase-peroxidase (KatG) (139, 140), which is composed of two peroxidase-like domains. The

N-terminal domain (KatGN) contains the heme-dependent bifunctional site and the C-terminal

domain (KatGC) which does not bind heme, has no catalytic activity, and is separated from the

active site by 30 A+. KatGN expressed separately possesses neither catalase nor peroxidase

activity (139, 140). However, separately expressed KatGc is able to restructure separately

expressed KatGN to its bifunctional conformation (141).










N-Terminal OxDC Single Domain Mutant (OxDC-N1) Does Not Catalyze the
Decarboxylation Reaction

This construct begins at the N-terminus and ends at glutamine-233. It includes a beta

strand which contributes to the C-terminal domain (Figure 5-5).





C-temrinal
.. as a we Damain












N-terminal
Domain Isee*14 =





Figure 5-5 Topology diagram of OxDC. Figure adapted from (72).

This mutant did not contain Mn as determined by ICPMS, but did contain up to 1.01 mole

Zn/mole of monomer (Table 5-4). It was possible, however, to incorporate up to 0. 14 moles

Mn/mole of monomer (0.45 moles Zn/ mole of monomer remaining) by the method described in

Chapter 2 for the preparation of the "apoenzyme" and reconstitution of wild type recombinant

OxDC (Table 5-4). This mutant was not found to possess OxDC activity by the OxDC-FDH

linked enzyme assay. A small but detectable amount of oxalate oxidase activity, however, was

detected in the manganese reconstituted sample by the dye oxidation assay described in the

Experimental Section.










Table 5-4 Metal content of single domain mutant preparations


Sample Spe. Act. Mn Fe Cu Zn Co


OxDC-N1 0 U/mg <0.01 <0.01 <0.01 1.01 <0.01

Recon OxDC-N1 0 U/mg 0.14 <0.01 <0.01 0.45 <0.01

OxDC-N1#2 0 U/mg 0.01 <0.04 <0.01 0.74 <0.01

Recon OxDC-N1#2 0 U/mg 0.06 0.02 <0.01 0.65 <0.01

OxDC-C 0 U/mg 0.08 0.02 <0.01 0.33 <0.01


EPR Characterization of Reconstituted N-terminal
N1)


OxDC Single Domain Mutant (OxDC-


Figure 5-6 shows the spectra of OxDC-N1 in 20 mM hexamethylenetetramine (HMTA)

HCI buffer, pH6.0, 0.5 M NaCl (storage buffer) (shown in black). The six line spectra is typical

of the Mn(II) centers in both the wild-type OxDC and in the E280Q mutant.




4 -- storage buffer
AB pH6.0
AB pH6.0 + oxalate pH6.0










2800 3000 3200 3400 3600 3800 4000
Magnetic Field [G]



Figure 5-6 Effect of buffer and oxalate on the g 2 X-band Mn(II) signal of reconstituted N-
terminal OxDC mutant (OxDC-N1)









A series of spectra were taken in order to make comparisons with the wild-type enzyme

(see Chapters 3 and 4). In an effort to reduce the pH into the range of OxDC activity for the

wild-type, the sample was made 50 mM sodium acetate, pH 5.2. Upon this treatment, some of

the protein precipitated and what appeared as the sharp lines of hexaaquo manganese(II)

appeared (spectrum not shown). Unlike the wild-type enzyme (but similar to the E2809Q

mutant data not shown), the spectrum changes very little if at all by the addition of sodium

acetate and/or oxalate at pH 6.0.

C-Terminal OxDC Single Domain Mutant (OxDC-C) Does Not Catalyze the
Decarboxylation Reaction

This construct begins with an engineered methione followed by leucine-231 and ends at

the end of the wild type protein. Although this construct did contain a small amount of Mn as

determined by ICPMS and by EPR at 324 GHz it was subj ected to the reconstitution procedure

described in Chapter 2 for the wild type full length enzyme. These spectra suggest that more Mn

is in the purified than the reconstituted sample (no ICPMS data for reconstituted sample). This

mutant was not found to possess neither OxDC activity by the OxDC-FDH linked enzyme assay

nor oxalate oxidase activity by the dye oxidation assay. Furthermore, the OxDC-C did not show

any signs of acetate or oxalate binding (data not shown).

Combining the N- and C-Terminal Single Domain Mutants Did Not Result in
Decarboxylase Activity

Neither combining reconstituted OxDC-N-1 and OxDC-C in storage buffer nor putting the

two single domain mutants through the reconstitution procedure together resulted in any

detectable decarboxylation activity.










Experimental Section

Expression and Purification of Site-Specific OxDC Mutants.

All site-specific OxDC mutants were constructed using the overlap extension method (142)

and the OxdC:pET-9a plasmid containing the gene coding for Bacillus subtilis OxDC (57). Thus,

primers (Table 5-4) for mutagenesis were designed such that the desired mutation was located at

the 5'-end. Primers overlapped 10 to 12 bases and included restriction sites to facilitate cloning

into pET9a. The 3'- and 5'- fragments were amplified independently and a third PCR combined

these two fragments to yield the full length gene. The resulting products were digested with

BamHI and Ndel and cloned into pET9a. Constructs were transformed into JM109 competent

cells, and transformants screened by restriction enzyme digestions using BamHI and Ndel.

Plasmids containing the desired clones were sequenced to confirm PCR fidelity and transformed

into BL21(DE3) competent cells.

Table 5-5 Primers used in the construction of Mn-binding mutants


Enzyme Type Primer Sequence

WT OxDC a Forward 5' -GGAGGAAACATCATATGAAAAAACAAAATG-3 '
WT OxDC b Reverse 5' -GCGGCAGGATCCTTATTTACTGCATTTC-3 '

E101A Forward 5' -GCTGCATGGGCTTATATGATTTACGG-3 '
E101A Reverse 5' -GCCCATGCAGCTTCTTTATGCCAGTG-3 '
E101D Forward 5' -GCTGACTGGGCTTATATGATTTACGG-3 '
E101D Reverse 5 '-GCCCAGTCAGCTTCTTTATGCTGCCAGTG-3 '
E101Q Forward 5' -GCTCAATGGGCTTATATGATTTACGG-3 '
E101Q Reverse 5' -GCCCATTGAGCTTCTTTATGCCAGTG-3 '
E280A Forward 5' -CCCACGCATGGCAATACTACATCTCC-3 '
E280A Reverse 5' -GCCATGCGTGGGTATTCGGGTGCC-3 '
E280D Forward 5' -GCTCATTGGGCTTATATGATTTACGG-3 '
E280D Reverse 5' -GCCCAATGAGCTTCTTTATGCCAGTG-3 '
E280Q Forward 5' -CCCACCAATGGCAATACTACATCTCC-3 '
E280Q Reverse 5' -GCCATTGGTGGGTATTCGGGTGCC-3 '

" Ndel restriction site engineered (residues shown in bold). D BamHI restriction site engineered (residues shown in
bold)









Expression of the OxDC mutants was carried out as for the wild type enzyme (Chapter 2).

After an initial purification using DEAE-Sepharose Fast Flow column chromatography, (Chapter

2), OxDC mutants were precipitated from 50 mM imidazole-C1, pH 7.0, containing 1.7 M

(NH4)2SO4. The precipitate was then centrifuged (10,000 rpm, 20 min, 4 oC), and re-suspended

in 20 mM hexamethylenetetramine-HC1, pH 6.0 containing 0.5 M NaCl to yield solutions of the

site-specific OxDC mutants at concentrations ranging from 3.5 to 19.6 mg/mL. This abbreviated

purification procedure gave mutant enzymes of > 90% purity, as evaluated by SDS-PAGE.

Oxalate Oxidase Assays

The level of oxalate oxidase activity for wild type OxDC and the series of OxDC mutants

at ambient temperatures (21-23 oC), using a continuous assay in which H202 prOduction was

coupled to the horseradish peroxidase (HRP) catalyzed oxidation of 2,2'-azinobis-(3-

ethylbenzthiazoline-6-sulphonic acid) (ABTS) (129). Reaction mixtures contained 25 U HRP, 5

mM ABTS, 50 mM potassium oxalate, wild type OxDC or the metal-binding OxDC mutants (at

concentrations up to 0.035 mg/mL) dissolved in 50 mM sodium acetate, pH 4.0 (total volume 1

mL). An extinction coefficient of 10,000 M^1 cm-l for the ABTS radical product was assumed in

these experiments. Control samples omitted HRP so as to differentiate between H202 prOduction

and any oxalate-dependent dye oxidation activity by wild type OxDC or the OxDC mutant.

Size-Exclusion Chromatography Measurements

The oligomeric state of the wild type enzyme was compared with that of the metal-binding

mutants by size exclusion chromatography using a BIOSEP-S2000 column (300 x 7.8 mm with

75 x 7.8 mm guard column) (Phenomenex, Torrance, CA) was equilibrated with 20 mM

hexamethylenetetramine hydrochloride, pH 6.0, containing 0.5 M NaCl (buffer C), and

calibrated using carbonic anhydrase (29.0 kDa), bovine serum albumin (66.0 kDa), alcohol

dehydrogenase (150 kDa), P-amylase (200 kDa), apoferritin (443 kDa), and thyroglobulin (669










kDa). The void volume was measured by inj ecting blue dextran. Samples of recombinant, wild

type OxDC or the site-specific OxDC mutants were then inj ected onto the column and eluted

with buffer C, at a flow rate of 1 mL/min with UV detection at 280 nm, to assign the oligomeric

form of the enzyme.

Circular Dichroism Studies

Recombinant, wild type OxDC was dialyzed into 25 mM potassium phosphate, pH 7.0

containing 100 mM NaC1, and the protein concentration adjusted to a final value of 185 Cpg/mL.

A similar procedure was performed for all 9 site-specific OxDC mutants. In cases where the

protein precipitated (8/10 samples), the precipitate was removed by microcentrifugation, The CD

spectrum of the protein was then obtained using an Aviv 215 spectrometer (Aviv Associates,

Lakewood, NJ) at wavelengths over the range of 190-250 nm (1 mm path length). All spectra

were corrected by subtracting the CD spectrum of the buffer over this range of wavelengths.

Electron Paramagnetic Resonance Spectroscopy

EPR spectra were determined using samples of wild type OxDC (12.3 mg/mL) or the

E280Q OxDC mutant (16.8 mg/mL) dissolved in 20 mM hexamethylenetetramine-HC1, pH 6.0,

containing 0.5 M NaCl (100 pL total volume). The metal contents of the wild type and mutant

enzymes were 1.63 and 0.73 Mn/monomer, respectively. All high-field EPR experiments were

performed using a custom-built spectrometer operating in transmission mode (124). Far IR

radiation was generated by a Gunn source at W-band (94-97 GHz or 105-110 GHz), which was

frequency tripled and/or quadrupled to achieve frequencies of 320 or 380 GHz with a radiation

power of 2-10 mW, and transmitted through an oversized waveguide so as to pass through the

sample once before being detected by an InSb hot-electron bolometer (QMC Instruments Ltd,

Cardiff, UK). The analog signal from the bolometer was fed into a Stanford Instrument SR830

lock-in detector, which was referenced to the field modulation at the sample. The magnetic field









sweep was carried out by either sweeping the main coil or a custom-built auxiliary 1000 G sweep

coil. Field calibration was performed using a piece of P-doped silicon, which has a g-value of

1.99854 and a hyperfine coupling constant of 117.507 MHz (143). Spectral simulations were

done with the Easy Spin toolbox (121) in the MATLAB computing environment (The

MathWorks, Natick, MA), and with in-house programs written Andrew Ozarowski. The time-

dependent EPR spectra required for the relaxation rate studies were taken with a Bruker Elexsys

E580 pulse/cw spectrometer equipped with a 5 mm Bruker Flexline dielectric resonator. The

Flexline resonator and the samples were cooled using cold helium gas in an Oxford CF935

cryostat, and the temperature during acquisition was controlled with an ITC4 temperature

controller and a VC40 gas flow controller (Oxford Instruments, Eynsham, UK). Standard pulse

sequences were employed in these experiments (144).

Expression of OxDC Single Domain Mutants

Three single domain mutants were independently amplified from pET9a plasmid

containing the gene coding for Bacillus subtilis OxDC (57). 1) OxDC-N1 (YDI) was amplified

using primers OxdC fwd and Domain-1 rev (Table 4-3). This construct begins at the N-terminus

and ends at glutamine-233. It includes a beta strand which contributes to the C-terminal

domain. 2) OxDC-N2 (DDI) was amplified using primers Domain-1 fwd and Domain-1 reverse

(Table 3-6). This construct does not contain the N-terminal beta strand that contributes to the C-

terminal domain. It begins with an engineered methionine followed by serine-53 and ends at

glutamine-233. 3) OxDC-C was amplified using primers Domain-2 fwd and OxdC rev. This

construct begins with an engineered methione followed by leucine-23 1 and ends at the end of the

wild type protein. The resulting products were digested with BamHI and Ndel and cloned into

the pET9a plasmid. Constructs were transformed into JM109 competent cells, and transformants

were screened by restriction enzyme digestions using BamHI and Ndel. Plasmids containing the









desired clones were sequenced to confirm PCR fidelity and were transformed into BL21(DE3)

competent cells. Expression of the OxDC mutants carried out as for the wild type enzyme.


Table 5-6 Primers used in the preparation of OxDC single domain mutants


Primer Name Primer sequence
OxdC fwd 5 '-GGAGGAAACAT CATAT GAAAAAAC AAAAT G-3 '
OxdC ** rev 5' -GCGGCAGGATCCTTATTTACTGCATTTC-3 '
Domain-1 fwd 5' -GGAGGAAACATATGTCTGATACTCATAACC-3 '
Domain-1 rev 5' -GCGGCAGGATCCCTATTGTTCAAGAAGGCG-3 '
Domain-2 fwd 5' -TTTACTTACCATATGCTTGAACAAGAGCCG-3 '
Ndel restriction site engineered (udrlnd
** BamHI restriction site engineered (udrlnd


Purification of Single Domain OxDC Mutant OxDC-N1

Expression of OxDC-N1 was confirmed by comparing cell lysates before and after

induction by 12% SDS PAGE. The appearance of a band in the induced cells at the calculated

molecular weight of 26.4 kd (http://www. scripps. edu/cgi-bin/cdputnam/protcalc3 ) confirmed

expression. Cells were lysed, extracted, and purified by DEAE column chromatography as

described for recombinant wild-type OxDC. Fractions containing OxDC-N1 as determined by

electrophoretic mobility were pooled and dialyzed for 4 h against 50 mM imidazole-HCI buffer,

pH 7.0 (2 L). The resulting sample was then applied to a Q-Sepharose Hi-Performance column

(2.5 x 18 cm) column equilibrated with 50 mM imidazole-HCI buffer, pH 7.0, and eluted using a

500 mL linear gradient from the column buffer to the same containing 1 M NaC1. Fractions

containing OxDC were pooled and exhaustively dialyzed against 20 mM

hexamethylenetetramine hydrochloride, pH 6.0, containing 0.5 M NaC1. Purified OxDC-N1 (>

90 % as determined by SDS PAGE) was concentrated to 5.3 mg/mL and stored as at -80 oC.









Purification of Single Domain OxDC Mutant OxDC-C

Expression of CTD was confirmed by comparing cell lysates before and after induction by

12% SDS PAGE. The appearance of a band in the induced cells at the calculated molecular

weight of 17.7 kd (http://www. scripps.edu/cgi-bin/cdputnam/protcalc3) confirmed expression.

OxDC-C was purified (>90% purity as determined by SDS PAGE) by the method described

above for OxDC-N1.









CHAPTER 6
CONCLUSIONS AND FUTURE WORK

We have demonstrated a linear dependence of oxalate decarboxylase specific activity on

the Mn incorporation. This observation is consistent with only three of the seven kinetic models

studied. The simplest model is that both Mn-binding sites are catalytically active. If the sites are

also independent, the absence of Mn in one domain would yield a mutant enzyme with 50% of

wild type activity (assuming insignificant structural changes and full metal incorporation).

OxDC Mn-binding mutants, however, exhibited much lower activity than expected based on

their Mn content (Figure 2-1). For example, the level of activity observed for the E280Q OxDC

mutant was significantly reduced in light of its Mn occupancy (0.73 Mn/monomer) even though

EPR experiments showed that Mn coordination by residues in the N-terminal domain was

unaffected. Size-exclusion chromatography is consistent with the assumption that the E280Q

OxDC mutant is correctly folded. This result implies that (i) either the C-terminal Mn site

mediates catalysis, or (ii) the N-terminal site catalyzes decarboxylation if, and only if, metal is

bound in the C-terminal site. That the E101D and E101Q OxDC mutants (in which Mn binding

to the N-terminal domain is disrupted) exhibit catalytic activity that is lower than that observed

for wild type OxDC containing an equivalent amount of bound Mn argues against the first of

these two hypotheses. This observation is therefore consistent with decarboxylation being

mediated by the N-terminal Mn site, unless activity in the C-terminal site is dependent on the

presence of metal in the N-terminal domain.

Kinetic simulations in which the activity of one active site is dependent on metal

occupancy of the other non-catalytic site, however, do not predict a linear relationship between

bound Mn and catalytic activity, with one exception (case 6) in which Mn binding in one site

causes a significant increase in Mn affinity of the second site while assuming that both sites have










equal affinities prior to metal binding (Figure 2-3). Although these data rule out the hypothesis

that both Mn binding sites can independently degrade oxalate, this mutagenesis strategy does not

permit us to define the location of the Mn(II) site that mediates catalysis.

A multi-frequency EPR approach has allowed us to spectroscopically distinguish two

Mn(II) species that are present in equal proportions in the resting state of oxalate decarboxylase

in storage buffer. The main difference between these two species is the value of the fine structure

parameters with DI = 1200 MHz and DuI = 2700 MHz. When the enzyme is placed in acetate

buffer pH5.2 or when format is added, DuI is reduced to 2150 MHz while DI remains the same

indicating that only one Mn(II) is solvent accessible. Based on published crystal structure data,

we suggest site I is the C-terminal Mn site while site II is the solvent-exposed N-terminal site

and, therefore, the site of small molecule (acetate and format) binding.

It would be of interest in terms of the catalytic mechanism to determine the redox

properties of OxDC. The observation that the Mn(II) EPR signal can be decreased with the

addition of sodium (meta) periodate and potassium hexachloroiridate with a concomitant

appearance of a carbon-based radical should be explored further and is significant in that it

demonstrates that oxidants can reach the manganese ions and that potentiometric titrations can be

carried out on OxDC. It would also be of interest to use an oxygen electrode to characterize the

oxygen dependence of the bacterial form of OxDC. Questions about the binding of substrate to

the Mn-binding site(s) could be addressed by crystallographic structure solution of the Co

substituted enzyme in the presence of oxalate.










APPENDIX A
KINETIC PARAMETERS USED IN GEPASI SIMULATIONS



Case 1 (site 2 unimportant or inactive)


1(a)
R1 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R2 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R3 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R4 (Mass action (rversible))
k1~ = .0000e+009
k2 = 5.0000e+000
R5 (Mass action (reversible))
k1 = 1.0000e008
k2 = 8.4000e+-005
R6 (Mass action (irreversible))
k= 5.3000e+001
R7 (Mass action (reversible))
k1 = 1.0000e+008
k2 = 8.4000e+005
R8 (M~ass action (reversible))
k1 = 1.0000e+008
k2 = 8.4000e+005
R9 (Mass action (irvrsible))
k= 5.3000e+001
R10 (MJass action (irreversible))
k = 5.0000e-06


1(b)
R1 (Mass action (reversible))
k1 = 1.0000e0009
kE2 = 5.0000e+003
R2 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R3 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.00001e+003
R4 (Mass action (rversible))
k1 = 1.0000e+409
k2 = 5.0000e+000
R5 (Mass action (reversible))
k1 = 1.0000c0008
kE2 = 8.4000e+005
R6 (Mass action (irreversible))
k= 5.3000e+001
R7 (Mass action (reversible))
k1 = 1.00010e+08
k2 = 8.4000e+005
RS (Mass action (reversible))
k1 = 1.0000e*008
k2 = 8.4000e+005
R9 (Mass action (irrevesbe)
k= 5.3000e+001
R10 (M~ass action (irreversible))
k = 5.0000e-006


1(c)
R1 (Mass action (reversible))
k1 = 1.0000e0009
k2 = 5.0000e+000
R2 (Mass action (reversible))
k1 = 1.0000e+009
k2= 5.0000e+003
R3 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000~e+000
R4 (Mass action (revrsible))
k1 = 1.0000e+009
k2 = 5.0000e+003
RS (Mass action (reversible))
k1 = 1.00000008
kr2 = 8.4000e+005
R6i (Mass action (irreversible))
k= 5.3000te401
R7 (Mass action (reversible))
k1 = 1.0000etD08
k2 = 8.4000e+005
R8 (Mass action (reversible))
k1 = 1.0000e+408
k2 = 8.4000etD05
R9 (Mulass action (irvrsible))
k= 5.3000e001
R10 (Ma8ss action (irreversible))
k = 5.0000et-006










Case 1' (site 2 unimportant or inactive)


1(a)'
R1 (M~ass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R2 Mass action (reversible))
k1 = 1.0000e*009
k2 = 5.0000e+000
R3 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R4 (IMass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R5 (Mass action (reversible))
k1 = 1.0000e+008
k2 = 8.4000e+005
R6 (Mlass action (irreversible))
k = 5.3000e001
R7 (Mass action (reversible))
k1 = 1.0000e+008
k2 = 8.4000e+005
R8 (Mlass action (reversible))
k1 = 1.0000e+008
k2 = 8.4000e+005
R9 (Mass action (irreversible))
k =2.6500e+001


R1 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000etDO3
R2 (Mass action (rwevsible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R3 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+003
R4 (Mass action (reversible))
k1 = 1.0000e+009
k2 = S.000~0e00
R5 (M~ass action (reversible))
k1 = 1.0000e+008
k2 = 8.4000etDOS
R6 (Mass action (irreversible))
k = 5.3000e+t001
R7 (Mass action (reversible))
k1 = 1.0000e+008
k2 = 8.4000e+005
R8 (Mass action (reversible))
k1 = 1.0000e+008
k2 = 8.4000e+005
R9 (Mass action (irreversible))
k = 2.6500e+001


1(c)'
R1 (Mass action (reverile))
k1 = 1.0000e+009
k2 = 5.0000e+4000
R2 (M~ass action (revesible))
k1 = 1.0000e4090
k2 = 5.0000e+003
R3 (Mass action (reversible))
k1 -= 1.0000e+009
k2 = 5.0000t4000
R4 (Mass action (rversible))
k1 = 1.0000e+009
kr2 = 5.0000e*D003
R5 (Mass action (reversible))
k1 = 1.0000e+008
k2 = 8.4000e+405
R6 (M~ass action (irreversible))
k = 5.3000e+001
R7 (Mass action (reversible))
k1 = 1.0000e+008
k2 = 8.4000e+005
R8 (Mass action (revesible))
k1 = 1.0000e+008
k2 = 8.4000e+005
R9 (Mass action (irreversible))
k = 2.6i500e+001
R10 (Mlass action (irreversible))
k = 5.00 ~e006


R10 (Mass action (irreversible)) R10 (Mass action (irreversible))
k = 5.0000e-006 k = 5.0000e-006











Case 2 (site 1 most active)


2(a)
R1 (Muass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+000
R2 (Muass action (reversible))
k1 =- 1.0000e+009
k2 = 5.0000e+000
R3 (1Mass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+000
R4 (Mulass action (reversible))
k1 = 1.0000e+009
k2 =- 5.0000e+000
R5 (Mass action (rversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R6 (Mass action (irreversible))
k = 5.3000e+001
R7 (Mas action (reversible))
k1 = 1.0000e+008
k2 = 8.4000e+005
R8 (Mrass action (reversible))
k1 = 1.0000e+008
k2 = 8.4000e+005
R9 (Miass action (irreversible))
k = 3.5000e+001
R10 (Mass action (irreversible))
k = 8.0000e+000


2b)
R1 (Mass action (reversible))
kl = 1.0000e+t009
k2 = 5.0000e+003
R2 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000et000
R3 (Mass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+003
R4 (Mass action (reversible))
k1 = 1.0000e009
k2 = 5.0000e+000
R5 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R6 (Mass action (irrevesible))
k = 5.3000e+001
R7 (Mass action (reversible))
k1 = 1.0000e+408
k2 = 8.4000e+005
R8 (Mass action (reversible))
k1 = 1.0000e+008
k2 = 8.4000e+005
R9 (Mass action (ireversible))
k = 3.5000ee+001
R10 (Mass action (irreversible))
k 8.0000e0000


2(c)
R1 (Mass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+000
R2 (M~ass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+003
R3 (Mass action (reversible))
kl = 1.0000ed09
k2= 5.0000e+000
R4 (MVass action (reversible))
k1 = 1.000~0e09
k2 = 5.000~0e03
R5 (MIass action (vreveible))
kl = 1.0000e+008
k2 = 8.4000t+005
R6 (Mass action (irrevensible))
k = 5.3000ete01
R7 (MVass action (reversible))
k1 = 1.0000e+08~
k2 8.4000e+005
R8 (Mlass action (reversible))
k1 = 1.0000e+008~~~~eeee~~~
k2 = 8.4000e+005
R9 (Mass action (irrevensible))
k = 3.5000e+e001
R10 (Mass action (irreversible))
k = 8.0000e0000











Case 3 (site 2 required, site 1 is active)


3(a)
R1 (Mlass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e000
R2 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R3 (Mass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+000
R4 (M~ass action (reversible))
kl = 1.0000e009
k2 = 5.0000e+000
R5 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R6 (Mass action (irrevesible))
k = 5.3000et001
R7 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R8 (Mlass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R9 (Mass action (irreesible))
k = 5.0000e-006


3b)
R1 (Mass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e*003
R2 (M/ass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R3 (Mass action (reversible))
kl = 1.0000e+009
k2 =5.00000e003
R~4 (Mass action (reversible))
kl = 1.0000e*009
k2 = 5.0000e+000
R5 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R6 (Mass action (irrevesible))
k = 5.3000e+001
R~7 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R8 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R9 (Mass action (ireversible))
k = 5.0000e-006


3(c)
R1 (Mass action (reversible))
kl = 1.0000e+009
k2 =- 5.0000e+000
R2 (Mlass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+003
R3 (Miass action (reversible))
kl = 1.0000e+009
k2 =5.0000e+000
R~4 (Mass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+003
R~5 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R6 (Mass action (irrevesible)
k = 5.3000e+001
R~7 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
~R8 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R9 (Mass action (irrevesible)
k 5.0000e-006
~R10 (~Mass action (irreversible))
k = 5.0000e-006


R10 (Mass action (irreversible)) R10 (Mass action (irreversible))
k = 5.0000e-006 k = 5.0000e-006










Case 4 (sites 1 and 2 have equal activity)


4(a)
R1 (Mass action (reversible))
kl = 1.0000e+-009
k2 = 5.0000e+-000
R2 (Mass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+000
R3 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R4 (Mass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+000
RS (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R6 (Mass action (irreversible))
k= 5.3000e+t001
R7 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R8 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R9 (Mass action (irreversible))
k = 2.16500et001
R10 (Mass action (irreversible))
k = 2.6500e+001


4(b)
R1 (Mass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+003
R2 (1Mass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+000
R3 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+003
R4 (Mass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+000
RS (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R6 (Mass action (irreversible))
k= 5.3000e+001
R7 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R8 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R9 (Mass action (irreversible))
k = 2.6500e+001
R10 (Mlass action (irreversible))
k = 2.6500e+001


4(c)
R1 (Miass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+000
R2 (M~ass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+003
R3 (M~ass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R4 (M~ass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+003
RS (M/ass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R6 (Mh~ass action (irrvrsible))
k= 5.3000e+001
R7 (M~ass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R8 (M~ass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R9 (M~ass action (irreversible))
k = 2.6500e+001
R10 (Miass action (irreversible))
k 2.6i500e+001











Case 5 (site 2 required, fully occupied enzyme twice as active)


5(a)
R1 (Mass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+000
R2 (Mass action (reversible))
kl = 1.0000e*009
k2 = 5.0000e+000
R3 (Mass action (reversible))
k1 = 1.0000e+~009
k2 = 5.0000e+000
R4 (Mass action (reversible))
k1 = 1.00008+009
k2 = 5.0000e+000
R5 (Mass action (reversible))
kl = 1.0000e~008
k2 = 8.4000e+005
R6 (Mass action (irreversible))
k = 5.3000et001
R7 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.40008+005
RS (Mass action (reversible))
kl = 1.0000e-t008
k2 = 8.4000e+005
R9 (Mass action (irreversible))
k = 2.6000e+001
R10 (Mass action (irreversible))
k = 5.0000e?-006


5(b)
R1 (M~ass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+003
R2 (Mass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+000
R3 (Mass action reversiblele)
k1 = 1.0000e+409
k2 = 5.0000e+-003
R4 (M~ass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R5 (1Mass action (reversible))
kl = 1.0000e+008
k2 = 8.40001e+005
R6~ (Mass action (irreversible))
k = 5.3000e+001
R7 (Mlass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
RS (M~ass action (reversible))
kl = 1.0000e+008
k2 = 8.40001e+005
R9 (Mass action (irreversible))
k = 2.60001e+001


5(c)
R1 (M~ass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+000
R2 (Mass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+003
R3 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R4 (M~ass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+003
R5 (M~ass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R6 (Mass action (irreversible))
k = 5.3000e+001
R7 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R8 (M~ass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R9 (Mass action (irreversible))
k = 2.6000e+001


R10 (Mass action (irreversible)) R10 (Mass action (irreversible))
k = 5.0000e-006i k = 5.0000e-006i










Case 6 (site 1 active, cooperative binding)


6(a)
R1 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+002
R2 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+002
R3 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R4 i(Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R5 (Mass action (reversible))
k1 = 1.OWI000+08
k2 = 8.4000e+005
R6 (Mass action (irreversible))
k = 5.3000e+001
R7 (Mass action (reversible))
k1 = 1.0000e+008
k2 = 8.4000e+005
RS (Mass action (reversible))
k1 = 1.0000e+008
k2 = 8.4000e+005
R9 (Mass action (irreversible))
k = 5.3000e+001


6(b)
R1 (Mlass action (reversible))
k1 = 1.0000e009
k2 = 5.0000e+002
R2 (Mass action (reversible))
k1 = 1.0000e4009
k2 = 5.0000e+000
R3 (M~ass action (reversible))
k1 = 1.0000e+009
k2= 5.0000e+000
R4 (M~ass action (reversible))
k1 = 1.0000e4009
k2 = 5.0000e-002
R5 (Mass action (reversible))
k1 = 1.0000E+008
k2 = 8.4000e+005
R6 (Mass action (irreversible))
k = 5.3000e+001
R7 (Mass action (reversible))
k1 = 1.0000e4GD8
k2 = 8.4000e+-005
RS (M~ass action (reversible))
k1 = 1.0000e+008
k2 8.4000e+005
R9 (Mass action (irreversible))
k = 5.3000e+001


6(c)
R1 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.000000000
R2 (Mass action (reversible))
k1 = 1.0000esoo9
k2 = 5.0000e+002
R3 (Mlass action (reversible))
k1 = 1.0000e0009
k2= 5.0000e-002
R4 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e0000
R5 (Mass action reversiblele)
k1 = 1.00000008
k2 = 8.4000et005
R6 (Mass action (irreversible))
k = 5.3000e+001
R7 (Mass action (reversible))
k1 = 1.0000e+408
k2 = 8.4000e+005
R8 (Mlass action (reversible))
k1 = 1.0000et008
kE2 = 8.4000~e+005
R9 (Mass action (irreversible))
k = 5.3000e+001
R10 (1Mass action (irreversible))
k = 5.0000e-006


R10 (Mass action (irreversible)) R10 (Mass action (irreversible))
k = 5.0000e-006 k = 5.0000e-006











Case 7 (only full enzyme active, cooperative binding)


7 (a)
~R1 (Mass action (reversible))
kl = 1.0000e+009
k2 =- 5.0000e0002
R2 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+002
R3 (Mass action (rversible))
k1 = 1.0000et009
k2 = 5.0000e+000
R4 (Mvass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+000
R5 (Mvass action (reversible))
k1 = 1.0000e+008
k2 = 8.4000e+005
R6 (Mass action (irreversible))
k = 5.3000e+001
R7 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R8s (Muass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
R9 (Mass action (irreversible))
k = 5.0000e006
R10o (1Mass action (irreversible))
k = 5.0000e-006


7 (b)
R1 (M~ass action (reversible))
kl = 1.0000e+009
k2 = 5.0000e+002
R2 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.0000e+000
R3 (Mass action (rversible))
k =e 1.0000e+009
k2 = 5.00000000 0
R4 (Mass action (reversible))
kl = 1.0000e*009
k2 = 5.0000e-002
R5 (Mass action (reversible))
k1 = 1.0000et008
k2 = 8.4000e+005
R6 (Mass action (irreversible))
k = 5.3000e+001
R7 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000e+005
RS (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.4000et005
R9 (Mass action (irreversible))
k = 5.0000e-006
R10 (Mass action (irreversible))
k = 5.0000e-006


7 (c)
R1 (Mass action (reversible))
kl = 1.0000e 009
k2 = 5.0000e+000
R2 (Mass action (reversible))
k1 = 1.0000e+009
k2 = 5.000e00
R3 (Mass action (rversible))
k1 = 1.0000e+009
k2 = 5.0000e-002
R4 (Mass action (revesible))
kl = 1.0000e+009
k2 = 5.0000e+000
R5 (Mass action (reversible))
k1 = 1.0000ev008~ee~~ee
k2 = 8.41000e+005
R6 (Mass action (irreversible))
k = 5.3000e+001
R7 (Mass action (reversible))
kl = 1.0000ietD08
k2 = 8.41000e*005
R8 (Mass action (reversible))
kl = 1.0000e+008
k2 = 8.41000e*D05
R9 (Mass action (irreversible))
k = 5.0000e-006
R10 (Mass action (irreversible))
k = 5.0000e-006










APPENDIX B
SIMULATIONS OF EPR SPECTRA AT DIFFERENT FIELD/FREQUENCY
COMBINATIONS OF OXALATE DECARBOXYLASE IN STORAGE BUFFER







SO -

410-





-20
-30
--40

200 250 300 350 400 450
Be [mT]

Figure B-1 X-band spectrum of OxDC in SB pH6.0 at T = 10 K. Experimental spectrum is
shown in black. Simulations for sites I and II are shown in blue and green,
respectively. The sum of the simulation of the two sites is shown in red.Experimental
parameters: Microwave frequency 9.48731 GHz, microwave power 0.64 mW,
modulation frequency 100 k time constant 41 ms, conversion time 41 ms, 1 sweep, 1.465 G/data point.Simulation
parameters for site I: giso = 2.000865, Aiso = 254 MHz, D = 1200 MHz, E = 252 MHz,
D-Strain = 0.24xD, E-Strain = 0.24xE, linewidthiso = 33 MHz.Simulation parameters
for site II: giso = 2.00094, Aiso = 248 MHz, D = 2750 MHz, E = 660 MHz, D-Strain =
0.20xD, E-Strain = 0.20xE, linewidthiso = 33 MHz.

























Figure B-2 V-band spectrum of OxDC in SB pH6.0 at T = 20 K. Experimental spectrum is
shown in black. Simulations for sites I and II are shown in blue and green,
respectively. The sum of the simulation of the two sites is shown in red.Experimental
parameters: Microwave frequency 49.200 GHz, microwave power corresponding to a
dector signal of 500 mV, modulation frequency 41.68 k G, lock-in sensitivity 200 CLV, time constant 300 ms, sweep speed 1 G/s, 1 sweep,
0.250 G/data point, center field 1.753 T.Simulation parameters for site I: giso =
2.000865, Aiso = 254 MHz, D = 1200 MHz, E = 276 MHz, D-Strain = 0.24xD, E-
Strain = 0.30 xE, linewidthiso = 33 MHz. Simulation parameters for site II: giso =
2.00094, Aiso = 248 MHz, D = 2700 MHz, E = 675 MHz, D-Strain = 0.25xD, E-
Strain = 0.20 xE, linewidthiso = 33 MHz.































3320 3330 3340 3350 3360 3370 3380 3390 3400 3410


Figure B-3 W-band EPR spectrum of OxDC in SB pH6.0 at T = 50 K. Experimental spectrum is
shown in black. Simulations for sites I and II are shown in blue and green,
respectively. The sum of the simulation traces of the two sites is given in red.
Experimental parameters: Microwave frequency 94.02141 GHz, microwave power
0.6 CLW modulation frequency 100 k dB, time constant 164 ms, conversion time 164 ms, 1 sweep, 1.172 G/data point.
Simulation parameters for site I: giso = 2.000865, Aiso = 254 MHz, D = 1200 MHz, E
= 252 MHz, D-Strain = 0.24xD, E-Strain = 0.24xE, linewidthiso = 33 MHz.
Simulation parameters for site II: giso = 2.00094, Aiso = 248 MHz, D = 2700 MHz, E =
648 MHz, D-Strain = 0.20 xD, E-Strain = 0.20 xE, linewidthiso = 33 MHz.


































7910 7920 793D 7940 7950
magnate lieks[mT)


7960 7970 7980


Figure B-4 Sub-mm EPR spectrum of OxDC in SB pH6.0 at T = 20 K and 222 GHz.
Experimental spectrum is shown in black. Simulations for sites I and II are shown in
blue and green, respectively. The sum of the simulation of the two sites is shown in
red. Experimental parameters: Microwave frequency 222.400 GHz, modulation
frequency 41.8 kHz, modulation amplitude 0.5 G, lock-in sensitivity 500 CLV, time
constant 100 ms, sweep speed 5.01 G/s, 1 sweep, 0.368 G/data point. Simulation
parameters for site I: giso = 2.000865, Aiso = 251 MHz, D = 1200 MHz, E = 252 MHz,
D-Strain = 0.24xD, E-Strain = 0.24xE, linewidthiso = 33 MHz. Simulation parameters
for site II: giso = 2.00094, Aiso = 247 MHz, D = 2700 MHz, E = 675 MHz, D-Strain =
0.20 xD, E-Strain = 0.20 xE, linewidthiso = 33 MHz.










324 Gli?.


Figure B-5 Sub-mm EPR spectrum of OxDC in SB pH6.0 at T = 20 K and 324 GHz.
Experimental spectrum is shown in black. Simulations for sites I and II are shown in
blue and green, respectively. The sum of the simulation traces of the two sites is
given in red. Experimental parameters: Microwave frequency 324.00 GHz,
modulation frequency 41.8 k CLV, time constant 100 ms, sweep speed 5.01 G/s, 1 sweep, 0.367 G/data
point. Simulation parameters for site I: giso = 2.000865, Aiso = 250 MHz, D = 1200
MHz, E = 252 MHz, D-Strain = 0.24xD, E-Strain = 0.24xE, linewidthiso = 33
MHz. Simulation parameters for site II: giso = 2.00094, Aiso = 247 MHz, D = 2700
MHz, E = 675 MHz, D-Strain = 0.20 xD, E-Strain = 0.20 xE, linewidthiso = 33 MHz.








413 GHz


Figure B-6 Sub-mm EPR spectrum of OxDC in SB pH6.0 at T = 20 K and 412.8 GHz.
Experimental spectrum is shown in black. Simulations for sites I and II are shown in
blue and green, respectively. The sum of the simulation traces of the two sites is
given in red. Experimental parameters: Microwave frequency 412.800 GHz,
modulation frequency 41.8 k CLV, time constant 100 ms, sweep speed 5.01 G/s, 1 sweep, 0.369 G/data point.
Simulation parameters for site I: giso = 2.000865, Aiso = 253 MHz, D = 1200 MHz, E
= 252 MHz, D-Strain = 0.24xD, E-Strain = 0.24xE, linewidthiso = 33
MHz. Simulation parameters for site II: giso = 2.00093, Aiso = 249 MHz, D = 2700
MHz, E = 675 MHz, D-Strain = 0.25 xD, E-Strain = 0.25 xE, linewidthiso = 33 MHz.









APPENDIX C
HIGH FIELD SPECTRA AND SIMULATIONS OF WT OXDC AND THE E280Q MUTANT

Figures C-1, C-2, and C-3 show the results of the best fits obtained for simulations of the

EPR spectra obtained for wild type OxDC and the E280Q mutant. All simulations were

performed with the Easy Spin toolbox (121) in the MATLAB computing environment (The

MathWorks, Natick, MA) by Dr. Ines Garcia-Rubio at ETH-Zurich. We assumed isotropic g-

and A-tensors while using an anisotropic fine structure tensor, and the fits improved considerably

by choosing a mixture of Lorentzian and Gaussian lineshapes. Nine independent fit parameters

were used for the main Mn(II) species in each spectrum. Given that only the region around g~2

was measured in these experiments, fine structure values and associated strain parameters have a

large uncertainty (conservatively estimated to be 150% of the fit values). On the other hand, the

hyperfine coupling constant could be extracted from the first with more confidence. Changing A

by a 1 MHz considerably worsened agreement between the simulated and the experimental

spectrum, and, similarly, the margin of error for the isotropic g-value is small being estimated as

approximately & 0.00001. Apparent linewidths are somewhat dependent on the choice of D and

E, although reducing both fine structure constants to almost zero only reduces the linewidth by a

few Gauss (0.85 mT and 1.1 mT for E280Q and wild-type enzyme respectively), and these

simulations suffer from lack of accuracy in the wings of the six lines.





























13760 13780 13800 13::20
Magnetic Field [mT]


Figure C-1 High field EPR of wild-type OxDC enzyme at 386. 116 GHz and 10 K. The
experimental and simulated spectra are displayed in blue and red, respectively.
Simulation parameters for the maj ority Mn(II) component are: g-factor = 2.00087, D
= 1200 MHz, E = 240 MHz, D-Strain: 40% of D, E-Strain: 40% of E, hyperfine
coupling, A: 250 MHz, A-Strain: 1% of A, linewidth: 1.5 mT, lineshape: 90%
Lorentzian, 10% Gaussian. A minority component was assumed to be present to
explain the low field shoulders on the main six-line spectrum, contributing
approximately 4% of the spectral intensity, with simulation parameters: g-factor =
2.00107, D = E = 0 MHz, hyperfine coupling, A: 245 MHz, linewidth: 1.8 mT,
lineshape: 100% Lorentian.






























11800 11820 11840 11860
Magnetic Field [mT]


Figure C-2 High field EPR of E280Q OxDC mutant at 33 1.2 GHz and 20 K. The experimental
and simulated spectra are displayed in blue and red, respectively. Simulation
parameters for the maj ority Mn(II) component are: g-factor = 2.00087, D = 850 MHz,
E = 85 MHz, D-Strain: 40% of D, E-Strain: 40% of E, hyperfine coupling, A: 255
MHz, A-Strain: 2% of A, linewidth: 0.9 mT, lineshape: 50% Lorentzian, 50%
Gaussian. A minority component was assumed to be present to explain the low field
shoulders on the main six-line spectrum, contributing approximately 7.5% of the
spectral intensity, with simulation parameters: g-factor = 2.00109, D = E = 0 MHz,
hyperfine coupling, A: 242 MHz, linewidth: 1.8 mT, lineshape: 100% Lorentian.




























136i40 13660 136i80 13700
Magnetic Field [mnTJ

Figure C-3 High field EPR ofE280Q OxDC mutant at 382.826 GHz and 10 K. The
experimental and simulated spectra are displayed in blue and red, respectively.
Simulation parameters for the maj ority Mn(II) component are: g-factor = 2.00087, D
= 850 MHz, E = 85 MHz, D-Strain: 40% of D, E-Strain: 40% of E, hyperfine
coupling, A: 253 MHz, A-Strain: 1% of A, linewidth: 1.0 mT, lineshape: 80%
Lorentzian, 20% Gaussian. A minority component was assumed to be present to
explain the low field shoulders on the main six-line spectrum, contributing
approximately 7.5% of the spectral intensity, with simulation parameters: g-factor =
2.00111, D = E = 0 MHz, hyperfine coupling, A: 240 MHz, linewidth: 1.8 mT,
lineshape: 100% Lorentian. This spectrum taken at 382 GHz did not have a field
standard, and to simulate the 382 GHz spectrum, the field axis was adjusted to yield
the same isotropic g-factor in the simulation in essence using the six-line spectrum as
the field standard for that particular frequency made possible using the calibrated
spectrum taken for the E280Q OxDC mutant at 331 GHz.










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BIOGRAPHICAL SKETCH

Ellen Moomaw was born in Jacksonville, FL in 1960. After earning a master' s of science

degree in biochemistry in the laboratory of Dr. Dale Edmondson (Emory University, 1984), she

worked in various biotechnology companies in San Diego. In 1987 Ellen was the 30th employee

hired at a young company called Agouron Pharmaceuticals, Inc. where she purified and

characterized a number of proteins including thymidylate synthetase, HIV reverse transcriptase

and RNase H, DNA polymerase b, and HCV protease. By the time she left Agouron in 1999 to

teach high school chemistry, Agouron (now part of Pfizer) had over 1200 employees and had

gotten the 4th HIV protease inhibitor on the market (ViraceptTM). Ellen started graduate studies

in the chemistry department of the University of Florida in 2003, where she j oined the research

group of Dr. Nigel G. J. Richards.





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1 MANGANESE CENTERS IN OXALATE DECARBOXYLASE By ELLEN WINGER MOOMAW A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Ellen Winger Moomaw

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3 To my parents and to my sisters

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4 ACKNOWLEDGMENTS There are many people that I would like to thank for their help and support throughout my doctoral research at the University of Florida. I am particularly indebted to my advisor Dr. Nigel G. J. Richards and EPR collaborator Dr. Alexande r Angerhofer for making this work a reality as well as for their guidance. I thank Dr. George Christou, Dr. Thomas J. Lyons, and Dr. Daniel L. Purich for their input and support as members of my doctoral dissertation committee. This study was supported by grants from the National Institutes of Health (DK61193 and DK61666) and by the University of Florida Chemistry Department. I am grateful to my coworkers and friends in the Richards research group, especially Dr. Patricia Moussatche, for the molecular biological training and collaboration that she provided. I also thank Kai Li, Ewa Wroclawska, Dr. Drazen ka Svedruzic, Dr. Christopher Chang, Cory Toyota, and Dr. Jemy Gutierrez for technical assistance, trai ning, and helpful discussions. I owe a debt of gratitude to my EPR collabora tors in addition to Dr. Alexander Angerhofer, Dr. Andrew Ozarowski and Dr. Jurek Kryztek of the National High Magnetic Field Laboratory, Dr. Ines Garcia-Rubio of the ETH Zurich, and Dr Ralph T. Weber of the Bruker Biospin Corp. I appreciate the technical support of Vijay Antharam and Omjoy Ganesh for the CD studies and of Witcha Imaram for the EPR spin-tapping experiments. I thank Dr. Ben Smith, Graduate Coordinator, and Lori Clark in the Graduate Student Office for their help and advice.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .........9 ABSTRACT....................................................................................................................... ............12 CHAPTER 1 INTRODUCTION..................................................................................................................14 Oxalic Acid in Biological Systems.........................................................................................14 Oxalate Degrading Enzymes..................................................................................................15 Oxalate Decarboxylase.......................................................................................................... .17 Biological Distribution....................................................................................................17 Oxalate Decarboxylase Belongs to the C upin Superfamily (DSBH) of Proteins...........18 Structural Features...........................................................................................................20 Mechanistic Information.................................................................................................23 Oxygen Dependence and the Formation of Hydrogen Peroxide.....................................25 Research Objectives............................................................................................................ ....26 2 CHARACTERIZATION OF THE MN-DEPENDENCE OF OXALATE DECARBOXYLASE ACTIVITY..........................................................................................27 Introduction................................................................................................................... ..........27 Results and Discussion......................................................................................................... ..27 Optimization of Expression of Recombinant Wild Type OxDC.....................................27 Effect of Addition of Other Me tals in the Growth Medium............................................28 Preparation of the OxDC Apoenzyme and Reconstitution of the Wild Type, MnContaining Enzyme......................................................................................................29 Gepasi Simulations..........................................................................................................31 Experimental Section........................................................................................................... ...34 Materials...................................................................................................................... ....34 Expression and Purification of Recombinant, Wild Type OxDC...................................34 Expression and Purification of Co-Substituted, Wild Type OxDC.................................35 Preparation of the OxDC Apoenzyme and Reconstitution of the Wild Type, MnContaining Enzyme......................................................................................................36 Metal Content Determination..........................................................................................36 Steady-State Kinetic Assays............................................................................................37 Gepasi Simulations..........................................................................................................38 3 SPECTROSCOPIC CHARACTERIZATION OF THE TWO MANGANESE CENTERS........................................................................................................................ .......42

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6 Introduction................................................................................................................... ..........42 Electron Paramagnetic Resonance Spectroscopy............................................................42 Electronic Configuration of Mn(II).................................................................................43 Electron Paramagnetic Resonance Properties of Mn(II).................................................43 Oxalate Decarboxylase EPR............................................................................................46 Results and Discussion......................................................................................................... ..47 X-band EPR.....................................................................................................................47 Field Dependence of the EPR Signal in Storage Buffer..................................................48 Spectral Simulations and Magnetic Parameters..............................................................49 Field Dependence of the EPR Signa l in Acetate Buffer, pH 5.2.....................................51 Experimental Section........................................................................................................... ...53 Oxalate Decarboxylase Sample Preparation...................................................................53 Electron Paramagnetic Resonance Spectroscopy............................................................54 4 SPECTROSCOPIC CHANGES OF THE MANGANESE CENTERS IN THE PRESENCE OF SUBSTRATE..............................................................................................55 Introduction................................................................................................................... ..........55 Results and Discussion......................................................................................................... ..56 X-band (9.5 GHz)............................................................................................................56 Chemical Oxidation of OxDC Observed at X-band.................................................59 X-band Spin-Trapping of an Oxygen Species Formed During Oxalate Decarboxylase Turnover.......................................................................................60 Q-band ( 35 GHz)............................................................................................................63 W-band (94 GHz)............................................................................................................63 324 GHz........................................................................................................................ ...64 690 GHz........................................................................................................................ ...64 Experimental Section........................................................................................................... ...65 5 SITE-DIRECTED MUTAGENESIS STUDI ES TO PROBE WHICH MANGANESEBINDING SITE(S) IS INVOL VED IN CATALYSIS...........................................................67 Introduction................................................................................................................... ..........67 Results and Discussion......................................................................................................... ..68 Design and Steady-State Char acterization of OxDC Mutants with Domain-Specific Modified Mn Affinity..................................................................................................68 Size-Exclusion Chromatography (SEC)..........................................................................70 Circular Dichroism Measurements..................................................................................71 Electron Paramagnetic Resonance Properties.................................................................73 Relaxation Enhancement Measurements.........................................................................75 Implications for the Location of the Catalytic Site(s) in OxDC......................................78 Motivation for the Preparation of Single Domain OxDC Mutants.................................79 N-Terminal OxDC Single Domain Mutant (OxDC-N1) Does Not Catalyze the Decarboxylation Reaction............................................................................................80 EPR Characterization of Reconstituted Nterminal OxDC Single Domain Mutant (OxDC-N1)..................................................................................................................81

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7 C-Terminal OxDC Single Domain Mutant (OxDC-C) Does Not Catalyze the Decarboxylation Reaction............................................................................................82 Combining the Nand C-Terminal Single Domain Mutants Did Not Result in Decarboxylase Activity................................................................................................82 Experimental Section........................................................................................................... ...83 Expression and Purification of Site-Specific OxDC Mutants.........................................83 Oxalate Oxidase Assays..................................................................................................84 Size-Exclusion Chromatography Measurements............................................................84 Circular Dichroism Studies.............................................................................................85 Electron Paramagnetic Resonance Spectroscopy............................................................85 Expression of OxDC Single Domain Mutants................................................................86 Purification of Single Domain OxDC Mutant OxDC-N1...............................................87 Purification of Single Doma in OxDC Mutant OxDC-C.................................................88 6 CONCLUSIONS AND FUTURE WORK.............................................................................89 APPENDIX A KINETIC PARAMETERS USED IN GEPASI SIMULATIONS.........................................91 B SIMULATIONS OF EPR SPECTRA AT DIFFERENT FIELD/FREQUENCY COMBINATIONS OF OXALATE DECARB OXYLASE IN STORAGE BUFFER...........99 C HIGH FIELD SPECTRA AND SIMULATI ONS OF WT OXDC AND THE E280Q MUTANT......................................................................................................................... ....105 LIST OF REFERENCES.............................................................................................................109 BIOGRAPHICAL SKETCH.......................................................................................................117

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8 LIST OF TABLES Table page 2-1 Effect of MnCl2 and CoCl2 in the growth medium on metal incorporation and specific activity of recombinant, wild type OxDC. .........................................................29 2-2 Metal content of apoenzyme and enzyme reconstituted with Mn. ...............................30 3-1 Magnetic parameters of OxDC species I and II. ..............................................................50 5-1 Mn incorporation and steady-state ki netic parameters for metal-binding OxDC mutants........................................................................................................................ .......68 5-2 Metal content of Mn-binding OxDC mutants....................................................................69 5-3 Estimates of size for the oligomeric fo rms of recombinant wild type OxDC and OxDC Mn-binding mutants obtained usi ng size exclusion chromatography....................71 5-4 Metal content of single do main mutant preparations.........................................................81 5-5 Primers used in the constr uction of Mn-binding mutants..................................................83 5-6 Primers used in the preparati on of OxDC single domain mutants.....................................87

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9 LIST OF FIGURES Figure page 1-1 Enzymes that catalyze oxalate degradation. ....................................................................16 1-2 Sequence alignments of OxDCs from Bacillus subtilis Bacillus clausii Collybia velutipes and Aspergillus oryzae showing the positions of the two conserved motifs (motif 1 in blue and motif 2 in red) in the two domains. .................................................19 1-3 Ribbon structures of the Bacillus subtilis OxDC monomer, trimer, and hexamer. .........21 1-4 Residues defining the Mn-binding sites in the N-terminal (1UW8) and C-terminal (1J58) domains of OxDC...................................................................................................22 1-5 Proposed catalytic mechanism for ox alate decarboxylase based on heavy-atom isotope effect measurements..............................................................................................24 2-1 The dependence of OxDC specific activity on the extent of Mn incorporation. .............31 2-2 Numerical simulations of the dependence of catalytic activity on the extent of Mn incorporation.................................................................................................................. ....32 3-1 Absorption of microwave irradiation by an unpaired electron in a magnetic field. ........42 3-2 Electron spin energy levels and hyperfin e splitting for Mn(II) in spherical symmetry. ............................................................................................................................... .............44 3-3 Overlay of the N-terminal (shown in magenta) and C-terminal (shown in green) manganese-coordinating ligands (PDB code: 1UW8) ...................................................46 3-4 X-band cw-EPR spectra of wild type Ox DC in storage buffer and in acetate buffer........48 3-5 Field dependence of the EPR spectra of OxDC in storage buffer (20 mM Hexamethylenetetramine HCl, pH 6.0) with 0.5 M NaCl.................................................49 3-6 W-Band (94 GHz) EPR spectra of OxDC.........................................................................50 3-7 Field dependence of the EPR spectra of OxDC in acetate buffer (50 mM, pH 5.2) with 0.5 M NaCl................................................................................................................51 4-1 Spectral changes of the g 2 signal at X-band upon addi tion of acetate and oxalate to OxDC........................................................................................................................ .....56 4-2 Spectral changes of the g 2 signal at X-band upon additi on of oxalate to OxDC in storage buffer at pH 5.2.....................................................................................................57

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10 4-3 Spectral changes of the g 2 signal at X-band upon additi on of oxalate to OxDC in storage buffer at pH 5.2 under anaerobic c onditions followed by th e reintroduction of air............................................................................................................................ ...........58 4-4 Mn(II) signal intensity and carbon-base d radical formation as a function of the concentration of sodium (meta) periodate.........................................................................60 4-5 EPR spectra of the spin-trapped radical formed during OxDC turnover. ........................61 4-6 EPR spectrum of a short-lived DMPO-oxygen species.....................................................62 4-7 Spectral changes of the Mn(II) signal at Q-band upon addition of acetate and oxalate to OxDC........................................................................................................................ .....62 4-8 Spectral changes of the Mn(II) signal at W-band upon addition of acetate and oxalate to OxDC........................................................................................................................ .....63 4-9 Spectral changes of the Mn(II) sign al at 324 GHz upon addition of acetate and oxalate to OxDC................................................................................................................64 4-10 Spectral changes of the Mn(II) sign al at 690 GHz upon addition of acetate and oxalate to OxDC................................................................................................................65 5-1 CD spectra of recombinant wild type OxDC and the Mn-binding OxDC mutants...........72 5-2 EPR spectra of the Mn(I I) signals in wild type OxDC (red) and the E280Q OxDC mutant (blue) at 10 K ........................................................................................................74 5-3 Inversion-recovery experiments on w ild type OxDC (12.3 mg/mL) and the E280Q OxDC mutant (16.8 mg/mL)..............................................................................................76 5-4 Data for the Hahn-echo decay experi ment on wild-type OxDC and the E280Q mutant. .................................................................................................................... .......77 5-5 Topology diagram of OxDC. ...........................................................................................80 5-6 Effect of buffer and oxalate on the g 2 X-band Mn(II) signal of reconstituted Nterminal OxDC mutant (OxDC-N1)..................................................................................81 B-1 X-band spectrum of OxDC in SB pH6.0 at T = 10 K........................................................99 B-2 V-band spectrum of OxDC in SB pH6.0 at T = 20 K......................................................100 B-3 W-band EPR spectrum of OxDC in SB pH6.0 at T = 50 K.............................................101 B-4 Sub-mm EPR spectrum of OxDC in SB pH6.0 at T = 20 K and 222 GHz.....................102 B-5 Sub-mm EPR spectrum of OxDC in SB pH6.0 at T = 20 K and 324 GHz.....................103

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11 B-6 Sub-mm EPR spectrum of OxDC in SB pH6.0 at T = 20 K and 412.8 GHz..................104 C-1 High field EPR of wild-type Ox DC enzyme at 386.116 GHz and 10 K. ......................106 C-2 High field EPR of E280Q OxDC mutant at 331.2 GHz and 20 K...................................107 C-3 High field EPR of E280Q OxDC mutant at 382.826 GHz and 10 K...............................108

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12 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy STUDIES OF THE MANGANESE CENT ERS IN OXALATE DECARBOXYLASE By Ellen Winger Moomaw August 2007 Chair: Nigel G. J. Richards Major: Chemistry Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO2 and formate by a mechanism that remains poorly understood. The bacterial form of the enzyme, present in Bacillus subtilis is composed of two cupin domains, ea ch of which contains a Mn(II) ion coordinated by four conserved re sidues. My work reports an in vivo strategy for obtaining recombinant, wild type OxDC in which manganese is substituted by coba lt, together with the first conditions for in vitro reconstitution of the apoenzyme with Mn(II). My work also examines the question of whether both Mn-binding sites in Bacillus subtilis OxDC can independently catalyze the decarboxylation reacti on by expressing and characterizing a series of OxDC mutants in which metal binding is pe rturbed. A linear relationship between Mn occupancy and catalytic activity is demonstrated. Electron Paramagnetic Resonance (EPR) measurements reveal that the a pparent line broadening observed for the Mn signals of wild type OxDC arises from dipolar coupling between nei ghboring Mn ions. These results are consistent with the proposal that there is only a single catalytic site in the enzyme. The similarity between the two Mn(II) sites has precluded previ ous attempts to distinguish them spectroscopically and complicated efforts to understand the catalytic mechanism. My research utilizes a multifrequency EPR approach to distinguish the two Mn ions on the basis of

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13 their differing fine structure parameters, and obs erved that acetate and formate bind to Mn(II) in only one of the two sites. The EPR evidence is consistent with the hypot hesis that this Mnbinding site is located in the N-terminal domain, in agreement with predic tions based on a recent X-ray structure of the enzyme.

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14 CHAPTER 1 INTRODUCTION Oxalic Acid in Biological Systems Oxalate producing plants, which include numerous crop plants, accumulate oxalate in the range of 3% to 80% (w/w) of their dry weight ei ther as the free acid, as sodium or potassium oxalate, or as an insoluble salt, most commonly calcium oxalate ( 1-4 ). A number of possible pathways for the biosynthesis of oxalic ac id in plants have been described ( 5 ). These pathways include the oxidation of glycol ate and glyoxylate (byproducts of photorespiration) by glycolate oxidase ( 6, 7 ) and the activity of isocitrate lyase on isocitrate ( 8, 9 ). Possible functions of soluble oxalate and calcium oxalate crystals in plants include protec tion against insects and foraging animals, ion regulation, and detoxification of heavy metals ( 2, 4, 10 ). Oxalic acid and its calcium salt accumu late in many fungi but knowledge of its biosynthetic pathway remains fragmentary. Oxalo acetase, which cleaves oxaloactetate to yield acetate and oxalate, is present in many fungal speci es and has been proposed as one biosynthetic route ( 5, 11, 12 ). Phytopathogenic fungi use oxalic acid at the site of infec tion to lower the pH which enhances the activity of lytic enzyme s such as polygalactur onase and cellulase ( 13, 14 ). Utilization of oxalate by wood rotting fungi to de grade lignin and cellulose has been the subject of much research ( 15-18 ). Bacteria capable of using oxala te as a sole carbon and ener gy source (oxalotrophic) play essential roles in soil fertility and retention an d/or recycling of elements necessary for plant growth and are important modulators in the biological carbon cycle ( 19, 20 ). Oxalotrophic bacteria inhabiting the gastroin testinal tracts of mammals provi de the only known route for the catabolism of dietary oxalate in these organisms.

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15 At high concentrations, oxalate causes death in humans and animals as a result of its corrosive properties. At lower concentrations oxalate leads to a variety of pathological disorders, including hyperoxaluria, pyridoxine deficiency, cardiomyopathy, cardiac conductance disorders, calcium oxalate stones and renal failure ( 20-24 ). The administration of oxalate degrading bacteria ( Oxalobacter formigenes) has been proposed as a treatment for hyperoxaluria ( 25 ). Several oxalate degrading enzymes have either actual or potential co mmercial significance with applications in medici ne, agriculture, and industry. Oxalate oxidase and oxalate decarboxylase are used in clinical as says of oxalate in blood and urine ( 26, 27 ). Transgenic plants have been engineered to express oxala te degrading enzymes as a means of protection against pathogens and to reduce the amount of oxalate present ( 28, 29 ). Structural, mechanistic, and biochemical information is needed in order to further the application of oxalate degrading enzymes in medicine, agriculture, and industry ( 28, 30, 31 ). Oxalate Degrading Enzymes Three major classes of enzymes have evolved to degrade oxalate. Plants employ oxalate oxidase ( 32 ), fungi ( 33 ) and soil bacteria ( 34 ) utilize oxalate decarboxylase, and bacteria exploit oxalyl-CoA decarboxylase ( 35 ) (Figure 1.1A). Oxalate oxid ases (OxOx), also known as germins, catalyze the oxygen-depe ndent oxidation of oxalate to ca rbon dioxide in a reaction that is coupled with the forma tion of hydrogen peroxide ( 32, 36, 37 ). Hydrogen peroxide formation is believed to serve as a defense me chanism against infection by pathogens ( 38, 39 ) and to contribute to cell wall crosslinking ( 40 ). X-ray crystallographic structure determination revealed that OxOx crystallizes as a hexamer ( 41 ) and electron paramagnetic resonance (EPR) studies demonstrated the presence of Mn(II) in the resting enzyme ( 36 ). The Mn ion is coordinated by four conserved residues (three His and a Glu) ( 42 ) and each monomer possesses the barrel

PAGE 16

16 topology characteristic of the c upin superfamily of enzymes ( 28, 43-46 ). EPR spectroscopic changes of the Mn signal upon the addition of oxal ate supports the hypothesis that the Mn ion is the site of catalysis ( 47 ). UV-visible spectroscop y, spin trapping studies, and structural studies have lead to proposed mechanisms that involve the binding of oxalate di rectly to Mn(II), the formation of Mn(III), and a ra dical intermediate species ( 36, 37, 48 ). Figure 1-1 Enzymes that catalyze oxalate degradation. (A) Oxal ate oxidases, found in plants. (B) Oxalate decarboxylases, pr esent in fungi and in some bacteria (C) Oxalyl-CoA decarboxylases, thiamin-dependent enzymes present in bacteria. Oxalyl-CoA decarboxylase (OXC) catalyzes th e cleavage of oxalyl-CoA to formyl CoA and carbon dioxide ( 37, 49, 50 ) (Figure 1-1C). In Oxalobacter formigenes OXC is coupled to formyl-CoA transferase, which catalyzes an acyl transfer from formyl-CoA to oxalate to yield formate and oxalyl-CoA ( 51 ). The overall coupled reactions convert oxalate to formate and carbon dioxide with the consumption of a proton. Insight into this system was provided by the isolation and characteriza tion of a membrane bound form ate-oxalate antiporter ( 52-54 ), which imports oxalate into the cell and exports formate, creating a proton gradient across the membrane which is then used to drive ATP synthesis ( 55, 56 ).

PAGE 17

17 Oxalate Decarboxylase Biological Distribution Oxalate decarboxylase (OxDC) catalyzes th e difficult carbon-carbon bond cleavage of oxalate to yield carbon dioxide and formate ( 57 ) (Figure 1-1B). This enzyme was first reported in the basidiomycete fungi Collybia (Flammulina) velutipes and Coriolus hersutus more than 50 years ago ( 33, 58 ). OxDC has since been reported in a number of fungal species including the following: Sclerotinia sclerotiorum ( 59 ) Coriolus (Trametes) versicolor ( 18 ) Mythrothecium verrucaria ( 60 ), Aspergillus niger ( 61, 62 ), Agaricus bisporus ( 63 ) and Postia placenta ( 64 ). Fungal OxDCs can be both an intracellula r enzyme ( 18 ) or excreted from the hyphal cells ( 18, 64, 65 ). Expression of OxDC in fungal cultures is induced by decreasing the pH and/or adding carboxylic acids such as oxalate glycolate, and citrate ( 18, 59-61, 66, 67 ). It has been proposed that differences in the regulation of OxDC expr ession may imply differences in the function of the enzyme in a specific organism ( 34, 68 ). Acid-induced OxDCs mi ght be involved in proton consumption whereas those induced by oxalate may protect the organism from the harmful metabolic effects of oxalate. The first prokaryotic OxDC was reported in 2000 when Bacillus subtilis, a soil bacterium, was shown to express a cytosolic OxDC. Bacteria l OxDC is induced by acid but not by oxalate which may suggest that its metabolic function is not related to its oxala te degrading activity ( 34, 69 ). This enzyme, encoded by the yvrK gene (renamed oxdC ) has been overexpressed in E. coli and further characterized ( 57, 70-74 ). Comparison of the OxDC fungal sequences with the B. subtilis genome ( 75 ), suggested that two other genes, yoaN (renamed oxdD ) and yxaG may also code for enzymes with OxDC activity ( 70 ). The yoaN gene product OxdD was shown to have low levels of OxDC activity ( 70 ) and the yxaG gene product has been repo rted to be a novel Fecontaining quercetin 2,3-dioxygenase ( 76-78 ).

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18 Oxalate Decarboxylase Belongs to the C upin Superfamily (DSB H) of Proteins The cupin superfamily of proteins have b een well recognized as possessing remarkable functional diversity with repr esentatives found in Archaea, Eubacteria, and Eukaryota ( 28, 4346 ). The identification of the cupin superfamily was originally based on the recognition that the wheat protein germin shared a nine amino acid sequence with another protein, spherulin, produced by the slime mold Physarum polycephalun during starvation ( 44 ). This sequence similarity was also observed in a number of s eed storage proteins calle d germin-like proteins (GLPs). Knowledge of the three dimensional structures of these proteins led to the collective name cupin on the basis of their barrel shape (cupa means small barrel in Latin) ( 46 ). Characteristic features of proteins with this fo ld include high thermal stability and resistance to proteases. These features are consistent w ith a high degree of subunit contacts, hydrophobic interactions, and short loops. The cupin domai n was originally described as two conserved motifs, each composed of two strands separated by a less c onserved region composed of another two strands separated by a loop of varying length ( 28, 45 ). Motif 1 was originally designated as G(X)5HXH(X)3,4E(X)6G (shown in blue in Figure 1-2) and Motif 2 as G(X)5PXG(X)2H(X)3H(X)3N (shown in red in Figure 1-2). With more sequences analyzed, it has become clear that the primary sequence of the two motifs is not as highly conserved as previously thought ( 42, 43 ). The cupin superfamily of proteins exemplif ies a general trend emerging from comparative genomics: classes of proteins are being expa nded beyond the presence of a set of conserved residues which had previously been the corn erstone of their identification. In 2003, Anantharaman et al. described the use of information from recently reported X-ray

PAGE 19

19 Figure 1-2 Sequence alignments of OxDCs from Bacillus subtilis Bacillus clausii Collybia velutipes and Aspergillus oryzae showing the positions of the two conserved motifs (motif 1 in blue and motif 2 in red) in the two domains. Alignment was made using the Clustal W method ( 79, 80 ). Asterisks indicate id entical residues, colons(:) indicate conservative substitutions, and periods (.) indicate semi-conservative substitutions. The Mn-binding residues of the Bacillus subtilis OxDC are underlined. crystallographic structures and sequences to ga in a perspective on the major principles that appear to have shaped the emergence of diverse enzymatic activities within structurally similar B. subtilis 1 --------------------------------------------------------MK B. clausii 1 -------------------------------------------------MKRGDNVKPLK C. velutipes 1 MFNNFQRLLTVILLSGFTAGVPLASTTTGTGTATGTSTAAEPSATVPFASTDPNPVLWNE A. oryzae 1 ------------------------------------MKAASLAFISFLPSVLGAVVHDKR B. subtilis 3 KQNDIPQPIRGD-KGATVKIPRNIERDRQNPDMLVPPETDHGTVSNMKFSFSDTHNRLEK B. clausii 12 GNPNIPQPIRADGAGGVDRGPRNLMRDLQNPNILVPPETDRGLIPNLRFSFSDAHMQLNH C. velutipes 61 TSDPALVKPERNQLGATIQGPDNLPIDLQNPDLLAPPTTDHGFVGNAKWPFSFSKQRLQT A. oryzae 25 SGFKDGQPISDNGKGAPLLGGTNKALDLQNPDNLGQPSTDNGFVPNLKWSFSDSKTRLFP : *. ***: **.* : ::.** :: :* B. subtilis 62 GGYAREVTVRELPISENLASVNMRLKP GAIRELH WH KEAE WAYMIYG SARVTIVDEKGRS B. clausii 72 GGWSREITQRDLPIATTLAGVNMSLTP GGVRELH WH KQAE WSYMLLG HARITAVDQNGRN C. velutipes 121 GGWARQQNEVVLPLATNLACTNMRLEA GAIRELH WH KNAE WAYVLKG STQISAVDNEGRN A. oryzae 85 ---VREQVIQDLPQSHDISGAQQHLKK GAIRELH WH RVAE WGFLYSG SLLLSGVDENGQF *: ** : :: .: *.:******: ***.:: :: **::*: B. subtilis 122 FIDDVGE GDLWYFPSGLPH SIQA LEEG---AEFLLVFDDGSFSEN-STFQLTDWLAHTPK B. clausii 132 FIADVGP GDLWYFPPGIPH SIQG LDDG---CEFLLVFDDGMFSDL-STLSLSDWMAHTPK C. velutipes 181 YISTVGP GDLWYFPPGIPH SLQA TADDPEGSEFILVFDSGAFNDD-GTFLLTDWLSHVPM A. oryzae 142 TTEKLEE GDIWYFPKGVAH NVQG LDDE---NEYLLVFDDGDFEKVGTTFMVDDWITHTPR : **:**** *:.*.:*. : *::****.* *.. *: : **::*.* B. subtilis 178 EVIAANFGVT-KEEISNLPGKEKYIFENQLPGSLKDDIVEGPNGEVPYPFTYRLLEQEPI B. clausii 188 DVLSANFGVP-ESVFATIPTEQVYIYQDEVPGPLQSQQINSPYGAVPQTFKHELLKQPPL C. velutipes 240 EVILKNFRAKNPAAWSHIPAQQLYIFPSEPPADNQPDPVS-PQGTVPLPYSFNFSSVEPT A. oryzae 199 DILAKNFGVD-ASVFDKVPEKFPYILNGTVSDEANNTPQGTLTGNSSYVYHTYKHPSEPV ::: ** :* : ** : : B. subtilis 237 ESEGGKVYIADSTNFKVSKTIASALVTVEP GAMRELH WH PNTHE WQYYISG KARMTVFAS B. clausii 247 VTPGGSVRIVDSRNFPVSKTIAAALVEVEP GAMREMH WH PNNDE WQYYLTG QARMTVFTG C. velutipes 299 QYSGGTAKIADSTTFNISVAIAVAEVTVEP GALRELH WH PTEDE WTFFISG NARVTIFAA A. oryzae 258 PGSGGTFRKIDSKNFPVSQTIAAALVELEP KGLRELH WH PNAEE WLYFHKG NARATVFLG **. ** .* :* :** :** .:**:****. .** :: .*:** *:* B. subtilis 297 DGHARTFNYQA GDVGYVPFAMGH YVEN IGD-EPLVFLEIFKDDHYADVSLNQWLAMLPET B. clausii 307 NGVARTFDYRA GDVGYVPFATGH YIQN TGN-ESVWFLEMFKSDRFEDVSLNQWLALTPTE C. velutipes 359 QSVASTFDYQG GDIAYVPASMGH YVEN IGN-TTLTYLEVFNTDRFADVSLSQWLALTPPS A. oryzae 319 DSKARTFDFTA GDTAVFPDNSGH YIEN TSETEKLVWIEIYKSDRVADISLAQWLALTPAD :. **:: .** .* ***::* .: : ::*::: *: *:** ****: B. subtilis 356 FVQAHLDLGKDFTDVLSKEKHPVVKKKCSK B. clausii 366 LVQHNIHVDSKFTNKLRKEKWPVVKYPTIC. velutipes 418 VVQAHLNLDDETLAELKQFATKATVVGPVN A. oryzae 378 VVATTLKVDIEVVKQIKKEKQVLVKGK--.* :.:. : :

PAGE 20

20 and evolutionarily related scaffolds ( 81 ). The absence or presence of various metals such as Ni, Fe, Zn, Mn, or Cu contribute to the functi onal diversity of the cupin superfamily ( 43, 78 ). The term cupin has been expanded into the Stru ctural Classification of Proteins (SCOP) (http://scop.berkele y.edu/data/scop.b.html ) database as the double-stranded -helix (DSBH) multicatalytic fold. The terms cupi n and DSBH are now used synonymously ( 82-84 ). Since OxDC has two of these characteristic DSBH domain s it is further classified as a bicupin. It has been suggested that OxDC evolved from OxOx by gene duplicati on and selection ( 28, 37, 45 ). This suggestion is consistent with cu rrent models of enzyme evolution ( 85, 86 ). Anantharaman et al. ( 81 ) propose that ancestral forms of the DSBH can be evolutionarily reconstructed as simple, small-molecule-binding domains that perhaps bound sugars and cyclic nucleotides ( 45, 81, 87 ) and that it is from these sugar-bi nding domains that sugar-modifying domains such as isomerases and epimerases arose. They further propose ( 81 ) that a set of conserved histidine residues employed in sugarbinding in the ancestral non-enzymatic domain evolved into the metal c oordinating histidine residue s observed in germin ( 88 ) and oxalate decarboxylase ( 72 ) and that another lineage of DSBH do mains acquired a new set of conserved residues with the ability to bi nd 2-oxoglutarate which gave rise to the iron-2-OG-dependent dioxygenases. Structural Features High resolution X-ray crystal structures of Bacillus subtilis OxDC ( 72, 73 ) have confirmed that the OxDC monomer is composed of two -barrel domains, each of which contains a metalbinding site (Figure 1-3A). Thes e metal ions are 26 angstroms apart from each other in the monomer. Evidence from inductively-coup led plasma mass spectrometry (ICP-MS) ( 89 ) and

PAGE 21

21 EPR spectroscopy initially s uggested that OxDC activ ity was Mn-dependent ( 70 ). This hypothesis is consistent with cr ystallographic observations ( 72, 73 ). Figure 1-3 Ribbon structures of the Bacillus subtilis OxDC monomer, trimer, and hexamer. (A) The Nand C-terminal cupin domains of the OxDC monomer ar e colored green and purple, respectively, and the N-terminal se gment that contributes to the secondary structure of the C-terminal domain is co lored red. The yellow spheres show the locations of the two Mn centers. (B) Stru cture of an OxDC trimer in which the monomers are colored red, green and purple. The locations of Mn ions are shown by the purple (N-terminal domain) and yello w spheres (C-terminal domain). (C) Structure of the OxDC hexamer in which one monomer is colored red to emphasize the role of -helical regions in mediating mono mer/monomer interactions. These structures were visualized using the CA Che Worksystem Pro V6.5 software package (Fujitsu America Inc., Beaverton, OR). (A) (B) (C)

PAGE 22

22 The quaternary structure of OxDC is hexameri c (Figure 1-3C) composed of two trimeric layers (Figure 1-3B) packed face to face that have 32 (D3) point symmetry. The OxDC trimer resembles the OxOx hexamer (37). The OxDC hexamer has a diameter of approximately 90 angstroms and a thickness of 85 angstroms. A large solvent channel (15 angstroms wide) runs through the hexamer along the 3-fold axis (72). The trimeric layers of the hexamer are stabilized by -helical protrusions of adjacent monomers (Figure 1-3C). Figure 1-4 Residues defining the Mn-binding sites in A) the N-terminal (1UW8) domain of OxDC and B) the C-terminal (1J58) doma in of OxDC. Residue numbering is for the enzyme encoded by the OxdC gene in Bac illus subtilis. For clarity, hydrogen atoms bound to carbon atoms are omitted. Atom colori ng: C, black; H, white; N, blue; O, red; Mn, silver. These structures were vi sualized using the CAChe Worksystem Pro V6.5 software package (Fujitsu America Inc., Beaverton, OR). Both of the Mn-binding sites in the OxDC monomer resemble the Mn-binding site of OxOx in that each Mn ion is coordinated by the si de chains of four conserved residues (Figure 14) in a distorted octahedral environment. The manganese-binding resi dues in the N-terminal domain are His95, His 97, His 140, and Glu101 a nd in the C-terminal domain are His273, His275, His319, and Glu280. In one of the availa ble X-ray crystallographi c structures (open conformation), the N-terminal Mn-binding site contains one water molecule and one formate A B

PAGE 23

23 molecule while the C-terminal Mn-bindi ng site contains two water molecules ( 72 ). In another available X-ray crystallographic structure (cl osed conformation), however, the N-terminal Mn-binding site contains two wa ter molecules and the C-terminal Mn-binding site contains a single water molecule in a penta-coordinated form ( 73 ). Not only do oxalate oxidase and oxalate deca rboxylase possess remarkably similar Mnbinding sites, the metal-binding cavity is also intr iguingly similar in that it is lined primarily by hydrophobic residues. Given these similarities and co mmon substrate, it appears that only subtle changes are necessary to promote different bioche mical activities. It has been proposed that the absence of a proton donor in th e active site of OxOx prev ents it from catalyzing the decarboxylation of oxalate ( 72 ). The putative proton donor(s) in OxDC have been proposed to be Glu162 in the N-terminal domain ( 73 ) and/or Glu333 in the C-terminal domain ( 72 ). Mechanistic Information Oxalate decarboxylase requires molecu lar oxygen for catal ytic turnover ( 57, 58, 61, 70 ) even though the production of formate and CO2 from oxalate involves no net oxidation or reduction. Furthermore, all of the OxDCs that have been characterized possess optimum activity at acidic pH and exhibit a high s ubstrate specificity for oxalate ( 37, 57, 58, 70 ). Efforts to elucidate the catalytic mechanism through th e use of heavy atom isotope effects ( 57 ), electron paramagnetic resona nce spectroscopy ( 71 ), density functional theory calculations ( 90 ), homology modeling with oxala te oxidase sequences ( 91 ), structural information and site-directed mutagenesis studies ( 72, 73 ) have led to a number of mechanistic proposals. Heavy-atom (13C and 18O) kinetic isotope effect (KIE) measurements ( 92 ) were used to probe the structure of the transition state for th e decarboxylation step for the recombinant, wild type OxDC from B. subtilis ( 57 ). Since V/K KIEs were measured in these competition experiments, no information can be obtained for the steps that o ccur after carbon-carbon bond

PAGE 24

24 cleavage ( 93 ). The pH dependence of the enzyme catalyzed reaction suggests that monoprotonated oxalate is the actual substrate for OxDC ( 57 ) and that the substrate likely binds directly to the Mn in the enzyme-substrate complex ( 37 ). Figure 1-5 Proposed catalytic mechanism for oxalate decarboxylase based on heavy-atom isotope effect measurements. Heavy-atom KIEs measured at pH 4.2 and 5.7 are consistent with a two step model in which a reversible step precedes carbon-carbon bond cleavage and decarboxylation (Figure 1-5) ( 37, 57 ). In this proposal, a reversible proton-coupled electron transfer ( 94, 95 ) yields a Mnbound oxalate radical anion, which then decarboxylates to form CO2 and a formate radical anion. Protonation of the Mn-bound format e radical anion produces format e which is then liberated from the enzyme. In this proposal, active site glutamate residue(s) serve as a general acid/base catalyst and active site arginine residue(s) act to polarize the oxalate carbonyl bond ( 57 ). The oxidation state in the above mechanism is purely hypothetical and remains to be demonstrated by

PAGE 25

25 experimental methods. Other proposals have invoked that the oxidation state of manganese alternates during catalysis, between Mn(II) and Mn(III) ( 70, 74 ) or Mn(III) and Mn(IV) ( 72 ). Only Mn(II) has been detected experimentally by either standard perpendi cular-mode or parallelmode EPR spectroscopy of the resting enzyme ( 70, 71, 74 ) or during turnover ( 71 ). Oxygen Dependence and the Form ation of Hydrogen Peroxide The first report of an oxalate decarboxylase observed that decarboxylation did not proceed under strictly anaerobic conditions and that the introduction of air into the manometric apparatus restored the activity to the original level ( 33 ). Subsequently reported OxDCs have shown a similar oxygen dependence ( 37, 57, 58, 60-62, 70 ) but vary with respect to the level of activity restored upon the reintroduction of oxygen. The role of dioxyge n in the catalytic cycle is unknown and cannot be replaced by othe r oxidizing agents such as H2O2, paraquinone, 2-methyl1,4-naphthoquinone, flavin adenine dinucleotide, flavin mononucleotide, and cytochrome c ( 58 ). The most extensive characterization of th e oxygen dependence of OxDC was carried out on the enzyme from Aspergillus niger using manometric techniques ( 62 ). In this study, the influence of the partial pressure of O2 on the enzyme was observed by replacing the air in the Warburg apparatus by mixtures of O2 and N2. In the absence of o -phenylenediamine (oPDA), maximal activity was obta ined at 0.04 atm of O2, whereas, in the presen ce of oPDA maximal activity was obtained at 0.2 atm. Pressures great er than optimal accelerated the irreversible inactivation of the enzyme even in the presen ce of oPDA. Inactivati on occurs, however, only during catalytic turnover since bubbling O2 through the enzyme solution prior to the addition of substrate did not affect product form ation. This suggests that if O2 binds directly to the metal, it does so after oxalate binding. Oxalate oxidase activity has been report ed for both fungal and bacterial oxalate decarboxylases. The rate of oxalate oxidation relative to decarboxylation is 1.5-

PAGE 26

26 3.0% for the A. niger enzyme ( 62 ) and 0.2% for the recombinant, wild type B. subtilis enzyme ( 70 ). Research Objectives The overarching goals of this research are motiv ated by the fact that the metal centers in oxalate decarboxylase and oxalate oxid ase are evolutionarily related ( 28 ) even though the chemical transformations catalyzed by the enzymes are different ( 32, 33 ). This research seeks to employ the techniques of bioinorganic chemistry, molecular spectroscopy, enzyme kinetics, and protein engineering to characteri ze oxalate decarboxylase. Increa sed knowledge of this enzyme may impact our general understand ing of metalloenzyme evolution and the role of the protein environment in modulating reactivity ( 86, 96 ). The specific objectives of the presented work were: 1) to optimize the expression and purification procedures to obtai n OxDC with high manganese occ upancy; 2) to characterize the manganese dependence of the enzyme; 3) to distinguish the manganese-binding sites spectroscopically; and, 4) to determine which ma nganese center is the si te(s) of catalysis.

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27 CHAPTER 2 CHARACTERIZATION OF THE MN-DEPENDENCE OF OXALATE DECARBOXYLASE ACTIVITY Introduction Three pieces of indirect evidence support th e idea that OxDC activity is Mn-dependent. First, Mn(II) is present in the resting form of recombinant, wild type Bacillus subtilis OxDC when the enzyme is expressed in Escherichia coli ( 70, 71 ). Second, the successful expression of correctly folded OxDC with reasonable catalytic activity specifically requires the presence of Mn(II) in the growth medium ( 70 ). Third, the X-ray scattering fact ors for Mn which were used in the refinement of the high-resolution structures of recombinant Bacillus subtilis OxDC were fully consistent with this metal being bound within both DSBH domains ( 72, 73 ). On the other hand, the native form of OxDC from Bacillus subtilis could not be purified in sufficient quantities for accurate metal analysis ( 34 ), and other enzymes in the bicupin family appear to be able to employ a variety of metals in catalysis ( 76, 78, 97 ). In order to characterize the Mndependence of recombinant B. subtilis OxDC, an in vivo strategy was employed for obtaining recombinant, wild type OxDC in which Mn is substituted by Co, and in vitro conditions for reconstituting the recombinant enzy me with Mn were developed. Results and Discussion Optimization of Expression of Recombinant Wild Type OxDC Expression conditions for obtai ning OxDC were optimized so that pure samples of the enzyme could be routinely obtained with a meta l content of 1.6-1.9 Mn/m onomer rather than the 0.86-1.14 Mn/monomer reported in prior studies of the enzyme ( 57, 70 ). At this level of Mn incorporation, recombinant OxDC exhibits a specif ic activity of 40-65 U/mg as measured in an endpoint assay employing formate dehydrogenase (FDH) ( 98 ). To obtain reproducibly high levels of Mn incorporation, protein expression was induced at a lower optical (0.6 at 600 nm)

PAGE 28

28 density than previously reported ( 57 ), and cells were grown at a post-induction temperature of 30oC so as to promote the transport of ma nganese ions into the bacterial cells ( 99, 100 ). Consistent with previous reports, we observe d the oxalate dependence of the decarboxylase activity followed Michaelis-M enton kinetics, with our Km value being lower than previously reported (8.4 vs. 15 mM) ( 70 ). Effect of Addition of Other Metals in the Growth Medium Having established conditions fo r obtaining recombinant OxDC with high specific activity, we next investigated whether in cluding other salt s in the growth medium might yield enzyme in which manganese had been replaced by ot her transition metals. Introducing FeCl2, FeCl3, or CoCl2 into to the growth medium in place of MnCl2 yielded samples of the recombinant, wild type enzyme with varying levels of Mn incorpora tion. All of these variants behaved similarly to the wild type, Mn containing OxDC on purification but exhibited activities that correlated best with their Mn content leading to the conclusion that metals such as Co or Fe do not support catalysis. When CoCl2 (2 mM) was used as a supplement, ICPMS analysis showed that samples of recombinant OxDC contained 0.80 Co/monomer and 0.05 Mn/monomer (Table 2-1). Given the very low specific activity of the Co-substituted Ox DC was similar to that expected solely on the amount of Mn present in the enzyme sample we assumed that enzyme-bound Co(II) did not catalyze the decarboxylation reaction and therefore investigated the effects of expressing the enzyme with mixtures of the chloride salts of both Co(II) and Mn(II) in the growth medium (Table 2-1). We anticipated that different con centration ratios of the e xogenous salts would yield OxDC samples substituted with different levels of Mn, and this proved to be the case although no obvious correlation was observed between the Mn:C o ratio and the extent to which Mn or Co was incorporated into the recombinant enzyme.

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29 Table 2-1 Effect of MnCl2 and CoCl2 in the growth medium on me tal incorporation and specific activity of recombinant, wild type OxDC. Metal content is expressed as the number of metal ions/OxDC monomer. n. d. indicates that the value was not determined. These samples contained <0.01 atoms/monomer Mg. When a 20:1 Mn:Co ratio was employed the two metals were incorporated into the enzyme in approximately equal amounts. Increasing the amount of MnCl2 relative to CoCl2 in the growth medium, however, did not yield wild type Ox DC containing more Mn than Co. This finding likely reflects the tight regulation of Mn metabo lism that is observed in bacteria such as Escherichia coli ( 100-103 ). Although a positive correla tion between Mn content and decarboxylation rate was evident on assaying the ac tivity of the Mn/Co-substituted enzymes, we could not definitely conclude th at decarboxylase activity was linear ly correlated with Mn content on the basis of these in vivo experiments because no expressi on conditions could be identified that gave OxDC samples containing 0.7-1.5 Mn/monomer. Preparation of the OxDC Apoenzyme and Reconstitution of the Wild Type, MnContaining Enzyme In order to correlate spec ific activity with metal content in the 0.7 1.5 Mn/monomer range, we examined alternate strategies to obtain samples of the OxDC apoenzyme ( 104-109 ), then prepare enzyme samples reconstituted with manganese. Following procedures that had been reported for removing the metals from other metalloenzymes, we tried a variety of chelating agents with an d without chaotropic agents. In co ntrast to the bicupin quercetin 2,3-OxDC Preparation MnCl2 (mM) CoCl2 (mM) Mn Co Zn Fe Cu Specific Activity (U/mg) 1 5 0 1.87 n.d. 0.51 0.07 0.01 61.2 2 5 0 1.87 n.d. 0.13 0.19 < 0.01 50.1 3 5 0 1.63 n.d. 0.08 < 0.01 < 0.01 40.9 4 0 2 0.05 0.80 0.14 < 0.01 < 0.01 2.2 5 0.25 2 0.27 1.03 0.22 0.18 < 0.01 8.0 6 1 1 0.12 1.32 0.26 0.13 < 0.01 4.5 7 5 0.25 0.56 0.68 0.22 0.13 < 0.01 13.5 8 5 0.05 0.47 0.09 0.19 0.06 < 0.01 19.0

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30 dioxygenase ( 76, 78, 97 ), it proved remarkably difficult to re move the metal ion from wild type OxDC. Many literature conditions either did not re move the metal or led to irreversible protein denaturation. The removal of Mn from recombinant OxDC and its subsequent reconstitution with Mn was eventually accomplished, however, following a protocol based on that used to obtain the apoenzyme of Mn-dependent supe roxide dismutase (Mn-SOD) ( 110-112 ). Table 2-2 Metal content of apoe nzyme and enzyme reconstituted with Mn. Metal content is expressed as the number of metal ions/OxDC monomer. n. d. indicates that the value was not determined. This multi-step procedure (see Experimental Methods section) involved partially unfolding the protein in 3.5 M guanidinium hydrochloride (GuHCl) with ethylenediaminetetraacetic acid (EDTA) present. Refolding samples into a buffe red solution without added metals resulted in a manganese-free apoenzyme (2 Zn/monomer) in whic h Zn(II) had replaced Mn(II) in wild type OxDC (Table 2-2). Alternate metal ions coul d also be introduced into the apoenzyme by refolding samples into a buffered solution containing salts such as MnCl2. These conditions were used to prepare samples of r ecombinant OxDC containing 0.64, 0.84, and 0.90 Mn/monomer (the remaining metal sites being oc cupied by Zn) for kinetic characterization. The specific activities of these samples were determined (Table 2-2). Combining these data with the data obtained adding CoCl2 to the growth medium resulted in a plot of manganese content vs. specific activity which suggests a linear co rrelation between decarboxylation rate and Mn incorporation (Figure 2-1) Enzyme Preparation Mn Co Zn Fe Cu Mg Specific Activity WT OxDC 1.87 n.d. 0.51 0.07 0.01 0.01 61.2 U/mg Apoenzyme 0.01 n.d. 2.0 0.01 0.01 0.01 0 U/mg Reconstituted OxDC 1 0.90 n.d. 0.79 0.14 0.01 0.01 25.1 U/mg Reconstituted OxDC 2 0.64 0.01 1.20 0.01 0.02 n.d. 14.4 U/mg Reconstituted OxDC 3 0.84 0.01 0.60 0.01 0.01 n.d. 19.5 U/mg

PAGE 31

31 Figure 2-1 The dependence of OxDC specific activ ity on the extent of Mn incorporation. The line shows the specific activity that would be expected assuming a linear correlation with Mn content. Gepasi Simulations The observation of a linear dependence of Ox DC specific activity on Mn incorporation places an important constraint on kinetic models for the number of active sites in OxDC that may mediate catalysis. The fact that OxDC is a bicupin capable of binding up to 2 Mn/monomer raises questions concerning the number and locati on of the catalytic site s that mediate C-C bond cleavage ( 72, 73 ). Previous efforts to address th ese questions by steady state kinetic characterization of OxDC mutants in which residues implicated in proton transfer (Glu-162 and Glu-333) were site-specifically modifi ed have given ambiguous results ( 72, 73 ). The steadystate behavior of seven kinetic models was, th erefore, simulated using the GEPASI simulation package ( 113, 114 ) to evaluate the effects of varying active site number and Mn-binding site affinity on the observed Mn-dep endence of catalytic activity. 0 10 20 30 40 50 60 70 00.511.52Mn/monomer Specific Activity (U/mg)

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32 Figure 2-2 Numerical simulations of the dependence of catalytic activity on the extent of Mn incorporation. Full details of the kinetic parameters used in the simulations are provided in Appendix A. All panels s how the amount of product formed after a reaction time of 5 s. Note th at site 1 and site 2 cannot be associated with a specific Nor Cterminal Mn-binding site.

PAGE 33

33 To date only three models have been identifi ed that are consistent with experimental observations (Case 1a, Case 4a,b,and c, and Case 6a)(Figure 2-2). In Ca se 1a one site (site 1 ) has catalytic activity that is inde pendent of metal occupancy of th e second, inactive Mn-binding site (site 2 ) and these two sites possess the same affinity for Mn. In Case 4 a linear relationship is observed independent of affinity fo r Mn as both sites have equal le vels of catalytic activity. Case 6 assumes that although catalytic activity is only associated with a single site (site 1 ), the affinity of Mn for site 2 is increased 100-fold when Mn occupies the active site (site 1 ). Kinetic models that seek to simu late an earlier proposal ( 8 ) in which the N-terminal site is responsible for the majority (if not all) of activity, with th e C-terminal site being primarily important in maintaining enzyme structure, only gave a non-linear relationship be tween activity and Mn incorporation (Cases 3, 5 and 7). A model in which both sites have equal activ ities (Case 4) reproduces the observed linear relationship between activity and Mn content. On the other hand, for models where it is assumed that only a single site (site 1) can mediate catal ysis, it is difficult to obtain a linear plot. For example, a model (Case 3) in which the catalytic activity of site 1 is turned on when Mn(II) occupies the second binding site (site 2) yields a non-linear relati onship between activity and Mn incorporation. Similarly, permitting differences in the affinity of the two sites for Mn while requiring that catalytic activity be localized with in a single site (site 1), irrespective of the occupation of the second site (s ite 2), results in non-linear behavi or (Cases 1b and 1c, Cases 1b and 1c). Linear behavior is antic ipated for this model only if the two sites initially have an equal affinity for Mn. It is possible, however, to obtain a linear dependen ce using a kinetic model (Case 6a) in which the affinities of the two binding sites are initially identical but Mn occupancy of site 1 results in an enhanced affinity of site 2 for the metal. We note that such a model

PAGE 34

34 corresponds to a recent sugge stion that structural interactions between the cupin domains may be important for yielding enzyme with full catalytic activity ( 73 ). Experimental Section Materials Unless otherwise stated, all chemicals and r eagents were purchased from Sigma-Aldrich (St. Louis, MO), and were of the highest available purity. Protein concentrations were determined using a modified Bradford assay (Pie rce, Rockford, IL) for which standard curves were constructed with bovine serum albumin ( 14 ). All DNA primers were obtained from Integrated DNA Technologies, Inc. (Coralville, IA), and DNA sequencing was performed by the core facility in the Inte rdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida. The metal content of wild type OxDC, and all site-direc ted OxDC mutants, was quantified at the University of Wisconsin Soil and Plant Analysis Laboratory on the basis of ICP-MS measurements ( 89 ). Expression and Purification of Recombinant, Wild Type OxDC. Recombinant wild-type Bacillus subtilis OxDC was expressed and purified using a modified literature procedure ( 57 ). Thus, Luria-Bertani br oth (50 mL) containing 50 g/mL kanamycin (LBK) was inoculated with oxdC:pET -9a/BL21(DE3) and incubated overnight at 37 oC. An aliquot (4 mL) of this stationary phase culture was then used to inoculate Luria-Bertani broth (5 x 400 mL) and the result ing cultures were incubated at 30 oC until reaching an OD600 value of 0.6. At this time, the ba cteria were heat-shocked at 42 oC for 10 minutes before the addition of isopropyl thiogalactoside and MnCl2 to final concentrations of 1 and 5 mM, respectively. The induced cel ls were then grown at 30 oC with shaking to ensure maximal aeration for 4 h. The cells were harves ted by centrifugation (6000 rpm, 20 min, 4 oC), and the pellets re-suspended in 50 mM imidazole-Cl, pH 7.0, (100 mL) before soni cation. The lysate was

PAGE 35

35 clarified by centrifugatio n (10,000 rpm, 20 min, 4 oC) and stored overnight at 4 oC. The lysis pellets were re-suspended in 50 mM imidazole-C l, pH 7.0, containing 1M sodium chloride, 10 M MnCl2, 0.1% Triton X-100, and 10 mM 2-mercaptoethanol (total volume 100 mL), and the resulting mixture stirred overnight at 4 oC. After centrifugatio n (10,000 rpm, 20 min, 4 oC), the solubilized extract was combined with the lysate and diluted 7-fold before being applied to a DEAE-Sepharose Fast Flow column (2.5 x 25 cm) equilibrated with 50 mM imidazole-HCl, pH 7.0 (buffer A). Elution was perfor med using a 500 mL linear grad ient from buffer A to buffer A containing 1 M NaCl. Fractions contai ning OxDC were pooled, and solid (NH4)2SO4 added to a final concentration of 1.7 M. The resulting solution was applied to a phenyl-Sepharose HiPerformance column (2.5 x 18 cm) (GE Healthca re, Piscataway, NJ) equilibrated with 50 mM imidazole-Cl, pH 7.0, containing 1.7 M (NH4)2SO4 (buffer B). Bound proteins were eluted using a 500 mL linear gradient from buffer B to bu ffer A, and fractions c ontaining purified OxDC were pooled, and concentrated by ultrafiltration in an Amicon stirred cell (Millipore, Billerica, MA) to a final volume of 10 mL before be ing exhaustively dialyzed against 20 mM hexamethylenetetramine hydrochloride, pH 6.0, containing 0.5 M NaCl. The dialyzed enzyme was then concentrated to approximately 9 mg/mL, and stored in aliquots at -80 oC. Expression and Purification of Co-Substituted, Wild Type OxDC The recombinant Co-containi ng, wild-type enzyme was obtai ned following the standard protocol for expressing the Mn-substituted enzyme, except that CoCl2 (Fisher Scientific, Pittsburgh, PA) or various CoCl2/MnCl2 mixtures were added to th e cell culture in place of MnCl2, after the heat shock step but prior to induc tion of OxDC expressio n. After cell lysis, and extraction of the recombinant protein from the cr ude lysate as described above for recombinant wild-type OxDC, the Co-containing enzyme wa s purified by DEAE column chromatography. Fractions containing OxDC were pooled and di alyzed for 4 h against 50 mM imidazole-HCl

PAGE 36

36 buffer, pH 7.0 (2 L). The resulting sample wa s then applied to a Q-Sepharose Hi-Performance column (2.5 x 18 cm) column equilibrated with buffer A, and eluted using a 500 mL linear gradient from buffer A to buffer A containi ng 1 M NaCl. Fractions containing OxDC were pooled and exhaustively dialyzed against 20 mM hexamethylenetetramine hydrochloride, pH 6.0, containing 0.5 M NaCl. The purified, Co-substitute d enzyme was concentrated and stored as described for recombinant, wild type OxDC. Preparation of the OxDC Apoenzyme and Reconstitution of the Wild Type, MnContaining Enzyme Recombinant Mn-containing, wild type OxDC was dialyzed against 3.5 M guanidinium hydrochloride (GuHCl), 20 mM Tris-HCl, and 10 mM EDTA, pH 3.1, for 8 h at 4 oC. A second round of dialysis against 2.5 M GuHCl, 20 mM Tris-HCl, and 10 mM EDTA, pH 7.0, was performed, and excess EDTA removed in a thir d dialysis against 2.5 M GuHCl containing 20 mM Tris-HCl, pH 7.0. Both of the latter steps were carried out for 8 h at 4 oC. At this stage, the protein could be re-folded by dialysis agai nst 20 mM hexamethylen etetramine-HCl, pH 6.0, containing 0.5 M NaCl, over 8 h at 4 oC, to yield a Mn-deficient form of OxDC (apoenzyme) that exhibited no catalytic activity when inc ubated with oxalate. Alte rnatively, a round of dialysis over 8 h, at 4 oC, against 20 mM Tris-HCl, pH 7.0, containing 10 mM MnCl2 could be used to re-introduce Mn(II) into the enzyme before exhaustive exchange into 20 mM hexamethylenetetramine-HCl, pH 6.0, containing 0.5 M NaCl 4 oC. The latter re-folding step gave samples of reconstituted OxDC containing Mn. Metal Content Determination Two methods were used to prepare samples a nd blanks for determination of metal content by ICPMS (University of Wisconsin Soil and Pl ant Analysis Lab). In the first method, approximately 0.2 mM enzyme samples (200 L of ~10 mg/mL) were made 1 mM

PAGE 37

37 ethylenediaminetetracetic acid (EDTA) and incuba ted on ice for 15 minutes. Samples were then desalted on a G-25 pasteur pipet column equilibrated with dH2O. The desalting column had been previously treated with EDTA. 200 L of storage buffer was put through an identical procedure for use as a blank. In the second method, di valent cations were removed from 20 mM hexamethylenetetramine hydrochloride, pH 6.0 containing 0.5 M sodium chloride by passing through a 1.5 x 16 cm column containing Chelex 100 (Bio-Rad) in the Na+ form. Purified protein samples were exchanged into the resul ting buffer by washing 2.5 mg samples three times with 10-fold volumes of the scrubbed buffe r in Centricon or Ce ntriprep 30 (Amicon) concentrators ( 104 ). The final filtrates were recovere d and used as blanks, which routinely possessed insignificant metal content. Samples were sent to the University of Wisconsin Soil and Plant Analysis Laboratory fo r the determination of metal content by ICPMS. Both methods yielded similar results, results repor ted here are from the second method. Steady-State Kinetic Assays Assay mixtures consisted of 50 mM NaOAc, pH 4.2, 0.2% Triton X-100, 0.5 mM o phenylenediamine, 1-50 mM potassium oxalate, and either the wild type OxDC (1-4 M) or OxDC mutant (80-120 M) (100 L total volume). Reactions were initiated by the addition of substrate, incubated at ambient temperature (21-23o C), and quenched by the addition of 1 N NaOH (10 L). The amount of formate product was determined by an end-point assay ( 98 ) consisting of 50 mM potassium phosphate, pH 7.8, 0.09 mM NAD+, and 0.4-1.0 U/mg of formate dehydrogenase (1 mL final volume). The absorbance at 340 nm was measured after overnight incubation at 37oC, and formate was quantitated by comparison to a standard curve generated by spiking pre-quenched OxDC a ssay mixtures with known amounts of sodium formate. Measurements were made at specific su bstrate and enzyme concentrations in duplicate,

PAGE 38

38 and data were analyzed to obtain the specific activity by standard computer-based methods ( 115 ). The initial rate of formate production is ex pressed in millimoles per liter per minute. Gepasi Simulations The rates used for the reactions for the various simulations (Figure 22) have been cut and pasted from the Gepasi output f iles into Appendix A. The equati ons used for the simulations are given in Figure 2-3. To ensure that the binding equilibria for the Mn ions are well established before catalysis takes place, the k1 values for the forward reacti on in equations [R1-4] were chosen to be fairly large, i.e., close to the diffusion limit: k1(R1-R4) = 1 x 109 (sM)-1, k2(R1-R4) = 5 (s)-1. Figure 2-3 Equations used for Gepasi simulations to describe the amount of product formed after 5 s as a function of Mn incorporation. The rate constants for dissociation we re small to ensure good binding. 5 s-1 represents a KD of 5 nM. For low affinity 5,000 s-1 was chosen, representing 5 M affinity with unchanged

PAGE 39

39 k1. As a result this ended up giving full sites for a complement of Mn ions present. The binding of substrate to enzyme is given by: k1(R5,R7,R8) = 1 x108 (s M)-1 k2(R5,R7,R8) = 8.4 x 105 (s)-1 These values were chosen to give a Km = ) 8 7 5 ( ) 10 9 6 ( ) 8 7 5 (1 1 2R R R k R R R k R R R k = 8.4 mM and kcat = k1(R6,R9,R10) = 53 (s)-1. In order to simulate inactive sites, the kcat value was reduced to 5 x 10-6 s-1. For less active sites the kcat value was changed to values between 17 and 35 s-1. For half active sites the kcat value was chosen to be 26.5 s-1. In case 3 where site 2 was mechanistically required all k1(R9,R10) = 5 x 10-6 s-1 and only k1(R6) = 53 s-1. This resulted in nonlinear behavior in all instances due to the fact that only the full enzyme is capable of catalysis. The linear relationships in Case 4 are due to the fact that each bound Mn participates equally in catalysis. This case is thus insensitiv e to the affinity of the two sites. To simulate cooperative binding (Case 6) positiv e feedback was assumed for the binding of the second site. Only site 1 is equally active in both the singl e Mn as well as the full enzyme. To simulate cooperativity of binding the second site was assume d to have a factor of 100 higher affinity once the first site was filled. To see what happens when only the full enzyme was active, case 7 was created which is otherwise equal to case 6. It shows essentially the same result as case 3 where only the full enzyme was capable of catalysis. In all cases, the starting conditions involved 1 mM apoenzyme concentration, up to 2 mM Mn concentration, and a saturating substrate concentration of 10 M. Case 1 (site 1 active, site 2 unimportant): In this case only site 1 is active and the presence or absence of site 2 is simply irrelevant. However, as the results show it is not quite so irrelevant if

PAGE 40

40 it is allowed to bind substrate (and thus sequester the enzyme in that state). Thus the dip seen in case 1c is artifactual since in reality one would expect the enzy me to also allow substrate to bind in the other site. Case 1 : To avoid the problem with the dip in ca se 1, a lower catalytic rate was assumed for the singly bound Mn, i.e. 26.5 s-1 for the single Mn case vs. 53 s-1 for the full enzyme. As it turns out the difference in activities between th e singly and doubly occupied enzyme starts to curve the former straight line pred icted for equal affinity of the Mn sites. This is expected since the model introduces some form of c ooperativity into the catalytic mechanism. Case 2 (site 1 most acti ve, site 2 less active) : is another modification of case 1, in which the rates were input as 53s-1 for the full enzyme, 35 s-1 for site 1, and 8 s-1 for site 2. Case 3 (site 1 active, and site 2 requ ired structurally or mechanistically) : The quadratic behavior of the case with equal affinity is confirmed and makes sense because the number of fully loaded enzymes is quadratic with the concentration of bound Mn. Case 4 (site 1 and site 2 have equal activity) : Case 4 doesnt show any di fference in its kinetics except a small difference in tota l bound Mn for the different data points, just as expected. Case 5 (site 2 required for fu ll activity of the enzyme) : This case is similar to case 3 but relaxes the necessity of the mechanistic site 2 a bit by allo wing for half activity of site 1 when site 2 in not occupied. Case 6 (site 1 is active, binding is cooperative with the affinity for the firs t Mn being different for the two sites) : Case 6 represents cooperat ive binding of the Mn sites. It is assumed that the subsequent Mn binds with a 100 fo ld higher affinity than the firs t one. However, the cases 6b and 6c differ, just like in all other cases treated before, by 3 orde rs of magnitude in their initial affinity for sites 1 and 2. In other words, when the first affinity is 5 nM the second affinity is

PAGE 41

41 now 50 pM, and when the first affinity is 5 M the second is now 50 nM. It should be noted that the activity of site 1 was assumed to be 53 s-1 for both the fully loaded enzyme as well as for the singly loaded one at site 1. Case 7 : Case 7 is similar to case 6, except that it assumes only the full enzyme containing both Mn to be active.

PAGE 42

42 CHAPTER 3 SPECTROSCOPIC CHARACTERIZATION OF THE TWO MANGANESE CENTERS Introduction Electron Paramagnetic Resonance Spectroscopy Electron paramagnetic resonance is a spectro scopic technique for detecting species containing unpaired electrons, gene rally organic radicals or transi tion metal ions with partially filled d orbitals. Since an unpaired electron possesses circular motion about its axis (spin angular momentum) and a charge, it has a magnetic moment. An external magnetic field interacts with the magnetic moment of the unpaired electron (Zeeman interaction) and can result in two possible orientations, parallel to the magnetic field or antiparallel to it. The two orientations (states) possess different energi es and the difference in energy (Zeeman energy) increases with increases in the magnetic field. Figure 3-1 Absorption of micr owave irradiation by an unpaired electron in a magnetic field. Figure adapted from http://www.bruker-biospin.com/cw.html

PAGE 43

43 Application of radiation at an appropriate frequency results in a transition between the two states (resonance condition, E = h = gBBo). In EPR spectroscopy, the sample is irradiated with a fixed frequency microwave energy and the magnetic field is gr adually increased. A line is generated in the EPR spectrum when the Zeeman energy matches the photon energy (Figure 3-1) ( 116 ). Electronic Configuration of Mn(II) The shape of the magnetization of Mn(II) in any given coordination environment dictates its EPR properties. This shape is determined by the distribution of unpa ired electrons around the Mn nucleus and is related to the types of ligands present a nd how they are geometrically arranged around the ion. As a free ion in the gaseous phase, Mn2+ possesses five 3 d electrons in a spherical distribution around the nucleus (an Sstate ion). Ligand in teractions with the d electrons in the condensed phases br eak both the degeneracy of the 3 d orbitals and their spherical symmetry which influences the EPR properties of Mn2+. The splitting that results from a noncubic environment is known as zero-field spl itting (zfs) or fine structure splitting ( 117 ). Electron Paramagnetic Resonance Properties of Mn(II) The characteristic six line EPR spectrum of Mn (II) arises from the interaction (hyperfine coupling) between the unpaired electrons ( S = 5/2) and the 55Mn nucleus ( I = 5/2). While this interaction decreases the sensitivity of the EPR m easurement by a factor of six, it provides a tool for distinguishing between octahedral and tetrahedral coordination states ( 118 ). Interactions between electron spin ( Ms) and nuclear spin ( MI) result in 36 states and 30 allowed transitions where Ms = 1 and MI = 0 (five EPR transitions, each split by hyperfine fine coupling to a sextet) (Figure 3-2 ) ( 117 ).

PAGE 44

44 Figure 3-2 Electron spin energy levels and hyperf ine splitting for Mn(II) in spherical symmetry. One sextet of the five-fold allowed transitions ( Ms = 1, MI = 0) is indicated by arrows. Figure adapted from ( 117 ). The schematic representation in Figure 3-2 depicts the electronic Zeeman and nuclear hyperfine interactions of a hypothe tical case in which there is a five-fold degeneracy of the Ms = 1 fine structure transitions. This situation would yield a sp ectrum of six well resolved EPR signals and is close to what is observed for hexaaquo mangane se in solution. For hexaaquo manganese there is, however, some inhomogenous br oadening of the EPR lines that is the result of an inexact superpos itioning of the five Ms = 1 fine structure transitions ( 117, 119 ). When Mn(II) is bound in a symmetry lowe r than cubic, the asymmetry of the ligand field (or crystal field) removes the degeneracy of the electronic sp in levels in the absence of an applied magnetic field. This means that the appr oximate degeneracy of the five Ms = 1 fine structure transitions is lifted resulting in a zero field sp litting. The three Kramer s doublets (/2, /2, /2) possess different energies in the absence of an external magnetic field.

PAGE 45

45 Four magnetic parameters are essential to define a paramagnetic species, these are g, A, E, and D The gyromagnetic ratio of an electron, ge, is the ratio of its magnetic dipole moment to its angular momentum. A free electr on has a g value of 2.002319304386 (which is ge, the electronic g-factor). When an unpaired electron is in an atom, it is affected by not only the external magnetic field, Bo, but also by any local magnetic fields. The effective field, Beff, felt by an electron is described by Beff = E = h = gBBo (1) where allows for the effects of the local fields The resonance condition is, therefore, E = h = geBBo(1) The quantity ge(1) is called the g -factor, given by the symbol g so E = h = gBBo The g -factor (or g -value) is determined in an EPR experiment by measuring the field, Bo, and the frequency, at which resonance occurs. If g is different than ge, the ratio of the electrons magnetic moment to its angular moment um has changed from the free electron value. Since the electrons magnetic moment (the Bohr magneton, B) is constant, it must have gained or lost angular momentum ( 116 ). As illustrated in Figure 3-2, the hyperfine in teraction is the interaction between the magnetic moment of an electron with the magnetic moment of the nucleus. The electron-nuclear interaction, depends on the projections of both electron and nuclear spins: Eelectron-nuclear = A MI Ms where A is the hyperfine coupling constant. A depends not only on the g-values for the electron and the nucleus but also on the distance between them and their orientation with respect to the external field.

PAGE 46

46 The fine structure parameters D and E reflect the deviation of the ligand field from spherical and axial symmetry, respectively. Dand Estrain reflect the i nhomogeniety of these values and depend on the metal-lig and distances and bond angles ( 117 ). Oxalate Decarboxylase EPR Since the two Mn(II) ions in th e resting monomer are in very similar coordination (Figures 1-4 and 3-3) environments, spectroscopic effort s to establish whether catalysis takes place in only one or both of the two metal sites have b een significantly complicat ed. In previous X-band studies, it has been shown that addition of small molecules like formate and oxalate have small but reproducible effects on the Mn (II) EPR spectra indicating the possibility of using EPR as a sensitive probe of the ligand environment (74) Distinguishing which metal signal(s) was perturbed, however, is difficult in X-band because the signals are very broad.3. Figure 3-3 Overlay of the N-terminal (shown in magenta) and C-terminal (shown in green) manganese-coordinating ligands (PDB code: 1UW8) The manganese ion is shown in blue. This image was prepared using the CAChe Worksystem Pro V6.5 software package (Fujitsu America Inc., Beaverton, OR).

PAGE 47

47 A multifrequency EPR approach has been employed to address the question of whether it is possible to distinguish the two Mn ions in Ox DC spectroscopically. Specifically this set of experiments was designed to (i) distinguish th e two Mn(II) sites and to (ii) determine their respective magnetic parameters. The rationale of using this appro ach is based on the fact that Mn(II) linewidths generally become narrower at higher fields (and thus higher frequencies) allowing for better spectral resolution of small diffe rences in g and A. Effects associated with differing fine structure parameters, however, are more prominent at low and intermediate fields (frequencies). Results and Discussion To avoid complications arising fr om the binding of ligands othe r than water to the two free ligand positions on each Mn, initial experi ments used OxDC dissolved in 20 mM hexamethylenetetramine (HMTA) HCl buffer at pH6.0, 0.5 M NaCl (storage buffer). HMTA is not expected to bind to Mn(II) b ecause it is positively charged and too bulky to fit into the Mnbinding pockets in the protein. OxDC has maxi mum activity at a pH value of around 4.0 and it is common practice in the literature to use vari ous types of negatively charged buffer molecules to control pH for spectroscopic and kinetic analysis ( 57, 72-74 ). It was of interest, therefore, to investigate the effect of acetate buffer at pH 5.2 on the EPR spectra. At this pH value the enzyme possesses substantial activity but is also highly soluble. X-band EPR The X-band EPR spectrum of OxDC (Figure 3-4) at low temperature is distributed over a wide field range with a clearly discernable but weak group of lines at half-field indicating substantial fine-structu re in the Mn(II) S=5/ 2 ions (71, 74)

PAGE 48

48 Figure 3-4 X-band cw-EPR spectra of wild type OxDC in storage buffer and in acetate buffer. Figure 3-4 shows that there may be a mixture of Mn(II) species with similar g-(2.001) and A-(250 MHz) values but with different zero-fi eld splitting constants D. When D is small compared to the EPR signals are mostly around g 2. However, for large D, the signal is spread over a broad field range with only parts staying near g 2. Field Dependence of the EPR Signal in Storage Buffer Figure 3-5 shows the g 2 region of the EPR spectra of OxDC in HMTA buffer pH 6.0 at frequencies ranging from X-band (corresponding to 0.34 T) to the sub-mm band (15 T). In Figure 3-5 the effect of incr easing field on the central + sextet of lines is clearly visible. They are substantially broadene d at low frequencies due to higher order c ontributions of the zero-field splitting (zfs) ( 117 ), but broadening is reduced as the sample is moved toward its highfield limit. No broadeni ng or field-dependent ( g ) or -independent ( A ) splitting of th e central lines was observed up to 15 T where the linewidth is at its narrowest. This indicates that g and A are in fact very similar for the Cand N-terminal Mn(II) ions.

PAGE 49

49 Figure 3-5 Field dependence of the EPR spectra of OxDC in storage buffer (20 mM Hexamethylenetetramine HCl, pH 6.0) with 0.5 M NaCl. To facilitate comparison of the + transitions spectra are shifted along the B0-axis. Field positions in T at the zero-points: 0.3340 (X-band, 9.4873 GHz), 1.7490 (V-band, 49.200 GHz), 3.3545 (W-band, 94.0214 GHz), 7.9427 (222.40 GHz), 11.567 (324.00 GHz), and 14.730 (412.80 GHz). All spectra were taken at temp eratures between 5 and 20 K. Reprinted from (120) with permission. Spectral Simulations and Magnetic Parameters Spectral simulations were performed with the easyspin toolbox for Matlab ( 121 ) by Ines Garcia-Rubio and are shown along with the experi mental settings in Appendix B. The main sextet lines could be simu lated considering a Mn(II) center with zfs parameters D = 1200 MHz and E = 283 MHz (site I in table 3-1). However, this did not account for th e weaker shoulders on the highand low-field sides of the sextet lines and a second Mn(II ) species was considered (site II in Table 3-1). Figure 3-6A shows the simula tion together with the experimental W-band spectrum of OxDC in HMTA buffer. Note that both sites are present in the same proportion and site II has a considerably highe r D-value (2700 MHz). This explai ns why its signal intensity is spread out over a broad field range and is seen only in the form of re latively weak shoulders on the narrow and intense lines of the site I signals, even at high frequencies. For this reason the

PAGE 50

50 multi-frequency approach was crucial to detect, identify, and characterize the signals from the Mn(II) ion in site II. Figure 3-6 W-Band (94 GHz) EPR spectra of Ox DC. A) HMTA buffer pH6.0. B) Acetate buffer pH5.2. C) HMTA buffer pH6.0 and 50 mM fo rmate. The right panel shows the simulation of site I (blue) and site II (g reen) with the magnetic parameters given in Table 3-1. The left panel shows the experime ntal spectra (black) and the sum of the simulations of the two sites in th e same proportion. Reprinted from ( 120 ) with permission. Table 3-1 Magnetic parameters of OxDC species I and II. Modified from ( 120 ) with permission. The best simulations required substantia l Dand E-strain (20-24%) which is not uncommon for transition metal ions in prot eins and in particular for Mn(II) ( 117 ). The E/D ratio was found to be approximately 25%, indicating considerable rhombicity of the distorted octahedral coordination environment of both Mn(II) ions. g A [MHz] D [MHz] E / D Dand E-Strain SB, site I 2.00087 253 1200 50 0.23 0.02 24% 24% SB, site II 2.00094 250 2700 50 0.25 0.02 20% 20% AB, site I 2.00086 252 1200 50 0.23 0.02 40% 40% AB, site II 2.00086 250 2150 50 0.05 0.02 33% 60% SB + formate, I 2.00087 253 1200 50 0.23 0.02 24% 24% SB + formate, II 2.00086 250 2150 50 0.05 0.02 33% 60%

PAGE 51

51 Field Dependence of the EPR Signal in Acetate Buffer, pH 5.2 The spectra recorded at vari ous frequencies from X-band to 420 GHz are shown in Figure 3-7. At high fields the wings that are characteristic for the fine structure are strongly suppressed compared to the main 6-line transitions. Therefore, the change in D upon acetate binding is less visible. The differences between this and the previous set of spectra are small and are most obvious in the intermediate to high frequency ra nges (W-band and up). They mainly involve the shoulders associated with site II. Figure 3-7 Field dependence of the EPR spectra of OxDC in acetate buffer (50 mM, pH 5.2) with 0.5 M NaCl. The spectra were shifted along the B0-axis. Field positions in T at the zero-points: 0.3339 (X-band, 9.4853 GHz), 1.7470 (V-band, 49.200 GHz), 3.353 (Wband, 94.0206 GHz), 7.9415 (222.40 GHz), 11.563 (324.00 GHz), and 14.7253 (412.80 GHz). Temperatures were set betw een 5 K and 20 K. Reprinted from ( 120 ) with permission. Figure 3-6B shows the experimental Wband signal of OxDC in acetate buffer pH5.2 with its simulation. The changes in the features of site II are mainly due to a decrease of D and E from 2700 to 2150 MHz, and 675 to 108 MHz, resp ectively as well as a small decrease in g (see

PAGE 52

52 Table 3-1). The magnetic parameters of site I were unchanged except for an increase in zfs parameter strain which was also seen for site II. Note that the rather dramatic change in E indicates a more axial ligand field environmen t for site II in the presence of acetate. The zfs parameters of Mn(II) have been dem onstrated to be sensitive to electrostatic charges in their vicinity ( 122 ). The replacement of one or two water molecules in the coordination sphere of Mn by acetat e is certainly expected to cha nge the electrostatic potential around the Mn-center and could lead to the observed changes in D and E The observation that only site II changes upon exposure to acetate buffer suggests that only site II is solventaccessible. When formate is added to OxDC in HMTA buffer the spectral changes observed for site II are very similar to those found for acetate (see Figure 3-2C). This is not surprising given that formate and acetate are alike in the polar parts of their structure and are expected to show the same coordination geometries with the metal ion. The simplest interpretation of these results points to a correlation between the two magnetic parameter sets and the two Mn-binding site s in the protein. The di fferences in the fine structure are due to subtle differe nces in the charge distribution in the Nand C-terminal binding sites while the almost identical g and A is due to similar octahedral coordination in both sites. The fact that both species are present in approxim ately the same concentra tion in all preparations investigated so far suppor ts this interpretation. The observation of changes in the fine structur e parameters of only s ite II upon addition of acetate buffer or formate is intriguing and suggests that small molecule binding mainly takes place at site II and not site I under ou r experimental conditions. Just et al ( 73 ) observed a channel leading from the N-terminal Mn binding site to the solvent which may be accessible by the hinge-motion of a flexible l oop region while they report no obvious solvent ch annel available

PAGE 53

53 for the C-terminal site. Moreover, formate was f ound coordinated to the N-terminal Mn(II) in the X-ray structure by Anand et al ( 72 ). Therefore, it seems reasona ble to identify site II with the N-terminal Mn-binding site, and site I with the C-terminal site. The open and closed conformations ( 72, 73 ) of OxDC show the C-terminal Mn-binding site in hexaand penta-coordinated forms, respectively. It is worth noting that D -values for penta-coordinated Mn(II) centers in MnSOD have been reported as one order of magnitude higher than what we found for site I ( 123 ). Therefore, our site I magnetic parameters are compatible with the hexa-coordinated Mn(II) ion in the C-terminal Mn site that is observed in the X-ray structure of OxDC published by Anand et al. ( 72 ). A multi-frequency EPR approach has allowed us to spectroscopically distinguish two Mn(II) species that are present in equal proportions in the resti ng state of the enzyme oxalate decarboxylase in HMTA storage buffer. The main difference between these two species is the value of the fine structure parameters with DI = 1200 MHz, DII = 2700 MHz, and E / D = 0.25. When the enzyme is placed in acetate buffer pH5.2 or when formate is added, DII is reduced to 2150 MHz and EII/ DII = 0.05 while DI and EI remain the same indicating that only one Mn(II) is solvent accessible. Based on published crystal structur e data, we conclude site I is the C-terminal Mn site while site II is the solvent-exposed N-terminal site and, therefore, the site of small molecule (acetate and formate) binding. Experimental Section Oxalate Decarboxylase Sample Preparation Several different batches of OxDC enzyme pr eparations were used. They were prepared according to the procedures listed in Chapter 2. Final concentrations ranged from 7.7 to 12.3 mg/mL. Samples in HMTA pH6.0 were used w ithout further modifications. Samples in 50mM acetate buffer (AB) pH5.2 were prepared from stoc k by addition of a concentrated acetate buffer

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54 solution (500 mM, pH5.2). Typically, 90 L of sample were mixed with 10 L of concentrated AB buffer. The volumes were scaled in the sa me ratio for experiments requiring smaller (Wband) or larger quantities (sub-mm bands). The same procedure was used for the addition of formate to the HMTA buffered samples. 10 L of 500 mM formate solution was added to 90 L of HMTA pH6.0 buffered sample to arrive at a final concentrati on of 50 mM formate. Electron Paramagnetic Resonance Spectroscopy X-band spectra (9.5 GHz) were recorded on an Elexsys E580 spectrometer (Bruker Biospin Corp.) and the OxDC samples were pl aced in 3 x 4 mm2 (IDxOD) homemade clear fused quartz tubes and frozen in liquid nitrogen be fore insertion into th e Oxford ESR900 cryostat which had been pre-cooled to ~10 K. W-band (94 GHz) spectra were recorded on an Elexsys E680 spectrometer (Bruker Biospin Corp.) and the samples were placed into 0.7 x 0.79 mm2 (IDxOD) clear fused quartz capillaries. The samples were then frozen in liquid nitrogen prior to insertion into the precooled Bruker ER4118CF-W cryostat. V-band and sub-mm bands (50, 200-420 GHz) spect ra were recorded with a home built instrument using a 15/17 T superconducti ng magnet as described by Hassan et al. ( 124 ). Samples were placed into 7.2 x 8.2 mm2 (IDxOD) home-made Teflon cups. The cups have a depth of 9.5 mm and were supplied w ith a Teflon stopper. Typically, 200 L of sample was inserted into the cup which was then closed with the stopper to protect the sample from contamination. The sample was pre-frozen in liqu id nitrogen, the field standard (P-doped Si sample) was then placed on top of the stopper before it was inserted into the sample holder. The sample holder was also pre-cooled to liquid nitrogen temperatures before it was inserted into the pre-cooled Oxford Spectrostat CF DY LT cryostat. Experimental settings and simu lations are givens in Appendix

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55 CHAPTER 4 SPECTROSCOPIC CHANGES OF THE MANGANESE CENTERS IN THE PRESENCE OF SUBSTRATE Introduction Previous EPR spectroscopic characterization of the Mn centers in oxalate decarboxylase by workers in this laboratory identified a ty rosyl radical formed dur ing oxalate turnover ( 71 ). Formation of this species requires OxDC, oxa late, and oxygen. The time course of radical formation and decay compared to the overall rate of enzyme turnover suggested that radical formation may be related to catalysis but is not on the catalytic pa thway. Furthermore, no spectroscopic signature for Mn(III) or Mn(IV) wa s observed in samples fr ozen during catalytic turnover ( 71 ). X-band EPR spectral perturbations of the Mn centers have been observed upon oxalate addition indicating that EPR spectroscopic characterization of the manganese centers in the presence of substrate may yield insights into the mechanistic role that they play during catalysis ( 74 ). Since, as noted in Chapter 3, the effect s of differing fine structure parameters are more prominent at low and intermediate fields and the fact that linewidths generally become narrower at higher fields allowi ng for better spectral resoluti on of small differences in g and A, multifrequency EPR characterization is a rationa l approach for characterizing the manganese centers in the presence of substrate. The scie ntific aim of the experi ments described in this chapter is to follow the decarboxy lation reaction of OxDC by mon itoring both the Mn as well as the formation of any intermediate radicals as th e reaction progresses. It is anticipated that experiments of this type will be essential to establishing the re dox state of the active Mn site before, during, and after catalysis.

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56 Results and Discussion X-band (9.5 GHz) Figure 4-1 shows spectral changes at X-band of the g 2 signal upon addition of acetate and oxalate to OxDC. The spectrum in storage buffer (HMTA, pH 6.0, 0.5 M NaCl) is shown in black. The sample is made 50 mM sodium acetate, pH 5.2 in order to decrease the pH to a level where OxDC is active yet still soluble enough to maintain the high protein concentration required (~10 mg/mL) to obtain high quality spectra. Figure 4-1 Spectral changes of the g 2 signal at X-band upon addition of acetate and oxalate to OxDC. The intensity of the signal increases upon acetate addition (sho wn in red). The spectral intensity decreases to its original value when oxalate is added to 50 mM in acetate buffer pH 5.2 (shown in green) and flash frozen in liquid nitr ogen. To detect the spectrum of the tyrosyl radical, the sample is thawed and allowed to react for 2 min, then freeze-quenched and reinserted into the EPR cryostat (s hown in blue). Formation of th e tyrosyl radical is accompanied by a further decrease in spectral intensity.

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57 To further explore the changes in spectral in tensity during radical formation buffers other than acetate were used to lower the pH of the st orage buffer. Buffers used were sodium citrate, pH 5.2, PIPES [piperazine-1,4-bis(2-ethanesulfonic acid)], pH 5.2, as well as the storage buffer adjusted to pH 5.2 (Figure 4-2). All tested buffe rs gave essentially the same spectra indicating that the effect of the change of the intensit y during and after radical formation was not buffer dependent. Figure 4-2 Spectral changes of the g 2 signal at X-band upon addition of oxalate to OxDC in storage buffer at pH 5.2. Figure 4-2 shows the spectral changes of the g 2 signal at X-band upon addition oxalate to OxDC in storage buffer at pH 5.2. Upon maki ng the sample 50 mM oxalate and flash freezing 2 minutes after mixing, the tyrosyl radical is obser ved (shown in green) as well as minor spectral changes and a reduction in the spectral intensit y. After spectral acquisition the sample was thawed and stored on ice for 3 hour s before being measured again (s hown in blue). At this point the Mn(II) signal as well as the radical signal was almost gone. Upon making the sample 5 mM

PAGE 58

58 dithionite almost all of the signal intensity was restored although spectral changes were observed, primarily in the wings of the sextet. Figure 4-3 Spectral changes of the g 2 signal at X-band upon addition of oxalate to OxDC in storage buffer at pH 5.2 under anaerobic c onditions followed by th e reintroduction of air. With respect to the half-field X-band signal (see Figure 3-1), oxalate almost completely destroys its multiplet pattern (d ata not shown) and leaves only a broad signal at 1560 G. Addition of dithionite does not rescue the low-field multiplet spectrum. The disappearance of the Mn(II) intensity and its almost complete restor ation with dithionite is strong but indirect evidence for the formation of Mn(III) or Mn(IV). When, however, X-band spectra were taken of these identical series of experiments using a parallel mode cavity (data not shown), no spectroscopic signature of Mn(III) was observed. The parallel polarization experiments, however, do not preclude the possibility of the formation of long-lived, high-valent manganese species. It is conceivable that any high-valent manganese species might possess such large zero

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59 field splitting parameters that th e anticipated signal in parallel mode would be unobservable at X-band or simply broadened beyond detection. Figure 4-3 shows spectral changes of the g 2 signal at X-band upon addition of oxalate to OxDC in storage buffer at pH 5.2 under anaerobi c conditions followed by the reintroduction of air. Very little spectral changes are observed when OxDC in storage buffer at pH 5.2 (shown in red) is made anaerobic by gently bubbling the EPR tube containing the sample with N2(g) for 4 minutes inside a glove box (shown in pink). A ddition of degassed oxalate decreases the Mn(II) signal dramatically (shown in br own) as in the case when oxygen is present (Figure 4-2), but no radical signal is observed. When the sample wa s thawed and air was allowed to enter the EPR tube, the Mn(II) signal in tensity decreased further and surpri singly no radical was formed (Figure 4-2, shown in turquoise). Finally, when air wa s bubbled through the sample the Mn(II) signal only decreased slightly (shown in purple) and there was still no ra dical formation. This sample was then assayed for decarboxylase activity and re markably showed an increase in specific activity from 51 U/mg to 88 U/mg. It was later confirmed that samples which have been made anaerobic then had oxygen reintrodu ced possessed an increase in sp ecific activity of at least 50%. Chemical Oxidation of OxDC Observed at X-band The addition of potassium ferricyanide to 50 mM (or hydrogen peroxide to 3%) to OxDC did not have any appreciable effect on the Mn(II) signal (data not shown). The intensity of the Mn(II) signals could be decrea sed, however, by the addition of the strong oxidizing agents potassium hexachloroiridate (data not shown) and sodium (meta) pe riodate (Figure 4-4). In these experiments, a series of additions were made to the enzyme in storage buffer. At each concentration of oxidant an X-band spectrum was ta ken at 7 K. The decrease in signal intensity was small up to 2 mM of either of the oxidants used. Both of the oxidants showed a marked

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60 decrease in signal intensity at about 4 mM with a concomitant app earance of a carbon-based radical. The linewidth of the radical signal is consistent with the previously described tyrosyl radical ( 71 ). The Mn(II) could be brought back with the addition of 5 mM dithionite (Figure 44). While it is difficult to inte rpret these intriguing observations with respect to the redox forms of the enzyme before, during, and after catalysis, th ese results indicate that molecules are able to enter into the Mn-binding sites facilitating future efforts to measure the reduction potentials of the two sites. Figure 4-4 Mn(II) signal intens ity and carbon-based radical form ation as a function of the concentration of sodium (meta) periodate. X-band Spin-Trapping of an Oxygen Speci es Formed During Oxalate Decarboxylase Turnover In the spin-trapping technique, a diamagnetic spin-trap (EPR silent) compound reacts with reactive short-lived free radicals to form a more persistent spin adduct. From the EPR spectrum of the spin adduct, the struct ure of the reactive free radical can be deduced indirectly. The spin trapping experiments described here showed that this technique can be used to detect radical

PAGE 61

61 species formed during OxDC turnover. These experiments employed the most commonly used nitrone spin trap 5,5-Dimethyl-1-pyrroline Noxide (DMPO). The spectral shape of the EPR signal of the trapped radical shown in the time course (Figure 4-5) s uggests that it may be a hydroxyl radical ( 125, 126 ), but this should be confirmed by analyzing the trapped product by mass spectroscopy. Figure 4-5 EPR spectra of the spin-trapped ra dical formed during OxDC turnover. The following blanks showed no significant signal: buffer + DMPO, buffer + DMPO + oxalate, and OxDC + buffer + DMPO. One problem with the use of DMPO as a sp in-trapping agent is th at a DMPO-superoxide adduct has a half-life on th e order of 1-2 minutes ( 125, 126 ) before decaying to the DMPOhydroxide adduct. Since the hypothetical mechanis m shown in Chapter 1 (Figure 1-5) proposes the formation of a manganese-bound superoxide radical, it was of interest to look at an earlier time point than those shown in Figure 4-5. A 2.5 minute time point is shown in Figure 4-6 and shows the appearance of a different signal that then decays to that shown in Figure 4-5 raising the possibility that it represents a DMPO-superoxide adduct. This should be explored further -2000 -1000 0 1000 2000 3000 4000 5000 349035003510352035303540 3.3 mg/mL OxdC 100 mM KAc, pH 4.2 5 uL [DMPO] initiated with 75 mM oxalate 4 min 7 min 11 min 15 min 19 min field

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62 using a spin trap with a longer-lived super oxide adduct species such as 2-ethoxycarbonyl-2methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO) or 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) ( 127 ). Figure 4-6 EPR spectrum of a short-lived DMPO-oxygen species. Q-band (3Hz) Figure 4-7 Spectral changes of the Mn(II) signal at Q-band upon addition of acetate and oxalate to OxDC. -3000 -2000 -1000 0 1000 2000 3000 4000 34803500352035403560 75 mM oxalate 10 dB att, 60 dB R/G first scan ~2.5 min field

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63 Q-band EPR (Figure 4-7) was able to reveal th e lowand high fieldwings that belong to the higher spin manifolds (trans itions outside the central + sextet of lines) at least for one of the Mn(II) species. The sample in storage buffer showed a true half field signal (data not shown) which becomes weaker when the sample is made pH 5.2 with the addition of acetate buffer. W-band (94 GHz) Figure 4-8 Spectral changes of the Mn(II) signal at W-band upon addition of acetate and oxalate to OxDC. Just as in X-band, the wild-type enzyme show s an effect of acetate binding on its six line Mn(II) signal in W-band g 2 region (Figure 4-8). At this higher frequency, where there are no contributions from zero field splitting, there is no half field signal and al l the signal is found at the g 2 region. The spectra taken in storage buffe r (shown in black) displa y a clear spliiting in their negative troughs which is absent in those taken in acetate buffer, pH 5.2. There is very little difference in the spectra taken in acetate bu ffer with (shown in green) and without oxalate, which is in strong contrast with what is obser ved at X-band. No radica l is observed at W-band.

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64 324 GHz Figure 4-9 shows the very clean 324 GHz spectrum of OxDC in storage buffer (black). In this spectrum only a single spec ies is visible (note the two line s of the phosphorus doped silicon field standard in the 5th line and between the 5th and 6th lines). Addition of acetate (red) leads to a broadening of the sextet lines especially in th e higher field portions with a clear splitting of the weaker component. The lines narrow again with the addition of substrate (green). Figure 4-9 Spectral changes of the Mn(II) signal at 324 GHz upon addition of acetate and oxalate to OxDC. 690 GHz The spectra shown in Figure 4-10 were acquire d at the highest field available, 690 GHz. The larger linewidth of all the spectra displaye d may be due to the fact that the Keck magnet (Bitter type magnet) is less homogeneous or this may be the first indication of an effect of g anisotropy. The two Mn(II) specie s are indistinguishable from each other in the spectrum taken in storage buffer (shown in black). Addition of acetate buffer broadens the six lines and begins to split the lower field lines (shown in red). The spectrum taken after th e addition of substrate

PAGE 65

65 (shown in green) splits approximately 50 % of its signal intensity off into additional sextet signals. The observed spectrum is consistent w ith the hypothesis that onl y one Mn-binding site is available for substrate binding. This experiment clearly demons trates that substrate binding to Mn(II) can be followed spectroscopically by very high field EPR. Figure 4-10 Spectral changes of the Mn(II) signal at 690 GHz upon addition of acetate and oxalate to OxDC. Experimental Section OxDC was purified as described in Chapter 2. Sample preparation and EPR spectroscopy was as described in Chapter 3. X-band spectra: microwave frequency 9.48731 GHz, microwave power 0.64 mW, modulation frequency 100 kHz, modulation amplitude 10 G, receiver gain 60 dB, time constant 41 ms, conversion time 41 ms, 1 sweep, 1.465 G/data point. Q-band spectra: microwave fre quency 34.05197 GHz, microwave power 17 W, modulation frequency 100 kHz, modulation amplitude 10 G, receiver gain 60 dB, time constant 10 ms, conversion time 41 ms, 1 sweep, 1.953 G/data point.

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66 W-band spectra: Microwave fre quency 94.02141 GHz, microwave power 0.6 W, modulation frequency 100 kHz, modulation amplitude 2 G, receiver gain 40 dB, time constant 82 ms, conversion time 82 ms, 1 sweep, 1.172 G/data point. 324 GHz spectra: Microwave frequency 324.00 GHz, modulation frequency 41.8 kHz, modulation amplitude 0.5 G, lock-in sensitivity 50 V, time constant 100 ms, sweep speed 5.01 G/s, 1 sweep, 0.367 G/data point.

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67 CHAPTER 5 SITE-DIRECTED MUTAGENESIS STUDIES TO PROBE WHICH MANGANESE-BINDING SITE(S) IS INVOLVED IN CATALYSIS Introduction The fact that the OxDC contains Mn in both the Nand C-terminal cupin domains raises the question of whether catalysis takes place in on ly one or both of the two Mn-binding sites. Multifrequency EPR studies (Chapter 3) suggest that only one Mn-binding site binds acetate and formate. Two structural observations can be ci ted as support for the hypothesis that the active site of the enzyme is located in the N-terminal domain. First, this dom ain appears to contain a channel along which oxalate can diffuse from solu tion, which can exist in an open or closed form as a result of the conformationa l rearrangement of residues 161-165 ( 73 ). Second, formate has been observed to coordinate the N-terminal Mn ion in one of the OxDC crystal structures ( 72 ). Site-directed mutagenesis studies of conserved arginine (A rg-92 and Arg-270) and glutamate (Glu-162 and Glu-333) residues in th e two Mn-binding sites (Figure 1-4) have, however, provided conflicting evidence for which of the two domains might mediate catalysis ( 72, 73 ). Interpretation of these studies is co mplicated by the presen ce of polyhistidine purification tags in the recombinant OxDC mu tant enzymes, and/or a lack of quantitative information on their Mn content ( 37, 73 ). Resolving the location of the active site(s) in OxDC is an important problem because if only a single site mediates the Ox DC-catalyzed reaction, legitimate issues are ra ised concerning the function (if any) of the second Mn-binding domain, and the extent to which local pr otein structure in each domain resu lts in the differential reactivity of the two metal centers.

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68 Results and Discussion Design and Steady-State Characterization of OxDC Mutants with Domain-Specific Modified Mn Affinity Given the possibility that both Mn sites catalyze the decarbo xylation reaction, a series of OxDC mutants were constructed designed to disr upt the Mn-binding capability of a given cupin domain by modifying the side chains of either Glu-101 and Glu-280, which coordinate the metal in the Nand C-terminal domains, resp ectively (Figure 1-4). Prior studies of Flammulina OxDC had shown that mutation of Mn-binding histidine residues in either cupin domain yielded only inactive enzyme ( 128 ). A series of site-specific OxDC mu tants was, therefore, constructed in which Mn-binding glutamate residues in each of the two domains (Figure 1-4) were replaced by alanine, aspartate and glutamine residues (Table 51). It was anticipated th at the affinity of the binding site containing the mutated residue would be severely reduced so as to yield enzyme in which Mn was incorporated preferentially into the other domain. If catalysis was mediated independently by both Mn-binding sites, we an ticipated these OxDC metal-binding mutants would exhibit activities reduced by approximately 50% from that of wild type enzyme (assuming metal incorporation proceeded to give 1 Mn/monomer). Table 5-1 Mn incorporation and steady-state kine tic parameters for metal-binding OxDC mutants Enzyme Spe. Act. Mncontent Km (mM) kcat (s-1) kcat/Km (M-1s-1) WT OxDC 61.2 U/mg 1.87 8.4 0.7 53 1.5 6309 E101A 0.05 U/mg 0.18 2.9 0.3 0.046 0.002 16 E101D 0.79 U/mg 0.09 3.4 0.1 0.49 0.01 144 E101Q 0.63 U/mg 0.11 4.0 0.2 0.62 0.01 155 E280A 0.03 U/mg 0.67 3.0 0.2 0.019 0.001 6 E280D 0.69 U/mg 0.64 5.4 0.4 0.14 0.01 26 E280Q 0.15 U/mg 0.73 10.1 0.6 0.62 0.01 61 E101Q/E280Q 0.01 U/mg 0.07 2.9 0.3 0.012 0.0003 4

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69 Contrary to this expectation, the steady-state kinetic parameters for the series of OxDC mutants showed that catalytic turnover was signif icantly lower than expected on the basis of Mn incorporation even though the oxalate Km values were not greatly perturbed (Table 5-1). In the case of the E280Q OxDC mutant, th e Mn content was 39% of that present in fully active, wild type OxDC yet the specific activ ity of the same mutant was onl y about 1% of the wild type activity. In other words, all Mn -binding mutants fell significantly below the line constructed in Figure 2-1 (the dependence of OxDC specific activity on the extent of Mn incorporation). In addition, none of the mutant enzymes were found to exhibit oxalate oxidase activity, at least as assayed with a dye oxidation method to monitor oxalate-dependent hydrogen peroxide formation ( 129 ). Table 5-2 Metal content of Mn-binding OxDC mutantsa. a Number of metal ions/OxDC monomer. b Value is relative to that of wild type OxDC. c Value was not determined. In designing these experiments, it was assu med that the absence of Mn in the domain lacking a key glutamate side chai n would not dramatically affect the three-dimensional fold of the -barrel domain structure. This assumption seems reasonable given the existence of stable, metal-free cupin domains that lack metal-binding residues ( 130, 131 ). Unexpectedly, these experiments showed that Mn incorporation at th e C-terminal binding site appears to require the presence of Mn in the N-terminal domain. Thus replacement of Glu-101 by alanine, aspartate or Enzyme Preparation Mn Co Zn Fe Cu Mg Specific Activity b WT OxDC 1.87 n.d.c 0.51 0.07 0.01 0.01 100% E101A 0.18 < 0.01 0.17 < 0.01 < 0.01 n.d.c < 0.1% E101D 0.11 < 0.01 0.08 < 0.01 < 0.01 n.d.c 1.0% E101Q 0.09 < 0.01 0.05 0.30 < 0.01 n.d.c 1.3% E280A 0.67 < 0.01 0.12 < 0.01 < 0.01 n.d.c < 0.1% E280D 0.64 < 0.01 0.07 0.04 < 0.01 n.d.c 0.3% E280Q 0.73 n.d.c 0.11 < 0.01 < 0.01 < 0.01 1.1% E101Q/E280Q 0.07 < 0.01 0.12 0.04 < 0.01 n.d.c < 0.1%

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70 glutamine gave OxDC mutants containing appr oximately 0.1-0.2 Mn/monomer (Table 5-1). That this was not merely an effect associ ated with expressing the OxDC mutant in Escherichia coli was demonstrated by the failure of efforts to introduce Mn into th e E101Q OxDC mutant using our well-defined in vitro conditions for metal subst itution and re-folding. OxDC molecules containing Mn bound only in the C-termin al domain may be absent in solution, and hence the activity of samples of recombinan t OxDC containing less than 2 Mn/monomer is associated with enzyme species containing Mn in both domains and/or one Mn in the N-terminal binding site. Size-Exclusion Chromatography (SEC) Because modification of the metal-binding glut amates gave OxDC mutants with catalytic activities far below those anticipated from thei r Mn content (assuming tw o independent catalytic sites) it was of interest to investigate whethe r changes to the metal bi nding glutamate residues might have caused large perturbations in enzyme structure. X-ray crystal structures show that Bacillus subtilis OxDC adopts a quaternary structure consisting of a hexamer in which two trimers are packed face to face so that the complex possesses 32 ( D3) point symmetry ( 72 ) (Figure 1-3). Because this crystallographic observa tion is consistent with PAGE studies of native OxDC ( 34 ), we employed size-exclusion chromatography to investigate the qua ternary structures adopted by the series of Ox DC mutants (Table 5-3). Although recombinant, wild type OxDC seemed to elute as a hexamer under our conditions, approximately 85% of the purified prot ein sample was present as oligomers of higher apparent mass, corresponding to complexes formed from approximately 12-18 monomers. Perhaps more importantly, however, replacement of either of the Mn-bin ding glutamate residues did not yield oligomeric forms of the OxDC muta nts that were significantly different to those

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71 adopted by the wild type enzyme, although small populations of dimers (based on their elution properties) were observed for several of the mutants. Table 5-3 Estimates of size for the oligomeric fo rms of recombinant wild type OxDC and OxDC Mn-binding mutants obtained using size exclusion chromatography. aValues shown are those given for the molecular weight standards. bEstimates obtained from size exclusion chromatography. As terix indicates the predomin ant species observed under elution conditions. cDouble mutant in which Glu-101 and Glu-280 are both replaced by glutamine residues. Actual MW (kDa)a Estimated MW (kDa)b Number of OxDC monomers Carbonic Anhydrase 29 27 Albumin 66 93 Alcohol Dehydrogenase 150 103 Amylase 200 259 Apoferritin 443 433 Thyroglobulin 669 579 Wt OxDC 596*, 310 14,7 E101A 589*, 208, 80 13, 5, 2 E101D 666 15 E101Q 500* 196 12, 5 E280A 603*, 272, 222, 105 14, 6, 5, 3 E280D 602 14 E280Q 617*, 216 14, 5 E101Q/E280Qc 572*, 189 13, 4 Circular Dichroism Measurements Circular dichroism (CD) measurements were em ployed to evaluate the extent of secondary structural changes resulting from site-speci fic replacement of the metal-binding glutamate residues (Figure 5-1). The utility of circular dichro ism in the analysis of proteins is derived from the fact that the polypeptide bac kbone is optically activ e in the far ultraviolet (170-250 nm) and that different secondary structures produce characteristic spectra ( 132 ). Since CD measurements give estimates of the fraction of residues in helical, sheet, -turn, and unordered conformations, the effects of mu tations, denaturants, and temper ature can be studied and the

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72 kinetics of protein folding and unfolding can be investigated much more efficiently than by high resolution methods such as X-ray crystallogra phy. Technical consider ations of CD data collection include sample preparat ion in a buffer which does not abso rb in the region of interest and balancing the factors of samp le concentration, background si gnal, and cuvette pathlength. As expected on the basis of the X-ray crysta l structures, the CD sp ectrum of wild type OxDC showed features cons istent with the largely -strand character of the cupin domains together with minima at 222 and 205 nm th at are presumably associated with the -helices that mediate monomer/monomer contact s (Figure 5-1). The three OxDC mutants containing perturbations in the C-terminal Mn-binding site (E280A, E280D and E280Q) exhibited very similar CD spectra, but which differed consid erably from that of wild type OxDC. Figure 5-1 CD spectra of recombinant wild type OxDC and the Mn-binding OxDC mutants. Because these three proteins still bind relatively large amounts of Mn/monomer, the simplest interpretation of the CD spectra is that they reflect the -strand character of the Nterminal (and possibly the C-terminal) cupin dom ain(s). Thus, the observed changes likely reflect changes in the amount of -helix, especially in light of the decreased molar el lipticities observed -8000 -6000 -4000 -2000 0 2000 4000 6000 8000 200210220230240250 Wavelength (nm)

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73 at wavelengths between 190 nm and 200 nm. For OxDC mutants in which Glu-101 was replaced by other residues (E101A, E101D and E101Q) the picture was complicated by the finding that only E101A and E101D exhibited similar CD sp ectra. Although there are differences in the absolute molar ellipticities observed in the CD spectra for E101A, E101D and the three Cterminal OxDC mutants, the overall shape of th e curves for the five enzymes were similar, suggesting a resemblance of their overall secondary structures. In the case of the E101Q OxDC mutant, however, the observed spectrum resembled that of the apo-form of Thermococcus litoralis phosphoglucose isomerase, a cupin for which activity is thought to be Fe-dependent ( 133 ). In many respects, the CD spectrum of the E 101Q OxDC mutant is consistent with a higher proportion of -helix, and similar levels of -strand, secondary structure, relative to wild type OxDC. Electron Paramagnetic Resonance Properties To gain additional insight into the struct ure of the Mn-binding pocket in the OxDC mutants, we compared the EPR properties (see Ch apter 3 for an introducti on to the technique) of the Mn(II) center in the E280Q Ox DC mutant with those of the w ild type enzyme (Figure 5-2). In spectra taken for aerobic soluti ons of the enzyme at 10 K, the six-line Mn signals for the two proteins showed a strong resemblance, suggestin g that the Mn(II) ions in both proteins were coordinated in similar environmen ts, and the isotropic g-factor ( giso = 2.00087) was identical for the two preparations within experimental limits (.00001). Differences were observed, however, in the signal linewidths (Figure 5-2) The linewidth for th e E280Q mutant was 0.85 mT and that for the wild type enzyme was 1.1 mT (see simulations in Appendix C). The spectra also revealed very little gand A-strain, becau se the peak-to-peak amplitude was approximately the same within each six-li ne pattern, and did not show any trace of transitions between the higher electron spin manifo lds. The latter is due to relatively large fine

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74 structure values of the order of about 1000 MHz (Table 3-1). Similarly large fine structure parameters have been observed for the Mn(II) centers in the superoxi de dismutases from Rhodobacter capsulatus and Escherichia coli (134) Taken together, these spectral features suggest that the Mn ions in both samples are lo cated in a very homogeneous ligand environment, supporting the proposal that the cupi n structure of the N-terminal domain of wild type OxDC is retained in the Mn-binding mutant enzyme (E280Q). Figure 5-2 EPR spectra of the Mn(II) signals in wild type OxDC (red) and the E280Q OxDC mutant (blue) at 10 K For the wild type enzyme, th ese experiments were performed at 386.116 GHz, with a modulation amplitude and modulation frequency of 2 G and 40 kHz, respectively. The main coil was slowly swept over a range of 13.75-13.81 T at 0.1 mT/s. In the case of the E280Q OxDC mutant, EPR experiments were performed at 382.826 GHz, with a modulati on amplitude and modulation frequency of 2 G and 43 kHz, respectively. The main coil was slowly swept over a range of 13.628-13.698 T at 0.1 mT/s. For ease of co mparison, the spectrum for the E280Q OxDC mutant (blue) is offset by 108.8 mT along the magnetic field axis. The additional sharp lines visible at 13796 and 13800 mT arise from the P-doped silicon field standard employed to calibrate the magnetic field sweep during acquisition of the spectrum for wild type OxDC (red).

PAGE 75

75 Relaxation Enhancement Measurements The significant difference in the spectral lin ewidth observed for the Mn signal in the two samples, however, suggested that a line broa dening mechanism was operative in wild type OxDC that was absent in the E280Q OxDC muta nt, perhaps because only one Mn-binding site was occupied in this protein. To answer this question, we measured the inversion recovery kinetics of the Mn signals for wild type OxDC and the E280QOxDC mutant of OxDC (Figure 53). Relaxation refers to the rec overy from a non-equilibrium state to an equilibrium state. The characteristic time is called the relaxation time and is strongly dependent on the electronic structure of the paramagnetic cente r and on its interactions with it s environment. The spin-lattice relaxation time, T1, is the time constant for equilibrati on of the populations of the two electron spin Zeeman energy levels. The transverse re laxation time (or spin-spin relaxation time), T2, is due to the variation in resonant fields that result from other spins in the vicinity. T1 is usually much longer than T2 ( 135 ). These relaxation studies were performed using echo-detected EPR spectra for the samples, which were acquired at 3700 G and 3675 G for the wild type and mutant enzyme, respectively. For both samples, the observed T1 decay was bi-exponential, with values of 9.25 and 55.4 sec being obtained for the Mn signal in wild type OxDC. In the cas e of the E280Q OxDC mutant, however, the corresponding T1 values were 16.3 and 92.5 sec, meaning that relaxation was enhanced in the sample of wild type OxDC by a factor of approximately 1.7.A similar enhancement was observed for Tm (T2) in the two samples (Figure 5-4).

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76 Figure 5-3. Inversion-recovery experiments on wild type OxDC (12.3 mg/mL) and the E280Q OxDC mutant (16.8 mg/mL). Samples were dissolved in 20 mM hexamethylenetetramine-HCl, pH 6.0, containing 0.5 M NaCl (100 L total volume), and both spectra were taken at 5 K with a microwave frequency of 9.70703 GHz. The fields for the wild type OxDC and the E280Q OxDC mutant samples were 370 mT and 367.5 mT, respectively. The pulse sequence (see inset) employed /2 and pulses of 16 ns and 32 ns, respectively, and a 2-phase CYCLOPS sequence was used to separate the echo from unwanted spurious ech oes. The data in the figure is the sum of 5 individual pulse tr ains per CYCLOPS phase, separa ted by a repetition time of 5.1 ms. Microwave attenuation was set to 11 dB. The echo amplitude from the Hahn readout sequence was integrated and plotted as a function of the pulse separation time T, with a pulse separation for the Hahn readout sequen ce of 140 ns. Both traces were simulated with bi-exponential recovery kinetics. The observed changes in both T1 and T2 for the Mn signals in the two spectra demonstrated that the relaxation kinetics for metal centers in the wild-type enzyme were enhanced when compared to those of the single Mn in the E 280Q OxDC mutant (Figures 5-3 and 5-4). The inter-monomer Mn-Mn distance of approximately 21 observed in the wild type OxDC hexamer is close enough for a dipolar interaction be tween the two metal ions to be observable in the EPR spectrum.

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77 Figure 5-4 Data for the Hahn-echo decay experime nt on wild-type OxDC and the E280Q mutant. Samples and fields were as for Figure 3-3. The experiment (inset) employed the usual /2Hahn echo pulse sequence with 16 and 32 ns pulses, respectively. A 2phase CYCLOPS sequence was used to sepa rate the echo from artifacts, and echo modulations were observed on the decay traces Simulations were performed using a mono-exponential model taking points after the decay modulations. Similar relaxation enhancements over distan ces of 15-30 have been seen in other proteins, including the bacterial photosynthetic reaction center (52) a nd metmyoglobin (53). Assuming that E280Q assembles in the same quate rnary structure, its cl osest Mn-Mn distance would be almost twice as great ( 39-40 ) as in the wild type he xamer, reducing the effect of the magnetic moment of one Mn ion on the relaxatio n dynamics of its nearest neighbor. Hence, it seems likely that the difference in the high-fiel d EPR linewidths in the two spectra is due to paramagnetic relaxation broadening.

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78 Implications for the Location of the Catalytic Site(s) in OxDC As discussed in Chapter 2, the observation of a linear relationship between Mn occupancy and activity is consistent with three kinetic m odels, the simplest of wh ich postulates that both Mn-binding sites are catalytically ac tive. If the sites are also inde pendent, then we anticipated the absence of Mn in one domain would yield a muta nt enzyme exhibiting 50% of wild type activity (assuming insignificant structural changes and full metal incorporation). This proved not to be the case, however, with OxDC mutants (even those containing up to 0.73 Mn/monomer) exhibiting much lower activity than expected base d on their Mn content (Fi gure 2-1). In fact, the level of activity observed for th e E280Q OxDC mutant was signifi cantly reduced in light of its Mn occupancy even though EPR experiments show ed that Mn coordination by residues in the Nterminal domain was unaffected. Size-exclusi on chromatography also supports the assumption that the E280Q OxDC mutant is correctly folded, and hence this result imp lies that (i) either the C-terminal Mn site mediates cat alysis, or (ii) the N-terminal site catalyzes decarboxylation if, and only if, metal is bound in the C-terminal site Unfortunately for the first of these two hypotheses, the E101D and E101Q OxDC mutant s (in which Mn binding to the N-terminal domain is disrupted) exhibit catalytic activity th at is lower than that observed for wild type OxDC containing an equivalent amount of bound M n. This observation is therefore consistent with decarboxylation being mediated by the N-terminal Mn site, un less activity in the C-terminal site is dependent on the presence of metal in the N-terminal domain. Kinetic simulations in which the activity of one active site is dependent on metal occupancy of the other non-catalytic site, however, do not predict a linear relationshi p between bound Mn and ca talytic activity, with one exception (case 6) in which Mn binding in one site causes a signifi cant increase in Mn affinity of the second site while assuming that both sites have equal affi nities prior to metal binding (Figure 2-2). Although these data rule out the hypo thesis that both Mn binding sites can

PAGE 79

79 independently degrade oxalate, this mutagene sis strategy does not permit us to define the location of the Mn(II) site th at mediates catalysis. Motivation for the Preparation of Single Domain OxDC Mutants As noted in Chapter 1, it has been suggested th at the two domains of oxalate decarboxylase arose from a gene duplication event ( 28, 37, 43-46 ). Two disparate sets of enzymological studies influenced the preparation of single doma in mutants of OxDC. Gerlt and Babbitt ( 136 ) raised the possibility that ( / )8-barrel fold proteins (unrelated to OxDC) may be derived from mixing and matching of ( / )4-half barrels as well as other ( / )8-barrels by divergent evolution. Crystallographic studies ( 137 ) indicated that imidazole glycerol phosphate synthase (HisF) from Thermatoga maritima is a ( / )8-barrel composed of two superimposable domains (HisF-N and HisF-C). To examine the possibility that Hi sF evolved from duplication and fusion from and ancestral half barrel, the Nand C-terminal ( / )4-half barrels of HisF (His-N and HisF-C) were produced in Escherichia coli purified and characterized ( 138 ). Separately, HisF-N and HisF-C are folded proteins, but are catalytica lly inactive. However, coexpression in vivo or joint refolding in vitro resulted in these two domains assembling into a stoichiometric and catalytically active HisF-NC complex ( 138 ). Another example of the expression of singl e domains in the literature was that of E. coli catalase-peroxidase (KatG) ( 139, 140 ), which is composed of two peroxidase-like domains. The N-terminal domain (KatGN) contains the heme-dependent bifunctional site and the C-terminal domain (KatGC) which does not bind heme, has no catalyt ic activity, and is separated from the activie site by 30 KatGN expressed separately possesses neither catalase nor peroxidase activity ( 139, 140 ). However, separately expressed KatGC is able to restructure separately expressed KatGN to its bifunctional conformation ( 141 ).

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80 N-Terminal OxDC Single Domain Mutant (OxDC-N1) Does Not Catalyze the Decarboxylation Reaction This construct begins at the N-terminus and ends at glutamine-233. It includes a beta strand which contributes to the C-te rminal domain (Figure 5-5). Figure 5-5 Topology diagram of Ox DC. Figure adapted from ( 72 ). This mutant did not contain Mn as determined by ICPMS, but did contain up to 1.01 mole Zn/mole of monomer (Table 5-4). It was possi ble, however, to incor porate up to 0.14 moles Mn/mole of monomer (0.45 moles Zn/ mole of monomer remaining) by the method described in Chapter 2 for the preparation of the apoenzyme and reconstitution of wild type recombinant OxDC (Table 5-4). This mutant was not found to possess OxDC activity by the OxDC-FDH linked enzyme assay. A small but detectable am ount of oxalate oxidase activity, however, was detected in the manganese reconstituted sample by the dye oxidation assay described in the Experimental Section.

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81 Table 5-4 Metal content of singl e domain mutant preparations EPR Characterization of Reconstituted N-te rminal OxDC Single Domain Mutant (OxDCN1) Figure 5-6 shows the spectra of OxDC-N1 in 20 mM hexamethylenetetramine (HMTA) HCl buffer, pH6.0, 0.5 M NaCl (storage buffer) (shown in black). The six line spectra is typical of the Mn(II) centers in both the wild-t ype OxDC and in the E280Q mutant. Figure 5-6 Effect of buffer and oxalate on the g 2 X-band Mn(II) signal of reconstituted Nterminal OxDC mutant (OxDC-N1) Sample Spe. Act.Mn Fe Cu Zn Co OxDC-N1 0 U/mg <0.01 <0.01 <0.01 1.01 <0.01 Recon OxDC-N1 0 U/mg 0.14 <0.01 <0.01 0.45 <0.01 OxDC-N1#2 0 U/mg 0.01 <0.04 <0.01 0.74 <0.01 Recon OxDC-N1#2 0 U/mg 0.06 0.02 <0.01 0.65 <0.01 OxDC-C 0 U/mg 0.08 0.02 <0.01 0.33 <0.01

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82 A series of spectra were taken in order to make comparisons with the wild-type enzyme (see Chapters 3 and 4). In an effort to reduce the pH into the range of OxDC activity for the wild-type, the sample was made 50 mM sodium a cetate, pH 5.2. Upon this treatment, some of the protein precipitated and what appeared as the sharp lines of hexaaquo manganese(II) appeared (spectrum not shown). Unlike the wi ld-type enzyme (but similar to the E2809Q mutant data not shown), the spectrum changes ve ry little if at all by the addition of sodium acetate and/or oxalate at pH 6.0. C-Terminal OxDC Single Domain Mutant (OxDC-C) Does Not Catalyze the Decarboxylation Reaction This construct begins with an engineered methione followed by leucine-231 and ends at the end of the wild type protein. Although this construct did contain a small amount of Mn as determined by ICPMS and by EPR at 324 GHz it was subjected to the reconstitution procedure described in Chapter 2 for the wild type full leng th enzyme. These spectra suggest that more Mn is in the purified than the reconstituted sample (no ICPMS data for reconstituted sample). This mutant was not found to possess neither OxDC activity by the OxDC-FDH linked enzyme assay nor oxalate oxidase activity by th e dye oxidation assay. Furthermore, the OxDC-C did not show any signs of acetate or oxalate binding (data not shown). Combining the Nand C-Terminal Single Domain Mutants Did Not Result in Decarboxylase Activity Neither combining reconstituted OxDC-N-1 a nd OxDC-C in storage buffer nor putting the two single domain mutants through the reconst itution procedure togeth er resulted in any detectable decarboxylation activity.

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83 Experimental Section Expression and Purification of Site-Specific OxDC Mutants. All site-specific OxDC mutants were constr ucted using the overlap extension method ( 142 ) and the OxdC:pET-9a plasmid containing the gene coding for Bacillus subtilis OxDC ( 57 ). Thus, primers (Table 5-4) for mutagenesis were designed such that the desired mutation was located at the 5-end. Primers overlapped 10 to 12 bases and in cluded restriction sites to facilitate cloning into pET9a. The 3and 5fragments were am plified independently and a third PCR combined these two fragments to yield the full length gene The resulting products were digested with BamHI and NdeI and cloned into pET9a. Constr ucts were transformed into JM109 competent cells, and transformants screened by restricti on enzyme digestions using BamHI and NdeI. Plasmids containing the desired cl ones were sequenced to confir m PCR fidelity and transformed into BL21(DE3) competent cells. Table 5-5 Primers used in the construction of Mn-binding mutants a NdeI restriction site engineered (residues shown in bold). b BamHI restriction site engineered (residues shown in bold) Enzyme Type Primer Sequence WT OxDC a Forward 5-GGAGGAAAC ATCATATG AAAAAACAAAATG-3 WT OxDC b Reverse 5-GCGGCA GGATCC TTATTTACTGCATTTC-3 E101A Forward 5-GCTGCATGGGCTTATATGA TTTACGG-3 E101A Reverse 5-GCCCATGCAGCTTCTTTATGCCAGTG-3 E101D Forward 5-GCTGACTGGGCTTATATGA TTTACGG-3 E101D Reverse 5-GCCCAGTCAGCTTCTTTATGCTGCCAGTG-3 E101Q Forward 5-GCTCAATGGGCTTATATGA TTTACGG-3 E101Q Reverse 5-GCCCATTGAGCTTCTTTATGCCAGTG-3 E280A Forward 5-CCCACGCATGGCAATACTACATCTCC-3 E280A Reverse 5-GCCATGCGTGGGTATTCGGGTGCC-3 E280D Forward 5-GCTCATTGGGCTTATATGA TTTACGG-3 E280D Reverse 5-GCCCAATGAGCTTCTTTATGCCAGTG-3 E280Q Forward 5-CCCACCAATGGCAATACTACATCTCC-3 E280Q Reverse 5-GCCATTGGTGGGTATTCGGGTGCC-3

PAGE 84

84 Expression of the OxDC mutants wa s carried out as for the wild type enzyme (Chapter 2). After an initial purification using DEAE-Sepharose Fast Flow column chromatography, (Chapter 2), OxDC mutants were preci pitated from 50 mM imidazole -Cl, pH 7.0, containing 1.7 M (NH4)2SO4. The precipitate was then centrifuged (10,000 rpm, 20 min, 4 oC), and re-suspended in 20 mM hexamethylenetetramine-HCl, pH 6.0 c ontaining 0.5 M NaCl to yield solutions of the site-specific OxDC mutants at c oncentrations ranging from 3.5 to 19.6 mg/mL. This abbreviated purification procedure gave mutant enzymes of > 90% purity, as evaluated by SDS-PAGE. Oxalate Oxidase Assays The level of oxalate oxidase activity for wild type OxDC and the series of OxDC mutants at ambient temperatures (21-23 oC), using a continuous assay in which H2O2 production was coupled to the horseradish peroxidase (H RP) catalyzed oxidati on of 2,2-azinobis-(3ethylbenzthiazoline-6-su lphonic acid) (ABTS) ( 129 ). Reaction mixtures contained 25 U HRP, 5 mM ABTS, 50 mM potassium oxalate, wild type OxDC or the metal-binding OxDC mutants (at concentrations up to 0.035 mg/mL) dissolved in 50 mM sodium acetate, pH 4.0 (total volume 1 mL). An extinction coefficient of 10,000 M-1 cm-1 for the ABTS radical product was assumed in these experiments. Control samples omitted HRP so as to differentiate between H2O2 production and any oxalate-dependent dye oxidation activity by w ild type OxDC or the OxDC mutant. Size-Exclusion Chromatography Measurements The oligomeric state of the wild type enzyme was compared with that of the metal-binding mutants by size exclusion chromatography us ing a BIOSEP-S2000 column (300 x 7.8 mm with 75 x 7.8 mm guard column) (Phenomenex, Torrance, CA) was equilibrated with 20 mM hexamethylenetetramine hydrochloride, pH 6.0, containing 0.5 M NaCl (buffer C), and calibrated using carbonic anhydr ase (29.0 kDa), bovine serum albumin (66.0 kDa), alcohol dehydrogenase (150 kDa), -amylase (200 kDa), apoferritin (443 kDa), and thyroglobulin (669

PAGE 85

85 kDa). The void volume was measured by injecting blue dextran. Samples of recombinant, wild type OxDC or the site-specific OxDC mutants were then inject ed onto the column and eluted with buffer C, at a flow rate of 1 mL/min with UV detection at 280 nm, to assign the oligomeric form of the enzyme. Circular Dichroism Studies Recombinant, wild type OxDC was dialyz ed into 25 mM potassium phosphate, pH 7.0 containing 100 mM NaCl, and th e protein concentration adjust ed to a final value of 185 g/mL. A similar procedure was performed for all 9 site -specific OxDC mutants. In cases where the protein precipitated (8/10 sample s), the precipitate was remove d by microcentrif ugation, The CD spectrum of the protein was then obtained usi ng an Aviv 215 spectrometer (Aviv Associates, Lakewood, NJ) at wavelengths over the range of 190-250 nm (1 mm path length). All spectra were corrected by subtracting the CD spectrum of the buffer over this range of wavelengths. Electron Paramagnetic Resonance Spectroscopy EPR spectra were determined using samples of wild type OxDC (12.3 mg/mL) or the E280Q OxDC mutant (16.8 mg/mL) dissolved in 20 mM hexame thylenetetramine-HCl, pH 6.0, containing 0.5 M NaCl (100 L total volume). The metal contents of the wild type and mutant enzymes were 1.63 and 0.73 Mn/monomer, respectively. All high-field EPR experiments were performed using a custom-built spectrometer operating in transmission mode ( 124 ). Far IR radiation was generated by a Gunn source at W-band (94-97 GHz or 105-110 GHz), which was frequency tripled and/or quadrupl ed to achieve frequencies of 320 or 380 GHz with a radiation power of 2-10 mW, and transmitted through an ov ersized waveguide so as to pass through the sample once before being detected by an InSb hot-electron bolometer (QMC Instruments Ltd, Cardiff, UK). The analog signal from the bolom eter was fed into a Stanford Instrument SR830 lock-in detector, which was referenced to the fi eld modulation at the sample. The magnetic field

PAGE 86

86 sweep was carried out by either sweeping the ma in coil or a custom-built auxiliary 1000 G sweep coil. Field calibration was performed usi ng a piece of P-doped silicon, which has a g -value of 1.99854 and a hyperfine coupling constant of 117.507 MHz ( 143 ). Spectral simulations were done with the EasySpin toolbox ( 121 ) in the MATLAB computing environment (The MathWorks, Natick, MA), and with in-house pr ograms written Andrew Ozarowski. The timedependent EPR spectra required for the relaxation rate studies were taken with a Bruker Elexsys E580 pulse/cw spectrometer equipped with a 5 mm Bruker Flexline dielectric resonator. The Flexline resonator and the samples were cooled using cold helium gas in an Oxford CF935 cryostat, and the temperature during acquisition was controlled with an ITC4 temperature controller and a VC40 gas flow c ontroller (Oxford Instruments, Eynsham, UK). Standard pulse sequences were employed in these experiments ( 144 ). Expression of OxDC Single Domain Mutants Three single domain mutants were inde pendently amplified from pET9a plasmid containing the gene coding for Bacillus subtilis OxDC ( 57 ). 1) OxDC-N1 (YD1) was amplified using primers OxdC fwd and Domain-1 rev (Table 43). This construct begins at the N-terminus and ends at glutamine-233. It includes a be ta strand which contribut es to the C-terminal domain. 2) OxDC-N2 (DD1) was amplified usi ng primers Domain-1 fwd and Domain-1 reverse (Table 3-6). This construct does not contain the N-terminal beta strand that contributes to the Cterminal domain. It begins w ith an engineered methionine fo llowed by serine-53 and ends at glutamine-233. 3) OxDC-C was amplified usin g primers Domain-2 fwd and OxdC rev. This construct begins with an engine ered methione followed by leucine231 and ends at the end of the wild type protein. The resulting products were digested with BamHI and NdeI and cloned into the pET9a plasmid. Constructs were transforme d into JM109 competent cells, and transformants were screened by restriction en zyme digestions using BamHI a nd NdeI. Plasmids containing the

PAGE 87

87 desired clones were sequenced to confirm PCR fidelity and were transformed into BL21(DE3) competent cells. Expression of the OxDC mutant s carried out as for the wild type enzyme. Table 5-6 Primers used in the preparation of OxDC single domain mutants Purification of Single Domain OxDC Mutant OxDC-N1 Expression of OxDC-N1 was confirmed by co mparing cell lysates before and after induction by 12% SDS PAGE. The appearance of a band in the induced cells at the calculated molecular weight of 26.4 kd (http://www.scripps.edu/cgibin/cdputnam/protcalc3 ) confirmed expression. Cells were ly sed, extracted, and purified by DEAE column chromatography as described for recombinant wild-type OxDC. Fr actions containing OxDC-N1 as determined by electrophoretic mobility were pooled and dialyzed for 4 h against 50 mM imidazole-HCl buffer, pH 7.0 (2 L). The resulting sample was then applied to a Q-Sepharose Hi-Performance column (2.5 x 18 cm) column equilibrated with 50 mM imidazole-HCl buffer, pH 7.0, and eluted using a 500 mL linear gradient from the column buffer to the same containing 1 M NaCl. Fractions containing OxDC were pooled and e xhaustively dialyzed against 20 mM hexamethylenetetramine hydrochloride, pH 6.0, containing 0.5 M NaCl. Purified OxDC-N1 (> 90 % as determined by SDS PAGE) was concentr ated to 5.3 mg/mL and stored as at -80 oC. Primer Name Primer sequence OxdC fwd 5-GGAGGAAACATCATATG AAAAAACAAAATG-3 OxdC ** rev 5-GCGGCAGGATCC TTATTTACTGCATTTC-3 Domain-1 fwd 5-GGAGGAAACATATGTCTGATACTCATAACC-3 Domain-1 rev 5-GCGGCAGGATCCCTATTGTTCAAGAAGGCG-3 Domain-2 fwd 5-TTTACTTACCATATGCTTGAACAAGAGCCG-3 NdeI restriction site engineered (underlined) ** BamHI restriction site engineered (underlined)

PAGE 88

88 Purification of Single Domain OxDC Mutant OxDC-C Expression of CTD was confirmed by compari ng cell lysates before and after induction by 12% SDS PAGE. The appearance of a band in th e induced cells at the calculated molecular weight of 17.7 kd (http://www.scripps.edu/cgibin/cdputnam/protcalc3 ) confirmed expression. OxDC-C was purified (>90% purity as dete rmined by SDS PAGE) by the method described above for OxDC-N1.

PAGE 89

89 CHAPTER 6 CONCLUSIONS AND FUTURE WORK We have demonstrated a linear dependence of oxalate decarboxylas e specific activity on the Mn incorporation. This obser vation is consistent with only th ree of the seven kinetic models studied. The simplest model is that both Mn-binding sites are catalyt ically active. If the sites are also independent, the absence of Mn in one dom ain would yield a mutant enzyme with 50% of wild type activity (assuming insi gnificant structural changes a nd full metal incorporation). OxDC Mn-binding mutants, however, exhibited much lower activity than expected based on their Mn content (Figure 2-1). For example, the level of activ ity observed for the E280Q OxDC mutant was significantly reduced in light of its Mn occupa ncy (0.73 Mn/monomer) even though EPR experiments showed that Mn coordinati on by residues in the N-terminal domain was unaffected. Size-exclusion chroma tography is consistent with the assumption that the E280Q OxDC mutant is correctly folded. This result im plies that (i) either the C-terminal Mn site mediates catalysis, or (ii) the N-terminal site catalyzes decarboxylation if and only if, metal is bound in the C-terminal site. That the E101D and E101Q OxDC mutants (in which Mn binding to the N-terminal domain is disrupted) exhibit catalytic activity that is lower than that observed for wild type OxDC containing an equivalent amount of bound Mn argues against the first of these two hypotheses. This observation is ther efore consistent with decarboxylation being mediated by the N-terminal Mn si te, unless activity in the C-term inal site is dependent on the presence of metal in the N-terminal domain. Kinetic simulations in which the activity of one active site is dependent on metal occupancy of the other non-catal ytic site, however, do not predic t a linear relationship between bound Mn and catalytic activity, with one exception (case 6) in which Mn binding in one site causes a significant increase in Mn affinity of the second site wh ile assuming that both sites have

PAGE 90

90 equal affinities prior to metal binding (Figure 2-3). Although th ese data rule out the hypothesis that both Mn binding sites can independently de grade oxalate, this mutagenesis strategy does not permit us to define the location of the Mn(II) site that mediates catalysis. A multi-frequency EPR approach has allowed us to spectroscopically distinguish two Mn(II) species that are present in equal proportions in the resti ng state of oxalate decarboxylase in storage buffer. The main difference between thes e two species is the valu e of the fine structure parameters with DI = 1200 MHz and DII = 2700 MHz. When the enzyme is placed in acetate buffer pH5.2 or when formate is added, DII is reduced to 2150 MHz while DI remains the same indicating that only one Mn(II) is solvent accessible. Based on published crystal structure data, we suggest site I is the C-terminal Mn site whil e site II is the solvent-exposed N-terminal site and, therefore, the site of small mo lecule (acetate and formate) binding. It would be of interest in terms of th e catalytic mechanism to determine the redox properties of OxDC. The observation that the Mn(II) EPR signal can be decreased with the addition of sodium (meta) pe riodate and potassium hexachlo roiridate with a concomitant appearance of a carbon-based radica l should be explored further a nd is significant in that it demonstrates that oxidants can reach the manganese ions and that potentiometric titrations can be carried out on OxDC. It would also be of intere st to use an oxygen electrode to characterize the oxygen dependence of the bacterial form of OxDC. Questions about the binding of substrate to the Mn-binding site(s) could be addressed by cr ystallographic structure solution of the Co substituted enzyme in the presence of oxalate.

PAGE 91

91 APPENDIX A KINETIC PARAMETERS USED IN GEPASI SIMULATIONS Case 1 (site 2 unimportant or inactive)

PAGE 92

92 Case 1 (site 2 unim portant or inactive)

PAGE 93

93 Case 2 (site 1 most active)

PAGE 94

94 Case 3 (site 2 require d, site 1 is active)

PAGE 95

95 Case 4 (sites 1 and 2 have equal activity)

PAGE 96

96 Case 5 (site 2 required, fully occu pied enzyme twice as active)

PAGE 97

97 Case 6 (site 1 active, cooperative binding)

PAGE 98

98 Case 7 (only full enzyme ac tive, cooperat ive binding)

PAGE 99

99 APPENDIX B SIMULATIONS OF EPR SPECTRA AT DIFFERENT FIELD/FREQUENCY COMBINATIONS OF OXALATE DECARB OXYLASE IN STORAGE BUFFER Figure B-1 X-band spectrum of OxDC in SB pH 6.0 at T = 10 K. Experimental spectrum is shown in black. Simulations for sites I and II are shown in blue and green, respectively. The sum of the simulation of the two sites is shown in red.Experimental parameters: Microwave frequency 9.48731 GHz, microwave power 0.64 mW, modulation frequency 100 kHz, modulation amplitude 10 G, receiver gain 60 dB, time constant 41 ms, conversion time 41 ms, 1 sweep, 1.465 G/data point.Simulation parameters for site I: giso = 2.000865, Aiso = 254 MHz, D = 1200 MHz, E = 252 MHz, D-Strain = 0.24D, E-Strain = 0.24E, lin ewidthiso = 33 MHz.Simulation parameters for site II: giso = 2.00094, Aiso = 248 MHz, D = 2750 MHz, E = 660 MHz, D-Strain = 0.20D, E-Strain = 0.20E, linewidthiso = 33 MHz.

PAGE 100

100 Figure B-2 V-band spectrum of OxDC in SB pH6.0 at T = 20 K. Experimental spectrum is shown in black. Simulations for sites I and II are shown in blue and green, respectively. The sum of the simulation of the two sites is shown in red.Experimental parameters: Microwave frequency 49.200 GHz, microwave power corresponding to a dector signal of 500 mV, modulation fre quency 41.68 kHz, modulation amplitude 4 G, lock-in sensitivity 200 V, time constant 300 ms, sweep speed 1 G/s, 1 sweep, 0.250 G/data point, center field 1.753 T.Si mulation parameters for site I: giso = 2.000865, Aiso = 254 MHz, D = 1200 MHz, E = 276 MHz, D-Strain = 0.24D, EStrain = 0.30E, linewidthiso = 33 MHz. Simulation parameters for site II: giso = 2.00094, Aiso = 248 MHz, D = 2700 MHz, E = 675 MHz, D-Strain = 0.25D, EStrain = 0.20E, linewidthiso = 33 MHz.

PAGE 101

101 Figure B-3 W-band EPR spectrum of OxDC in SB pH6.0 at T = 50 K. Experimental spectrum is shown in black. Simulations for sites I and II are shown in blue and green, respectively. The sum of the simulation tr aces of the two sites is given in red. Experimental parameters: Microwave frequency 94.02141 GHz, microwave power 0.6 W modulation frequency 100 kHz, modula tion amplitude 2 G, receiver gain 40 dB, time constant 164 ms, conversion time 164 ms, 1 sweep, 1.172 G/data point. Simulation parameters for site I: giso = 2.000865, Aiso = 254 MHz, D = 1200 MHz, E = 252 MHz, D -Strain = 0.24 D E -Strain = 0.24 E linewidthiso = 33 MHz. Simulation parameters for site II: giso = 2.00094, Aiso = 248 MHz, D = 2700 MHz, E = 648 MHz, D -Strain = 0.20 D E -Strain = 0.20 E linewidthiso = 33 MHz.

PAGE 102

102 Figure B-4 Sub-mm EPR spectrum of OxDC in SB pH6.0 at T = 20 K and 222 GHz. Experimental spectrum is shown in black. Simulations for sites I and II are shown in blue and green, respectively. The sum of the simulation of the two sites is shown in red. Experimental parameters: Micr owave frequency 222.400 GHz, modulation frequency 41.8 kHz, modulation amplitude 0.5 G, lock-in sensitivity 500 V, time constant 100 ms, sweep speed 5.01 G/s, 1 sweep, 0.368 G/data point. Simulation parameters for site I: giso = 2.000865, Aiso = 251 MHz, D = 1200 MHz, E = 252 MHz, D -Strain = 0.24 D E -Strain = 0.24 E linewidthiso = 33 MHz. Simulation parameters for site II: giso = 2.00094, Aiso = 247 MHz, D = 2700 MHz, E = 675 MHz, D -Strain = 0.20 D E -Strain = 0.20 E linewidthiso = 33 MHz.

PAGE 103

103 Figure B-5 Sub-mm EPR spectrum of OxDC in SB pH6.0 at T = 20 K and 324 GHz. Experimental spectrum is shown in black. Simulations for sites I and II are shown in blue and green, respectively. The sum of th e simulation traces of the two sites is given in red. Experimental para meters: Microwave frequency 324.00 GHz, modulation frequency 41.8 kHz, modulation amplitude 0.5 G, lock-in sensitivity 50 V, time constant 100 ms, sweep sp eed 5.01 G/s, 1 sweep, 0.367 G/data point.Simulation parameters for site I: giso = 2.000865, Aiso = 250 MHz, D = 1200 MHz, E = 252 MHz, D -Strain = 0.24 D E -Strain = 0.24 E linewidthiso = 33 MHz.Simulation parameters for site II: giso = 2.00094, Aiso = 247 MHz, D = 2700 MHz, E = 675 MHz, D -Strain = 0.20 D E -Strain = 0.20 E linewidthiso = 33 MHz.

PAGE 104

104 Figure B-6 Sub-mm EPR spectrum of OxDC in SB pH6.0 at T = 20 K and 412.8 GHz. Experimental spectrum is shown in black. Simulations for sites I and II are shown in blue and green, respectively. The sum of th e simulation traces of the two sites is given in red. Experimental parame ters: Microwave frequency 412.800 GHz, modulation frequency 41.8 kHz, modulation amplitude 4 G, lock-in sensitivity 500 V, time constant 100 ms, sweep speed 5.01 G/s, 1 sweep, 0.369 G/data point. Simulation parameters for site I: giso = 2.000865, Aiso = 253 MHz, D = 1200 MHz, E = 252 MHz, D -Strain = 0.24 D E -Strain = 0.24 E linewidthiso = 33 MHz.Simulation parameters for site II: giso = 2.00093, Aiso = 249 MHz, D = 2700 MHz, E = 675 MHz, D -Strain = 0.25 D E -Strain = 0.25 E linewidthiso = 33 MHz.

PAGE 105

105 APPENDIX C HIGH FIELD SPECTRA AND SIMULATIONS OF WT OXDC AND THE E280Q MUTANT Figures C-1, C-2, and C-3 show the results of th e best fits obtained for simulations of the EPR spectra obtained for wild type OxDC and the E280Q mutant. All simulations were performed with the Easy Spin toolbox ( 121 ) in the MATLAB computing environment (The MathWorks, Natick, MA) by Dr. Ines Garcia-Rubi o at ETH-Zurich. We assumed isotropic gand A-tensors while using an anisotropic fine stru cture tensor, and the fits improved considerably by choosing a mixture of Lorentzian and Gaussian lineshapes. Ni ne independent fit parameters were used for the main Mn(II) species in each spectrum. Given that only the region around g~2 was measured in these experiments, fine structure values and associated strain parameters have a large uncertainty (conservatively estimated to be 50% of the fit values). On the other hand, the hyperfine coupling constant could be extracted fro m the first with more confidence. Changing A by 1 MHz considerably worsened agreement between the simulated and the experimental spectrum, and, similarly, the margin of error for th e isotropic g-value is sm all being estimated as approximately 0.00001. Apparent linewidths are somewhat depe ndent on the choice of D and E, although reducing both fine stru cture constants to almost zero only reduces the linewidth by a few Gauss (0.85 mT and 1.1 mT for E280Q and wild-type enzyme respectively), and these simulations suffer from lack of accuracy in the wings of the six lines.

PAGE 106

106 Figure C-1 High field EPR of wild-type Ox DC enzyme at 386.116 GHz and 10 K. The experimental and simulated spectra are di splayed in blue and red, respectively. Simulation parameters for the majority Mn(II) component ar e: g-factor = 2.00087, D = 1200 MHz, E = 240 MHz, D-Strain: 40% of D, E-Strain: 40% of E, hyperfine coupling, A: 250 MHz, A-Strain: 1% of A, linewidth: 1.5 mT, lineshape: 90% Lorentzian, 10% Gaussian. A minority co mponent was assumed to be present to explain the low field shoulders on the main six-line spectrum, contributing approximately 4% of the spectral intensit y, with simulation parameters: g-factor = 2.00107, D = E = 0 MHz, hyperfine coupling, A: 245 MHz, linewidth: 1.8 mT, lineshape: 100% Lorentian.

PAGE 107

107 Figure C-2 High field EPR of E 280Q OxDC mutant at 331.2 GHz and 20 K. The experimental and simulated spectra are displayed in blue and red, respectively. Simulation parameters for the majority Mn(II) co mponent are: g-factor = 2.00087, D = 850 MHz, E = 85 MHz, D-Strain: 40% of D, E-Stra in: 40% of E, hyperf ine coupling, A: 255 MHz, A-Strain: 2% of A, linewidth: 0.9 mT, lineshape: 50% Lorentzian, 50% Gaussian. A minority component was assumed to be present to explain the low field shoulders on the main sixline spectrum, contributing approximately 7.5% of the spectral intensity, with simulation parame ters: g-factor = 2.00109, D = E = 0 MHz, hyperfine coupling, A: 242 MHz, linewidth: 1.8 mT, lin eshape: 100% Lorentian.

PAGE 108

108 Figure C-3 High field EPR of E280Q OxDC mutant at 382.826 GHz and 10 K. The experimental and simulated spectra are di splayed in blue and red, respectively. Simulation parameters for the majority Mn(II) component ar e: g-factor = 2.00087, D = 850 MHz, E = 85 MHz, D-Strain: 40% of D, E-Strain: 40% of E, hyperfine coupling, A: 253 MHz, A-Strain: 1% of A, linewidth: 1.0 mT, lineshape: 80% Lorentzian, 20% Gaussian. A minority co mponent was assumed to be present to explain the low field shoulders on the main six-line spectrum, contributing approximately 7.5% of the spectral intensit y, with simulation parameters: g-factor = 2.00111, D = E = 0 MHz, hyperfine coupling, A: 240 MHz, linewidth: 1.8 mT, lineshape: 100% Lorentian. This spectrum taken at 382 GHz did not have a field standard, and to simulate the 382 GHz spectru m, the field axis was adjusted to yield the same isotropic g-factor in the simulati on in essence using the six-line spectrum as the field standard for that particular frequency made possible using the calibrated spectrum taken for the E280Q OxDC mutant at 331 GHz.

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117 BIOGRAPHICAL SKETCH Ellen Moomaw was born in Jacksonville, FL in 1960. After earning a masters of science degree in biochemistry in the laboratory of Dr. Dale Edmondson (Emory University, 1984), she worked in various biotechnology companies in San Diego. In 1987 Ellen was the 30th employee hired at a young company called Agouron Phar maceuticals, Inc. where she purified and characterized a number of protei ns including thymidylate syntheta se, HIV reverse transcriptase and RNase H, DNA polymerase b, and HCV proteas e. By the time she left Agouron in 1999 to teach high school chemistry, Agouron (now part of Pfizer) had over 1200 employees and had gotten the 4th HIV protease inhibitor on the market (Vir acept). Ellen started graduate studies in the chemistry department of the University of Florida in 2003, where sh e joined the research group of Dr. Nigel G. J. Richards.