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Kinetic and Structural Effects of Interfacial Interruption and Protein Nitration in Human Manganese Superoxide Dismutase

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PAGE 1

KINETIC AND STRUCTURAL EFFECTS OF INTERFACIAL INTERRUPTION AND PROTEIN NITRATION IN HUMAN MANG ANESE SUPEROXIDE DISMUTASE By PATRICK QUINT 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 2006

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Copyright 2006 by PATRICK QUINT

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iii ACKNOWLEDGMENTS Sincere thanks go to my parents, brothers and sisters, and all those with whom I have shared the joy and frustration of this work. However, this di ssertation would not have been possible without the patient enc ouragement of my wife Ingvild, the impish smile of my son Anders or the expert mentorship of Drs. Silverman and McKenna.

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iv TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES.............................................................................................................vi LIST OF FIGURES..........................................................................................................vii ABSTRACT....................................................................................................................... ix CHAPTER 1 INTRODUCTION........................................................................................................1 Superoxide Formation and Reactions...........................................................................1 Classes of Superoxide Dismutase.................................................................................2 Human Manganese Superoxide Dismutase..................................................................2 Active Site and Hydrogen Bond Network.............................................................2 Manganese Superoxide Dismutase a nd its Reaction with Superoxide..................3 The Subunit Interfaces of Human Ma nganese Superoxide Dismutase.................4 Peroxynitrite.................................................................................................................4 Tyrosine Nitration.........................................................................................................5 Research Goals.............................................................................................................6 Interfacial Mobility at the Dimeric and Tetrameric Interfaces of Human Manganese Superoxide Dismutase....................................................................7 Replacement of a Key Dimeric Interfacial Residue..............................................7 Structure of Nitrated Human MnSOD...................................................................8 2 STRUCTURAL MOBILITY IN HUMAN MANGANESE SUPEROXIDE DISMUTASE...............................................................................................................9 Introduction................................................................................................................... 9 Materials and Methods...............................................................................................11 Labeling with Fluorotyrosine, Expres sion in E. coli, and Purification...............11 Site-Directed Mutagenesis of Fluorin e Manganese Superoxide Dismutase.......12 19F Nuclear Magnetic Resonance Spectroscopy................................................13 Differential Scanning Calorimetry......................................................................14 Results........................................................................................................................ .14 Assignment of Fluorine Resonances...................................................................14 Thermal Stability.................................................................................................17 Temperature Dependence of Fluorine Resonances.............................................17

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v Discussion...................................................................................................................19 3 ROLE OF A GLUTAMATE BRID GE SPANNING THE DIMERIC INTERFACE OF HUMAN MANGANESE SUPEROXIDE DISMUTASE............24 Introduction.................................................................................................................24 Materials and Methods...............................................................................................25 Results........................................................................................................................ .29 Discussion...................................................................................................................37 4 STRUCTURE OF NITRATED HU MAN MANGANESE SUPEROXIDE DISMUTASE.............................................................................................................45 Introduction.................................................................................................................45 Materials and Methods...............................................................................................47 Preparation of Nitr ated Human MnSOD.............................................................47 Crystallization......................................................................................................48 Data Collection and Processing...........................................................................48 Structure Determination and Refinement............................................................49 Results........................................................................................................................ .50 Discussion...................................................................................................................53 5 CONCLUSIONS AND FUTURE DIRECTIONS.....................................................58 Conclusions.................................................................................................................58 The Tetrameric Interface in Human MnSOD......................................................58 The Dimeric Interface and Different ial Roles of Glu162 in Human MnSOD and Glu170 in E. coli MnSOD.........................................................................59 A Structural Explanation for Abolished Catalysis of Nitrat ed Human MnSOD.60 Future Directions........................................................................................................60 The Dimeric Interface of Human MnSOD..........................................................60 Redox properties of E162 mutants......................................................................61 Catalytic Properties of Nitrated MnSOD............................................................61 Future of Therapeutic Studies.............................................................................61 LIST OF REFERENCES...................................................................................................63 BIOGRAPHICAL SKETCH.............................................................................................71

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vi LIST OF TABLES Table page 3-1 Maximal rate constants for the catalysi s and inhibition of human wild type MnSOD and mutants................................................................................................33 3-2 Diffraction data and refinement st atistics for human E162D and E162A MnSOD. ..................................................................................................................36 3-3 Maximal values for kcat/Km and k0/[E] for the catalysis of human wild-type MnSOD and mutants................................................................................................40 4-1 X-ray crystallographic stru cture statistics of unmodifi ed and nitrated human MnSOD ..................................................................................................................53 4-2 Distance geometries () in the active-si tes of unmodified and nitrated human MnSOD ..................................................................................................................54

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vii LIST OF FIGURES Figure page 1-1 Hydrogen-bond network of human MnSOD. Shown in cyan are intervening residues from the adjacent, non-crystallographic subunit..........................................3 1-2 Tetrameric human MnSOD. The dimeric interface is shown in green and blue and the tetrameric interface is shown in blue and orange. ........................................6 2-1 Structure of human MnSOD showing the dimeric and tetrameric interfaces..........10 2-2 19F NMR spectrum (470 MHz) of wild-type human MnSOD in which all nine tyrosines were replaced with 3-fluorotyrosine.........................................................16 2-3 Stacked fluorine NMR spectra for wild -type human MnSOD in which tyrosines 9, 11, 34, 45, 166, 176 and 193 have been mutated to phenlylalanine....................16 2-4 The temperature dependence of five 19F chemical shifts of wild-type human MnSOD in which all tyrosine residues are replaced with 3-fluorotyrosine.............18 2-5 The temperature dependence of 19F linewidths at half height for wild-type human MnSOD in which all tyrosine residues are replaced with 3fluorotyrosine...........................................................................................................18 2-6 H/D exchange map for human MnSOD...................................................................20 3-1 Active-site structure including the hydr ogen bond network for human wild-type MnSOD (Borgstahl et al., 1992)..............................................................................25 3-2 pH profile for molar absorptivity at 480 nm for human wild-type Mn3+SOD, 162D Mn3+SOD and E162A Mn3+SOD...................................................................30 3-3 Change in absorbance at 420 nm over a millisecond timescale after generation of 5.6 M superoxide....................................................................................................32 3-4 pH profile for k1 ( ), k2 ( ), and ( ) k3 in catalysis of the disproportionation of superoxide by E162D MnSOD................................................................................34 3-5 pH profile for Kcat/Km for E162D MnSOD.............................................................34 3-6 Structure of E162D MnSOD (shown in green) superimposed on wild-type human MnSOD (shown in blue) in th e region of the mutated residue....................35

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viii 3-7 Structure of E162A MnSOD (shown in green) superimposed on wild-type human MnSOD (shown in blue) in th e region of the mutated residue....................37 3-8 Structure of E. coli MnSOD (green) superimposed on wild-type human MnSOD (blue) in the dimeric interface..................................................................................43 4-1 Scheme for nitration of tyrosine in the presence of peroxynitrite showing nitration through the pathway of equation 2............................................................46 4-2 Structure of the active site of the nitrated human MnSOD. ....................................52 4-3 The structure of the active-site region of nitrated (yellow) superimposed onto unmodified human MnSOD (green)........................................................................52

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ix 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 KINETIC AND STRUCTURAL EFFECTS OF INTERFACIAL INTERRUPTION AND PROTEIN NITRATION IN HUMAN MANG ANESE SUPEROXIDE DISMUTASE By Patrick Quint August 2006 Chair: David Silverman Major Department: Biochemi stry and Molecular Biology This dissertation investigated interfacial attributes of human manganese superoxide dismutase (MnSOD), a homotetramer with two structurally unique interfaces. A conserved dimeric interface is formed from the junction of two subunits and the tetrameric interface is formed from the interaction of two dimer pairs. Using 19F NMR, observation of chemical shift and linewidth changes for fluor ine-labeled tyrosines in the dimeric and tetrameric interfaces of human MnSOD (Tyr169 and Tyr45 respectively) indicated greater rigidity in th e dimeric interface compared to the tetrameric interface. Replacement of the dimeric, interfacial resi due Glu162 with aspartate or alanine did not significantly alter the crystal structure of human MnSOD, though an interaction with a histidine ligand of the activ e site manganese was truncat ed. This interaction was mediated in E162D by an intervening solven t molecule. Catalytic activity for E162D and E162A was 5-20% that of wild-type enzy me and differential scanning calorimetry indicated a role for E162 in thermal stabilit y. Nitration of Tyr34, a dimeric, interfacial residue involved in a hydroge n-bond network emanating from the active-site manganese, abolishes activity in human MnSOD. In v itro nitration of human MnSOD yielded 74%

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x nitration of Tyr34 with moderate nitration of other aromatic residues. A 2.4 structure of nitrated MnSOD aligned well with wild type though an NO2 group covalently linked to Tyr34 is observed. The struct ure of nitro-MnSOD indicates that alteration of the hydrogen-bond network as well as steric blockade and electros tatic repulsion of substrate all account for the loss in catalytic activity associated with nitration. Taken together, these findings provide a role for the tetram eric interface in stability and the dimeric interface in catalysis. In addi tion, this dissertation provides a structural explanation for abolished catalytic activity associated w ith nitration of Tyr34 in MnSOD. Future therapeutic studies on this enzyme will i nvolve the study of resi dues that form the dimeric interface of MnSOD.

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1 CHAPTER 1 INTRODUCTION Superoxide Formation and Reactions Superoxide is an oxygen radical formed from the reaction of free electrons with O2 to form O2 -. There are several enzymatic sources of superoxide including xanthine oxidase, NADPH-oxidase within phagocytes, and other oxidases and an important endogenous source of superoxide are the mito chondria. Leaks in the electron transport chain allow for the addition of a single electron to O2 to form O2 .(Kalra et al. 1994; Haliwell, 1995). Though superoxide is itself toxi c to the cell, its prim ary mode of damage to cellular structures occurs through the Ha ber-Weiss reaction to fo rm the highly unstable hydroxyl radical (Haber and Weiss, 1934). In addition, superoxide reacts with nitric oxide to form the nitrating agent peroxynitrite, which dissoci ates to form hydroxyl radical and nitrite radical (Beckman et al., 1990; B eckman et al., 1992). Superoxide and its breakdown products can cause damage to several biomolecules including lipids, DNA and proteins and can affect their normal f unction. Its reaction with proteins, lipids and DNA has implicated superoxide in several pathological states in cluding reperfusion injury (Becker, 2004), degenerative diseases like amyotrophic lateral sclerosis and muscular dystrophy and has been implicated in damage induced aging of cells (Harman, 1956). To avoid the deleterious effects of th e superoxide anion a nd hydroxyl radical, cells have evolved a mechanism to scavenge and catalyze the disproportionation of superoxide.

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2 Classes of Superoxide Dismutase Organisms that thrive in aerobic environm ents utilize superoxide dismutase (SOD) to scavenge and detoxify super oxide radicals. There are four isoforms of SOD that utilize for catalysis the metals copper and zi nc, manganese, iron, and nickel. A copper containing protein with dismutase activity was first discovered in 1968 by McCord and Fridovich and a year later id entified as a copper-containi ng SOD (McCord and Fridovich, 1968; McCord and Fridovich, 1969). The sa me group reported the discovery of a structurally unique SOD in E. coli that utilized manganese in its active site (Keele, McCord and Fridovich, 1970). It was later di scovered that both Cu/ZnSOD and MnSOD are utilized by eukaryotes, t hough their localizations are diffe rent; Cu/ZnSOD is localized primarily in the cytosol while MnSOD is localized exclusively within the innermitochondrial matrix (Weisinger and Fr idovich, 1973). Its localization in the mitochondrial matrix suggests a role for MnSOD in the protection of mitochondrial DNA, lipids and proteins. Eukaryotic organism s also express an extracellular SOD which utilizes copper and zinc for catalysis (M arklund, 1982). Like eukaryotic MnSOD, it is tetrameric in solution. Two other classes of SO D also exist: FeSOD, which is structurally similar to MnSOD with an id entical active-site structure (Slykhouse and Fee, 1976) and the structurally unique NiSOD (Chodhury et al,. 1999; Barondeau et al., 2004). Human Manganese Superoxide Dismutase Active Site and Hydrogen Bond Network Human manganese superoxide dismutase (MnSOD) is a homotetramer of 22 kDa subunits that catalyzes the dispropo rtionation of superoxide into O2 and H2O2 (Fridovich et al., 1989). Localized in the mitoc hondria, MnSOD is an enzyme with kcat/Km = 8 x 108 M-1 sec-1 (Hsu et al., 1996). Structural studies done by Borgstahl et al. in 1992 reveal a

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3 trigonal, bipyramidal geometry about the active site manganese composed of three histidines (His26, 74 and 163), one aspartate (Asp159) and one Figure 1-1 Hydrogen-bond networ k of human MnSOD. Shown in cyan are intervening residues from the adjacent, non-crystallographic subunit. solvent molecule (Figure 1-1). A hydrogen bond network extends from the active site metal to the coordinating solvent molecule through Gln143 to Tyr34, through a solvent to His30, and finally to Tyr166 from the adjacent subunit (Figure 1-1). Manganese Superoxide Dismutase a nd its Reaction with Superoxide Human MnSOD catalyzes th e rapid disproportionation of superoxide through a two-step reaction in which the active-site ma nganese is first reduced and then oxidized with formation of products O2 and H2O2 (see equation 1 and 2). Mn(III)(OH)SOD + O2 .+ H+ k1 Mn(II)(H2O)SOD + O2 (1) Mn(II)(H2O)SOD + O2 .+ H+ k2 Mn(III)(OH)SOD + H2O2 (2) Mn(II)(H2O)SOD + O2 .k3 Mn(III)(X)SOD (3) Mn(III)(X)SOD + H+ + H2O k4 Mn(III)(OH)SOD + H2O2 (4)

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4 The scheme shown above indicates that the coordinated solvent of MnSOD is protonated upon reduction (Eq. 1) (Miller et al., 2003) and that one proton is donated from the coordinating solvent to form product H2O2 (Eq. 2). Catalysis is further complicated by the reversible formation and dissociation of a produc t inhibited complex, shown in equations 3 and 4 (McAdam et al., 197 7; Bull et al., 1991). It is speculated that inhibition of the enzyme occurs th rough the oxidative addition of O2 .to Mn(II), shown in equation 3, forming a peroxo-bound complex of Mn (III) with the revers e of this reaction, shown in equation 4, yielding activ e enzyme (Bull et al., 1991). The Subunit Interfaces of Human Manganese Superoxide Dismutase Human MnSOD contains two interfaces, th e dimeric interface, conserved between prokaryotes and eukaryotes, and the tetramer ic interface generally associated with the eukaryotic enzyme (Figure 1-2) (Borgsta hl et al., 1992). The dimeric interface is composed of several residues that participate in hydroge n bond interactions and the location of the hydrogen bond network (Fig 1-1 and Fig 1-2). The tetr americ interface is formed from the dimerization of dimers in the eukaryotic enzyme, creating a novel fourhelix bundle, first described by Borgstahl et al. in 1992. The role of the tetrameric interface is unknown, though it likely plays a ro le in stabilizing the enzyme, thus providing an evolutionary advantage. The tetrameric interface does not appear to participate in catalysis. The scheme shown a bove indicates that the coordinating solvent of MnSOD is protonated upon reduction (Eq. 1) (Miller et al., 2003) and that one proton is donated from the coordinating solvent to form product H2O2 (Eq. 2). Peroxynitrite An important mechanism for catalytic i nhibition is through th e reaction of MnSOD with peroxynitrite. Peroxynitrite is a nitrati ng agent formed from the diffusion-controlled

PAGE 15

5 reaction of superoxide with nitric oxi de (Beckman et al., 1990). Upon protonation, peroxynitrite dissociates to form nitrate (70%) and hydroxyl and n itrite radicals (30%) with a first order rate constant of 0.17 sec-1 (Beckman et al., 1990). The reaction of hydroxyl radicals with biomolecules results in the one-electron oxidation of lipids, proteins and DNA. However, hydrox yl radical formation is likely to occur at acidic pH when peroxynitrite is in its protonated form, but at physiological pH its reaction with CO2 becomes important. At pH 7.8, peroxynitrite reacts with CO2 to form nitrosoperoxycarbonate and out competes the spontaneous dissociation of peroxynitrous acid (Bonini et al., 1999). The short-live d nitrosoperoxycarbonate intermediate dissociates into nitrate (~65%) and car bonate radicals (~35%) which mediate the formation of 3-nitrotyrosine. Tyrosine Nitration Electron paramagnetic resonance studies ha ve shown that tyrosine nitration by peroxynitrite is increase d in the presence of CO2, and membrane inlet mass spectrometry studies have shown that CO2 catalyzes the isomerization of peroxynitrite into nitrate through a nitrosoperoxycarbonate intermediate (Santos et al., 2000; Tu et al., 2004). The predominant pathway for tyrosine nitration is through the reaction of carbonate radical with tyrosine to form tyrosyl radical. Though hydroxyl radical is capable of forming tyrosyl radical, it is a more pr omiscuous oxidative agent than CO3 .-. It has been shown previously that catalytic i nhibition of MnSOD results prim arily from the nitration of Tyr34 (MacMillan-Crow et al., 1996; Yamakur a et al., 2001). The mechanism for this substantial decrease has been elucidated by a 2.4 stru cture of nitrated human MnSOD indicating that steric hindran ce or bulk blockade of the substrate access channel may be

PAGE 16

6 responsible for the change in activity (Quint and Reutzel et al., 2005). These results are discussed in chapter 4. Figure 1-2 Tetrameric human MnSOD. The di meric interface is shown in green and blue and the tetrameric interface is shown in blue and orange. Magenta spheres are active site manganese. Research Goals The data and conclusions presented in this thesis focus on the role of the subunit interfaces in human MnSOD and the structural effects of nitration of Tyr34. Techniques utilized include 19F NMR, x-ray crystallography, diffe rential scanning calorimetry, mass spectrometry, site-directed mutagenesis and pulse radiolysis. The firs t aim was to define the contribution of the two subunit interfaces to enzymatic stability. Building on the conclusions of the first study, the involvemen t of the dimeric inte rface in stability and catalysis was probed through replacement of Glu162. Finally, a structure of nitrated human MnSOD was determined.

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7 Interfacial Mobility at the Dimeric and Tetrameric Interfaces of Human Manganese Superoxide Dismutase The tetrameric interface is generally asso ciated with eukaryotic enzymes. The advantage of a tetrameric structure over a dimeric structure in human MnSOD is not yet understood. The goal of this study was to unders tand differences in st ability exhibited by the two structurally unique interfaces of human MnSOD. Using fluorine labeled Tyr169 as a reporter for the dimeric interface and fluorine labeled Tyr45 as a reporter for the tetrameric interface, 19F NMR was utilized to probe the conformational mobility of the two interfaces. The data indicate that the dimeric interface is significantly less conformationally mobile than the tetrameric in terface, suggesting a role for the tetrameric interface in thermal stability. This study will provide a better understand ing of the role of both interfaces in stability and may aid in our understanding of the evolutionary role of the tetrameric interface. Replacement of a Key Dimeric Interfacial Residue Following the thermostability work of th e previous study, an important dimeric interfacial residue, Glu162, was replaced with aspartate a nd alanine. Glu162 interacts through a hydrogen bond with Glu162 and His163 of the adjacent subun it (Fig 1-1). The goals of this study were twofold: determine the role of Glu162 in structure and catalysis and compare the human E162A to the equivalent mutation in E. coli (Whittaker and Whittaker, 1998). The Glu170A mutant in E. coli is less stable, catalytically inactive and exhibits an altered metal specificity. Replacement of Glu162 in the human enzyme resulted in diminished catalysis and a highe r degree of product inhibition than wild-type enzyme. A crystal structure of both E162A a nd E162D indicated an abolished interaction between the side chains of residues 162 a nd 163 though an intervening solvent molecule

PAGE 18

8 bridged the interaction between Asp162 and Hi s163 in the E162D mutant. Both mutants were tetrameric in solution and metal specif icity was not altered for either mutant. Structure of Nitrated Human MnSOD The third goal was to establis h a structural explanation fo r the decrease in catalysis observed when human MnSOD is nitrated at position 34. Previous studies have shown that nitration of Tyr34 in human MnSOD is associated with abolished catalysis, and recently this lab published the structure of nitrated MnSOD (Quint and Reutzel et al., 2005). Chapter 4 describes the first structur e of a nitrated MnSOD. A 2.4 crystal structure of nitrated human MnSOD indicates exclusive nitration of Tyr34 though mass spectrometry indicates nitr ation of other positions. The orientation of the NO2 group on 3-nitrotyrosine-34 suggests that steric blockade and potenti ally electronic repulsion of substrate could both cause catalytic inhibition.

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9 CHAPTER 2 STRUCTURAL MOBILITY IN HU MAN MANGANESE SUPEROXIDE DISMUTASE Introduction The presence of a tetram eric interface in human MnSOD suggests enhanced stability; the E. coli dimeric MnSOD has a melting temperature of 76oC compared to 90oC for human, tetrameric MnSOD. A polymorphism in the tetrameric interface of human MnSOD, I58T, is associated with 50% decreased activity compared to wild type MnSOD and its melting temperature is decreased to 76o C (Borgstahl et al., 1996). In addition, the heat extremophile Thermus thermophilus utilizes a tetrameric MnSOD (Wagner et al., 1993). This also suggests a ro le for the tetrameric interface in thermal stability. The properties of the dimeric and tetram eric interfaces in human MnSOD have been investigated using 19F NMR. Human MnSOD has been prepared with all nine tyrosine residues of each subunit replaced by 3-fluorotyrosine (abbreviated FluoroMnSOD) (Fig 2-1). The use of 19F labels allows the observati on of specific, well-resolved NMR signals of labeled-tyrosine residues at the dimeric and tetrameric interfaces. The NMR frequency of 19F resonances are nearly as high as 1H, thus producing about the same signal-to-noise as 1H. Moreover, they have a much la rger chemical shift range than 1H, making them considerably more sensit ive to local electronic environment. The replacement of hydrogen by fluorin e in 3-fluorotyrosine is a mi nor steric change since the van der Waals radius of a fluorine is just 0.15 larger than the hydrogen it replaces

PAGE 20

10 (Bondi, 1993), and the C-F moiety is a ra ther weak hydrogen bond acceptor (Jeffrey, 1997). Figure 2-1 Structure of human MnSOD showin g the dimeric and tetrameric interfaces. Also shown are the positions of five tyrosine residues that give sharp 19F resonances when labeled with fluorine. Tyr45 is located at the tetrameric and Tyr169 at the dimeric interfaces. The replacement of all tyrosines with 3-fluorotyrosine in human MnSOD has no observed effect on the structure of the enzyme as determined by X-ray crystallography at 1.5 resolution (Ayala et al., 2005); the fluorinated and unf luorinated structures are closely superimposable with the root-mean-square deviation for 198 -carbon atoms at 0.3 (Ayala et al., 2005). We point out th at the crystal structure of Fluoro-MnSOD showed a single side chain rotamer for each of the nine 3-fluorotyrosines (Ayala et al., Tyr9/11 Tyr169 Tyr193 Tyr45 Tyr176 Tyr166 Tyr165 Tyr34

PAGE 21

11 2005). The catalytic activity of Fluoro-MnS OD was lower than that of MnSOD by a factor of 25 (Ren et al., 2005). This decrea se could not be attr ibuted to a single 3fluorotyrosine residue, and was not primarily due to 3-fluorotyrosine at residue 34, which is in the active site. The 19F NMR show that Tyr169 at the dimeric interface of human MnSOD (Figure 2-1), has significantly less conformational freedom or mobility than does Tyr45 at the tetrameric interface. Consistent with these results, differential scanning calorimetry of human MnSOD showed that replacement by s ite-specific mutagenesis of Tyr169 at the dimeric interface decreased ther mal stability and replacement of Tyr45 at the tetrameric interface did not. These results are discussed in terms of catalysis and stability of MnSOD. Materials and Methods Labeling with Fluorotyrosine, Expressi on in E. coli, and Purification E. coli that express wild-type and the site-directed muta nts of human MnSOD were grown for 17 hours at 37C in 50 mL of mi nimal media. The minimal medium (M9), which consisted of 0.06 M phosphate buffe r at pH 8.2, 8.6 mM NaCl, and 0.02 M NH4Cl, was sterilized by autoclaving. The overni ght culture was supplemented with 0.1 mM CaCl2, 1 mM MgSO4, 11 mM glucose, 1 g/mL of thiamine, 0.2 mg/mL of amino acids (except the aromatic amino acids), 1 mM tryptophan, 1 mM phenylalanine, and ampicillin. The overnight growth was then tr ansferred to 7.5 L of minimal media and supplemented in the same manner as the overnight culture plus the addition of MnSO4 to 18 M. The cells were allowed to grow for approximately 5 hours until an OD595 of 0.30.4 was reached. At this point, the cells were induced with 0.3 mM IPTG and supplemented with 1 mM 3-fluorotyrosine (or unlabeled L-tyrosine as a control) and were

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12 allowed to grow for an additional 4 hours. Due to the low solubility of L-tyrosine and its fluorinated analog in water, these compounds we re added as solids to the growing media. The cells were placed at 4C overnight a nd harvested the next day by centrifugation. The resulting pellet was frozen at -70C overnigh t and the pellet was then lysed the following day. Depending on the particular sample prep aration, the amount of 3-fluorotyrosine incorporated into MnSOD was 67% to 76%, as determined by amino acid analysis composition (Protein Chemistry Laboratory, Texas A&M University, College Station, TX) and corroborated by hybrid LCQToF (QSTAR) mass spectrometry (ICBR, University of Florida, Gainesville, FL). Site-Directed Mutagenesis of Fluori ne Manganese Superoxide Dismutase Site-directed mutants of human FluoroMnSOD were constructed for the purpose of assigning 19F spectra of enzyme containing 3-fluor otyrosine. Each tyrosine of the enzyme was replaced individually by phenylal anine. An exception was the double mutant Y9F-Y11F; since these residues are near in se quence and tertiary structure we replaced these together. These mutants were generate d with the Stratagene QuikChange SiteDirected Mutagenesis Kit (La Jolla, CA) in a Perkin Elmer GeneAmp PCR System 2400 (Foster City, CA). The plasmid of wild t ype MnSOD contained in the pTrc99A vector was used as the template. PCR was perfor med using specific oligonucleotides (SigmaGenosys, The Woodlands, TX) containing the desired mutations as primers. The PCR products were digested with the restriction enzyme Dpn I and transformed into supercompetent XL-1 cells for selection. The plasmid containing the mutation of interest was isolated using the plasmid mini prep kit from Qiagen and the mutation was corroborated by DNA sequencing of the enti re coding region (ICBR, University of

PAGE 23

13 Florida, Gainesville, FL). The plasmid containing the desired mutation was then transformed into QC774 cells from E. coli This particular strain lacks the genes that encode for endogenous FeSOD ( SodB-) and MnSOD ( SodA-). Amide Hydrogen/Deuterium Exchange Kinetics. We employed amide H/D exchange mass spectrometry to examine backbone dynamics for human MnSOD (not fluorinated). By measuring the rate of amide H/D exchange over defined regions of human MnSOD, we were able to develop a comprehensive map of backbone dynamics that was complementary to the NMR studies. On-exchange experiments of amide backbone hydrogens with deuterium were performed in triplicate and involved exposing na tive human MnSOD to solvent 80% D2O for 0, 1, 15, 300, 900, 1800, 3600, and 12000 s prior to quenching of amide hydrogen exchange by rapidly lowering the solution pH and temp erature. After quenching, the protein was digested by exposure to pepsin, and the resu ltant peptide pool was examined by LC-MS. The uptake of deuterium for MnSOD pepsin -derived peptides was determined by measuring the increase in number-average m / z values of the ion is otopic distributions for each peptide from an on-exchange time point (deuterated peptide) when compared to the same peptide from t = 0 (nondeuterated peptide). The percent deuterium incorporation was determined for each peptide by dividing the measured number of deuterium atoms incorporated by the calculated number of exchangeable amide hydrogen atoms for that peptide 19F Nuclear Magnetic Resonance Spectroscopy The 19F NMR spectra of fluorinated samples were recorded on a Bruker Avance 500 MHz spectrometer. A 1H 5mm TXI probe tuned for 19F at 470 MHz was employed. We were not able to use a fluorine specific pr obe; the probe used displayed a very broad

PAGE 24

14 background 19F resonance upon which the peaks of our enzyme were superimposed. This arrangement precluded measurements of T2 and we report linewidths instead. Due to this broad fluorine background, a T2 filter was utilized. This allows for the broader signals of the spectrum to decay before the start of th e data collection. The enzyme concentrations were 0.5 mM in phosphate buffer at pH 7.8, unless otherwise specified, and 10% (by volume) D2O for an internal lock. Chemical shif ts were referenced to the internal standard trifluoroacetate (TFA ) at 0 ppm; high-field or shie lded values with respect to TFA are taken as negative. Temperature was varied from 17o C to 62o C by the flow of heated nitrogen gas. Spectra were acquired by averaging 4000 scans w ith a scan rate of 8000 per hour. Differential Scanning Calorimetry Proteins were prepared in potassium phosphate buffer (20 mM, pH 7.8) at a concentration of 1 mg/ml. A solution of 20 mM potassium phosphate (pH 7.8) was used as a buffer reference. Both the sample and re ference were degassed for 10 minutes before scanning from 250C to 1200C at a rate of 10C per minute (Microcal VP-DSC). A buffer blank was subtracted from the final protein scan and a cubi c baseline was fit to the profile. Changes in heat capacity ( Cp) for the unfolding peaks we re corrected by fitting a non-two state model with a single component. Baseline correction and peak fitting were performed using Origin (Microcal Software, Northampton, MA). Results Assignment of Fluorine Resonances There are nine 3-fluorotyrosine residues in each subunit of the tetramer in human wild-type Fluoro-MnSOD; five appear as di stinct major peaks spanning about 8 ppm in the 19F NMR spectrum (Figure 2-2). The integrated intensity of each individual peak was

PAGE 25

15 approximately the same and represents one fluorine atom each. The assignment of the peaks in Figure 2 was achieved by measuring the 19F NMR spectra of individual sitespecific mutants in which each tyrosine wa s replaced by phenylalanine (Figure 2-2). Since residues 9 and ll are near each other in sequence, we saved effort by preparing the double mutant; hence, the NMR resonance assign ments are not yet verified. However, in the crystal structure of Fluor o-MnSOD (Ayala et al., 2005; PDB # 1XDC) the side chain of Fluor-Tyr9 is buried with the pheno lic hydroxyl hydrogen bonded to the backbone carbonyl of residue 78 and in near van de r Waals contact with Pro8, suggesting the downfield shifted 19F resonance of residue 9 with resp ect to Fluoro-Tyr11. Tyr11 is more exposed to the solvent than Tyr9. Thus we a ssign the more downfield resonance at -57.6 ppm to Tyr9 and the resonance at -60.1 ppm to Tyr11 (Figure 2-3). For reference, the 19F resonance for monomeric tyrosine (pH 7.8, 25 oC) is -61.4 ppm, and the chemical shift of the single large resonance of collapsed Fluoro-MnSOD collapses at 62o C is -62.3 ppm. Of the nine 3-fluorotyrosine residues of each monomer, four are not observed in the 19F NMR spectrum under the conditions of Figure 2-2. These are residues 34, 165, 166, and 176, the side chains of which are located at distances less than 9 from the manganese (manganese to hydroxyl distance). All of the observed resonances, residues 9, 11, 45, 169, and 193, are located at distances greater than 13 from the manganese. Thus it is a reasonable sugges tion that the four residues of 3-fluorotyrosine not observed are broadened by the paramagnetic manganese, in addition to broadening by the overall slow motion of the homotetramer. At the pH 7.8 of these studies it is not expected that any of the tyrosine residues ar e ionized, consistent with lite rature on structure (Borgstahl et al., 1996) and catalysis (Hsu et al., 1996).

PAGE 26

16 Figure 2-2 19F NMR spectrum (470 MHz) of wild-type human MnSOD in which all nine tyrosines were replaced with 3-fluorot yrosine. Residue assignments were made by replacement of individual 3-fluor otyrosine residues with Phe and are written above the peaks. Chemical shif ts referenced to TFA as internal standard. Figure 2-3 Stacked fluorine NMR spectra for wild-type human MnSOD in which tyrosines 9, 11, 34, 45, 166, 176 and 193 have been mutated to phenlylalanine. -126 -128 -130 -132 -134 -136 -138 -140 -142 ppm -124 Tyr166 WT Tyr34 Tyr193 Tyr45 Tyr169 Tyr176 Tyr9/11

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17 Thermal Stability Differential scanning calorimetry was used to determine changes in thermal stability for two unfluorinated mutant s of human MnSOD at positions 45 and 169 measuring the main unfolding transition of th e enzyme. The mutant with Tyr45 replaced by Ala exhibited an unfolding temperature of 94.3o C compared to 90.7o C for the wild type MnSOD (Hsu et al., 1996; Greenleaf et al., 2004)(standard deviation estimated at 0.3 oC). The unfolding temperature for the muta nt with Tyr169 replaced with Ala was decreased to 86.5o C. These site-specific mutants, one with Tyr45 replaced by Ala and the second with Tyr169 also replaced by Ala, s howed no significant ch ange in catalytic decay of superoxide measured by pulse radi olysis at Brookhaven National Lab (data not shown). Analysis by native polyacrylamid e gel electrophoresis indicated that both mutants, Y45A and Y169A, remained tetrameric in solution. Temperature Dependence of Fluorine Resonances The change as temperature was increased from 17o to 57o C in the chemical shifts of each of the five assigned resonances was uniform with no significant changes in slope for individual peaks over this temperature range (Figure 2-3). The two residues that had the largest downfield 19F chemical shift, Fluoro-Tyr45 and Fluoro-Tyr9, also showed the largest changes in chemical shift (Figure 2-3) and in linewidth at half height (Figure 2-4) as temperature increased. The remaining assigned residues Fluoro-Tyr11, 169, and 193 showed smaller changes in chem ical shifts and in linewidths with changes in temperature over the range of temperatures in Figures 24 and 2-5. It is notabl e that Fluoro-Tyr169, iun the dimeric interface, showed almost no change in linewidth with temperature (Figure 2-5).

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18 Figure 2-4 The temperature dependence of five 19F chemical shifts of wild-type human MnSOD in which all tyrosine residues are replaced with 3-fluorotyrosine. Values are normalized to show a single chemical shift at 17o C in order to compare trends. Conditions are as described in Figure 2-2. ( ) Tyr45; ( ) Tyr9; ( )Tyr169; ( ) Tyr193; ( )Tyr11. Figure 2-5 The temperature dependence of 19F linewidths at half height for wild-type human MnSOD in which all tyrosine residues are replaced with 3fluorotyrosine. Conditions are as described in Figure 2-2. () Tyr45; () Tyr9; ()Tyr169; ( ) Tyr193; ( )Tyr11. -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 17222732374247525762 Temperature ( oC) (ppm)Tyr45 Tyr9 Tyr169 Tyr193 Tyr11 0.15 0.25 0.35 0.45 0.55 0.65 0.75 1217222732374247525762 Temperature (oC)Linewidth (ppm)Tyr45Tyr9 Tyr169Tyr193 Tyr11

PAGE 29

19 To corroborate these findings, hydrogen /deuterium exchange studies were performed. Using this approach, amide hydr ogen exchange kinetics of 29 peptides (comprising approximately 78% of the human MnSOD protein) were determined (Figure 2-6). For each peptide, the percentage of de uterium uptake versus time for the seven onexchange time intervals was plotted with erro r bars (plots not shown) representing the mean standard deviation of the deuterium incorporation percentages determined from triplicate experiments. The rate of deuterium incorporation va ried in different regions of the protein (Figure 2-6). Peptides co rresponding to regions 25-40, 58-77, and 94-113 displayed significant protection from amide H/ D exchange as demonstrated by very low levels of deuterium incorporation with thei r maximum levels being below 35% (percent of the maximum on-exchange possible corr ected for percent deuterium exposure and back exchange) at the longest on-exchange time point. Peptides corresponding to regions 1-20, 78-96, 114-135, and 155-173 showed moderate protection from amide H/D exchange with levels of deuterium incor poration between 50% a nd 60% at the longest on-exchange time point. Discussion Emphasized here are the properties of Fl uoro-Tyr45 and 169, which are located in the tetrameric and dimeric interf aces, respectively (Figure 2-1). The 19F resonance of Fluoro-Tyr45 showed a large increase in chem ical shift as temperat ure increased (Figure 2-4), moving toward the position of the 19F peak for denatured enzyme, and showed a large decrease in linewidth as temperatur e increased (Figure 2-5). These features characterize a region at the tetrameric in terface with increased conformational and dynamic mobility as temperature increases. Ba sed on previous studies of the motions of

PAGE 30

20 Figure 2-6 H/D exchange map for human Mn SOD. Each block represents a pepsinderived fragment of MnSOD detected by LC-MS and monitored during onexchange time periods to determine th e degree of deuterium incorporation. The gradations within each block re present the seven on-exchange time periods used with the shor test period being on top. Th e deuteration level, as a percentage of the theoretical maximum, for each peptide at each time period is color-coded. fluorotyrosine rings in prot eins (Hull and Sykes, 1975; Hull and Sykes, 1975B), we anticipate that the dominant spin-lattice re laxation mechanism is dipolar with a contribution from chemical shift anisotropy, a nd that the decrease in linewidths observed in 19F resonances can be largely attributed to motional narrowing. We point out that the crystal structure shows just one rotamer fo r the side-chains of each 3-fluorotyrosine residue in Fluoro-MnSOD (Ayala et al., 2005); although we cannot exclude a contribution of chemical exchange to line br oadening, neither the crys tal structure nor the observed 19F spectrum suggest that such a mechanism is predominant.

PAGE 31

21 The 19F resonance of Fluoro-Tyr169 is not able because it does not change appreciably in linewidth over the temperature range from 17o to 57 oC (Figure 2-5). The other 19F resonances have linewidths that de crease with increasing temperature, suggesting increasing motional processes; Fl uoro-Tyr169 does not show a detectably higher degree of motional freedom as temper ature increases. Als o, Fluoro-Tyr169 shows a chemical shift change as temperature increas es that is modest compared with that of Fluoro-Tyr45 (Figure 2-4). The linewidth data especially indicate that the environment of this side chain is rather stable with little change in mobility over the temperature range studied. Residue 169 is located at the dimeric interface and its side chain appears in near van der Waals contact with the hydrocarbon side chain of Gln168. Considering residue 169 as a reporter for the dimeric interface, these data indicate stability and lower motional freedom for the region of Fluoro-Tyr169. Of the remaining observed 19F resonances, those of residues 11 and 193 had chemical shifts closest to monomeric 3-fluor otyrosine or to part ially denatured FluoroMnSOD. With increasing temperature, their ch emical shifts moved toward that of the denatured enzyme and their linewidths narro wed somewhat (Figures 2-4, 2-5). These features are consistent with the partially constrained posit ions of Tyr11 and 193 in the structure. Fluoro-Tyr9 was different in showi ng a rather substantial temperature effect in its 19F linewidth (Figure 2-5), al though showing a rather mode st temperature effect on 19F chemical shift (Figure 2-4). Deutrium exchange studies on native Mn SOD corroborate the findings of fluorine NMR. The most rapidly ex changing MnSOD peptides, or regions of MnSOD that demonstrate little or no protec tion to amide H/D exchange, we re in the 40-58 region that

PAGE 32

22 showed 96% deuterium incorporation at the 3600 s time point. The exchange kinetics for a region of a protein is depe ndent in part on the extent of localized hydrogen bonding as amide hydrogens are protected from exch ange while involved in hydrogen bonding. For an amide hydrogen involved in a hydrogen bond to become exchange competent, localized unfolding must occur to break the hydrogen bond and allow exchange with solvent protons or deuterons. Therefore, sl owly exchanging regions of a protein are considered less dynamic in part due to si gnificant hydrogen bonding. Taken together, the data indicate that the regi ons corresponding to the tetram eric domain (40-58) and the dimeric domain (159-174) display different amide H/D exchange kinetics with the tetrameric domain affording rapid exchange and the dimeric domain affording moderate protection from amide H/D exchange (Figure 2-6) The thermal unfolding data appear consiste nt with these conclusions. Specifically, differential scanning calorimetry showed that replacement of Tyr169 with Ala destabilized human MnSOD with the majo r unfolding transition decreased about 4o C compared with wild type, while the replacemen t of Tyr45 did not de stabilize but actually enhanced stability somewhat. The observation that the melting temperature for Y169A is decreased unlike the Y45A mutant suggests th at the dimeric interf ace is stabilized by specific residue interactions wh ereas the tetrameric interface is stabilized by several nonspecific interactions. Evolution clearly shows a dimeric MnSOD of primitive species, with tetrameric MnSOD a different development (Purrelo et al., 2005). In fact, crossing the dimeric interface are residues such as Glu162 and Tyr166 that extend into the active site of the adjacent subunit and are significant contributo rs to catalysis (Whittaker and Whittaker,

PAGE 33

23 1998; Hearn et al., 2004). In human MnSOD, th e reporter residue at the dimeric interface Tyr169 showed less conformational mobility an d greater contribution to stability than Tyr45 at the tetrameric interface. This may in part reflect the observation that residues at the dimeric interface are closer to the active si te, likely play a greater role in supporting catalysis, and hence require a greater degree of conformational stiffne ss than residues at the tetrameric interface.

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24 CHAPTER 3 ROLE OF A GLUTAMATE BRIDGE SPANN ING THE DIMERIC INTERFACE OF HUMAN MANGANESE SUPEROXIDE DISMUTASE Introduction The previous chapter elucidated the roles of the tetrameric and dimeric interfaces and their contribution to enzyme stability. R ecognizing the role of the dimeric interface in catalysis, this chapter focuses on a mutation in the dimeric interface and the subsequent effects on catalysis and stability. This study reports the role of Glu162 in human MnSOD, the side chain carboxyl of which forms a hydrogen bond with the imidazole side chain of His163 of the adjacent subunit, a ligand of the metal (Figure 3-1). Using both X-ray crystallography and pulse radiol ysis, reported here are the effects of mutation of Glu162 to aspartate and alanine. The X-ray data for E162D and E162A reveal no significant structural changes compared with wild type other than the lost interaction with His163, and in the case of E162D an intervening water molecule maintains a hydrogen-bond link between Asp162 and His163. However, mutatio n of Glu162 introduces a pH dependence in catalysis and in the visi ble absorption spectrum of Mn3+SOD near pKa 8.5 not observed in human wild-type MnSOD. Mutation of Glu162 to either aspartate or alanine results in a greater degree of product inhibition compar ed to wild-type MnSOD. Differential scanning calorimetry indicat es that the hydrogen bond between Glu162 and His163 contributes to the stab ility of MnSOD. These data emphasize the role of the dimeric interface of human MnSOD in catal ysis and thermal stability.

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25 Figure 3-1 Active-site struct ure including the hydrogen bond network for human wildtype MnSOD (Borgstahl et al., 1992). The manganese is designated as a purple sphere and solvent molecules as red spheres. Dotted lines indicate a hydrogen-bonded network emanating from the metal-bound solvent molecule to Tyr166 from an adjacent subunit. Materials and Methods Site Directed Mutagenesis Mutants were generated with the St ratagene QuikChange Site-Directed Mutagenesis Kit (La Jolla, CA) in a Perk in Elmer GeneAmp PCR System 2400 (Foster City, CA). The plasmid of wild type MnSOD contained in the pTrc99A vector was used as the template. PCR was performed using specific oligonucleotid es (Sigma-Genosys, The Woodlands, TX) containing the desired mutations as primers. The PCR products were digested with the restriction enzyme Dpn I and transformed into supercompetent XL-1 cells for selection. The plasmid contai ning the mutation of interest was isolated using the plasmid mini prep kit from Qi agen and the mutation was corroborated by DNA sequencing of the entire coding region (I CBR, University of Florida, Gainesville, His163 Tyr34 His74 His26 Asp159 Gln143 His30 S2 S1Mn Trp123 Glu162 (adjacent subunit) Tyr166 (adjacent subunit)

PAGE 36

26 FL). The plasmid containing the desired mutation was then transformed into QC774 cells from E. coli This particular strain lacks the genes that encode for endogenous FeSOD ( SodB-) and MnSOD ( SodA-). Expression and Purification of Human MnSOD The pTrc99A plasmid containing the mutant MnSOD template was transformed into SodA-/ SodBE. coli QC774 cells. Cells were grown in luria broth media supplemented with 6 mM MnCl2 and ampicillin for selection. Cultures were grown to 0.8 absorbance units and then induced with IPTG. Cells were centrifuged and then lysed. The lysate was heat treated at 60o C for 15 minutes to select for MnSOD, which is thermostable to 70o C. Following heat treatment, the lysate was spun and the supernatant was dialysed against three excha nges of 20 mM Tris pH 8.2 and 50 M EDTA. The dialysate was purified using a Q-sepharose anion exchange co lumn (Pharmacia). Protein concentrations were determined by UV spectrometry using a Beckman Coulter DU 800 spectrometer at 25o C and pH 7.8 (280 = 40,500 M-1 cm-1) (Greenleaf et al., 2004). Manganese and iron content for each enzyme sample were determined using flame atomic absorption spectroscopy (flame AA) and division by the total protein concentration gives metal occupancy for the enzyme. Visible Absorption The visible spectrum for human MnS OD shows a broad absorption with a maximum at 480 nm (480 = 610 M-1 cm-1) (Hsu et al., 1996). Pr ofiles for pH dependence were determined by measuring the absorpti on at 482 nm at pH varying from 6.5 to 11.5. Enzyme samples were diluted 1:1 (~500 M enzyme) in a buffer containing 200 mM

PAGE 37

27 MES and 200 mM TAPS and pH was adjusted using 5 M KOH. The pH was measured using a Fisher Accumet 610 pH meter with a Co rning semi-micro combo electrode. Pulse Radiolysis Pulse radiolysis experiments were perf ormed at Brookhaven National Lab using a 2 MeV van de Graff generator to produce supe roxide directly in solution. Superoxide radicals were formed by exposing aqueous, airsaturated solutions to a high-dose electron pulse according to methods described by Schwarz (Schwarz, 1981). Up to 45 M superoxide was produced in solution. En zyme solutions contained 2 mM buffer containing either MOPS pH 6.5-8.0, TAPS 8.0-9.0, or CAPS 9.0-10.0 depending on the desired pH, 50 M EDTA, and 30 mM formate to scavenge hydroxyl radicals. The reactions were monitored spectrophotometri cally using a Cary 210 spectrophotometer at 25o C by following changes in the absorbance of superoxide (260 = 2000 M-1 cm-1) (Rabini and Nielson, 1969) or by followi ng enzyme absorbance at 420 nm or 480 nm (Cabelli et al., 1999). Manganese and Iron Content Determination Flame atomic absorption spectroscopy was used to determine total metal content in all enzyme solutions. A Perkin Elemer 308 Flame Atomic Absorption Spectrometer was utilized to determine manganese concentrati on. A multi-ion lamp with a 3-slit burner was used and absorption was measured at 279 nm. Manganese occupancies were determined by dividing the total metal c oncentration by the enzyme concentration to yield a percentage of total active sites containing manganese. Typical occupancies for metal in the active site range from 70% to 90%. Iron content was measured by ABC Research Corp (Gainesville, FL) and was determined to account for less than 2% of the total metal

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28 in solution E162D and E162A MnSOD. The ma nganese concentration was used as the active enzyme concentration for all pulse radiolysis measurements. Crystallography Hexagonal crystals were grown from a solution of 3 M ammonium sulfate containing 100 mM imidazole and 100 mM malate at pH 7.8-8.2 using the vapor diffusion method. Crystals approximately 0.2 x 0.2 x 0.3 mm grew w ithin one week and were magenta in color. Diffraction data were collected from single crystals wet mounted in quartz capillaries (Hampton Research) on an R-AXIS IV++ image plate (IP) system with Osmic mirrors and a Rigaku HU-H3R CU rotating anode operating at 50 kV and 100 mA (Rigaku/MSC). Diffracti on data was collected at room temperature. A 0.3 mm collimator was used with a crysta l to IP distance of 220 mm and the 2 angle fixed at 0o. The frames were collected using a 0.3o oscillation angle with an exposure time of 5 min/frame at room temperature. Both data sets were indexed using DENZO and scaled and reduced with SCALEPACK software (O twinowski, 1997). Diffr action intensities were visible to 2.3 resolution for E162D and 2.5 for E162A. Diffraction and refinement statistics are given in Table 3-2. To prevent model bias, human MnSOD mutants were phased using the human, wild-type MnSOD structure (Q uint and Reutzel et al., 2006 ; PDB accession 2ADQ) from which the residue at position 162 was repla ced with an alanine and the active-site manganese had been removed. The structures were phased and refined using the software package CNS (Brunger et al., 1998). Refine ment cycling (using rigid body, simulated annealing for the first cycle, minimization, a nd individual B-factor refinement) was done

PAGE 39

29 in conjunction with rounds of manual model building using the program COOT for molecular modeling (Emsley and Cowtan, 1998). Differential Scanning Calorimetry Enzyme samples were buffered in 20 mM potassium phosphate pH 7.8 at a concentration of 1 mg/ml. A solution of 20 mM potassium phosphate pH 7.8 was used as a buffer reference and was subtracted from th e protein scan prior to baseline correction and model fitting. Both the sample and refere nce were degassed for 10 minutes before scanning from 25o C to 110o C at a rate of 1o C per minute (Microcal VP-DSC). A buffer blank was subtracted from the final protein scan and a cubi c baseline was fit to the profile. Changes in heat capacity ( Cp) for the thermal unfolding peaks were corrected by fitting a reversible, non-two state model with two components. Baseline correction and peak fitting were performed using the program Origin (Microcal Software, Northampton, MA). Results Visible Spectrometry Atomic absorption spectroscopy for the mu tants of MnSOD studied here showed that the metal occupancy for E162D was 88% Mn with <1% Fe, and the metal occupancy for E162A was 54% Mn and <1% Fe. Both mutants were tetramers as determined by non-denaturing PAGE. Human wild type Mn3+SOD as well as E162D and E162A Mn3+SOD exhibit a characteristic visible ab sorbance with a maximum at 480 nM (480 = 610 M-1 cm-1) corresponding to Mn3+ in the active site (Hsu et al., 1996). The pH profile for molar absorptivity for wild-type human Mn3+SOD fits a single ionization with a pKa of 9.2 0.1 (Figure 3-2) (Hsu et al., 1996; Greenleaf et al., 2004). Mutation to aspartate

PAGE 40

30 resulted in a diminished pKa (pKa = 8.7 0.2) whereas repl acement with alanine increased the pKa (pKa = 10.1 0.1) (Figure 3-2). 300 400 500 600 681012pH480 ( M-1 cm-1) Figure 3-2 pH profile for molar absorptivity at 480 nm for ( ) human wild-type Mn3+SOD, ( ) E162D Mn3+SOD and ( ) E162A Mn3+SOD. Data were fit to a single ionization with values of pKa 8.7 0.2, 9.2 0.1, and 10.1 0.1 for E162D, wild type, and E162A Mn3+SOD respectively. Solutions contained 200 mM MES and TAPS at 25o C and pH was adjusted using 5 M KOH. Catalysis MnSOD catalyzes the disproportionation of superoxide through a two-step process in which the active-site metal cycles between the Mn3+ and Mn2+ states concomitant with oxidation and reduction of superoxide (shown in eqs. 3-1 and 3-2) (Hearn et al. 2001; McAdam et al., 1977; Cabelli et al., 1999). Mn3+(OH)SOD + O2 .+ H+ k1 Mn2+(H2O)SOD + O2 (3-1) Mn2+(H2O)SOD + O2 .+ H+ k2 Mn3+(OH)SOD + H2O2 (3-2) Mn2+(H2O)SOD + O2 .k3 Mn3+(O2 2-)SOD (3-3) Mn3+(O2 2-)SOD + H+ + H2O k4 Mn3+(OH)SOD + H2O2 (3-4)

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31 The scheme shown here reflects the obs ervation that the solvent ligand of Mn3+(OH)SOD takes up a proton upon reduction of the metal as shown in equation 3-1. Equations 3-3 and 3-4 represent the forma tion and dissociation of a product inhibited complex characterized by zero-order catalysis (Hearn et al., 2001; McAdam et al., 1977). Estimation of the rate constants for eq s 3-1 through 3-4 were carried out by measuring the rate of change of absorbance of O2 and of enzyme species after the generation of O2 by pulse radiolysis (Hearn et al., 2001; Cabelli et al., 1999). Decrease in absorption at 260 nm (260 = 2000) (Rabini an d Nielson, 1969) corresponds to the disappearance of superoxide catalyzed by w ild type, E162D, and E162A MnSOD, which is characterized by a first-order phase of catal ysis that is relatively uninhibited followed by a zero-order, product inhibi ted phase (Hsu et el., 1996; Bull, Yoshida and Fee, 1991). When enzyme and superoxide are similar in concentration, th e progress curve for superoxide decay is dominated by the reacti on in eq 3-1, and a rate constant for k1 can be determined from a first-order fit to th e decay. Another method for determining k1, described by Hearn et al., 1999 (Borgstahl et al., 1996), involves measuring changes in absorption at 480 nm under single turnover c onditions when there is a molar excess of enzyme. Decrease in absorption at 480 nm results from the conversion of Mn3+SOD to Mn2+SOD shown in eq 3-1. This is a complementary method for determining k1 and values for the two methods are in agreement. After addition of a molar equivalent of H2O2 to reduce the active-site manganese to Mn2+, observation of the increase in absorption at 480 nm yields an estimate for k2 at earlier time points and k4 at the later part of the curve. The product-inhi bited complex has a characteri stic absorption at 420 nm (Bull, Yoshida and Fee, 1991) and measur ing absorption increase at 420 nm after

PAGE 42

32 reduction of the enzyme with H2O2 allows for an estimate of the rate constant k3 (Hearn et al., 2001). Figure 3-3 shows typical data gene rated for the calculation of k3. 0 1 2 3 4 0.000.100.200.30 Time (msec)mAbs (420 nm) Figure 3-3 Change in absorbance at 420 nm over a millisecond timescale after generation of 5.6 M superoxide by pulse radi olysis in a solution containing 120 M E162D MnSOD buffered by 2 mM TAPS at pH 8.35 with 50 M EDTA and 30 mM sodium formate at 25 oC. Prior to pulsing, the sample was reduced with 300 M H2O2. Increase in absorbance at 420 nm was fit to a first-order process giving the ra te constant 14.4 ms-1. The values of k1-k4 describing catalysis by hum an wild-type MnSOD are independent of pH in the range of pH 7 to 9.5 with a slight decrease above pH 9.5 (Greenleaf et al., 2004; H earn et al., 2001). Values of the rate constants k1-k3 for catalysis by E162D MnSOD were pH dependent (Figure 34) and could be fit to a single ionization with maxima given in Table 3-1 and values of pKa in the range of 8.0 to 8.7 given in the legend to Figure 3-4. The rate constant k4 describing the dissociation of the productinhibited complex was independent of pH t hough it was diminished three-fold compared

PAGE 43

33 to wild-type enzyme. E162A MnSOD exhibited no pH dependence for k1-k3 with values given in Table 3-1 concomitant with oxida tion and reduction of superoxide (shown in eqs. 3-1 and 3-2) (Hearn et al., 2001; Cabelli et al., 1999; McAdam et al., 1977). However, above pH 8.0, the rate constant k4 was decreased 10-fold, from 30 3 sec-1 to 3 1 sec-1. Table 3-1 Maximal rate constants for the ca talysis and inhibition of human wild type MnSOD and mutants. The maximal value for the steady-state rate constant kcat/Km for E162D was 290 M-1 sec-1 compared to 800 M-1 sec-1 for wild-type enzyme, and decreased in a pH dependent manner with a pKa of 8.8 (Figure 3-5). The maximal value for kcat/Km for E162A was diminished 8-fold (120 M-1 sec-1) independently of pH (Table 3-3). The rate constant k0/[E] describes the product-inhibited, zero-order region of catalysis. The maximal value of k0/[E] for E162D MnSOD was 270 sec-1 and was mostly pH independent, though there was a decrease at higher pH (190 sec-1 at pH 8.3) (Table 3-3). The E162A mutant exhibited a more extens ive decrease with pH. The value for k0/[E] at pH 7.7 was 190 sec-1 while at pH 8.4 it decreased to 18 sec-1. For comparison, the value WTb 1500 1100 1100 120 E162Da 355 33 133 16 215 20 40 1 E162Aa 63 4 50 4 87 3 30 3 H30Nc 210 400 680 480 Y166Fd 0.2 0.2 0.2 270 E. colie 1000 800 150 60 (a) 2mM TAPS pH 7.7, 50mM EDTA, 30 mM fo rmate (see methods for pulse radiolysis) (b) Ramilo et al., 1998 (c) Hearn et al., 2003 (d) Hearn et al., 2001 ( e ) un p ublishe d k1 k2 k3 k4 (M-1 sec-1) (M-1 sec-1) (M-1 sec-1) (sec-1)

PAGE 44

34 of k0/[E] is near 500 s-1 and is pH independent (Hearn et al., 2001; Hsu et al., 1996; Greenleaf et al., 2004) (Table 3-3). Figure 3-4 pH profile for k1 ( ), k2 ( ), and ( ) k3 in catalysis of the disproportionation of superoxide by E162D MnS OD. Solutions contained 120 M enzyme, 2 mM buffer (see methods), 50 M EDTA and 30 mM sodium formate at 25o C. A single ionization was fit to the data with values for pKa of 8.7 0.2 for k1 and 8.1 0.1 and 8.0 0.1 for k2 and k3 respectively. Figure 3-5 pH profile for Kcat/Km for E162D MnSOD. Solutions contained 2mM MOPS (pH 6.5-8.0), TAPS (8.0-9.0) or CAPS (9.0-10.0) 50 M EDTA and 30 mM sodium formate at 25o C The line represents a th eoretical curve for a single ionization with value for pKa of 8.8. 0 100 200 300 400 500 7.588.599.510pHk ( M-1 sec-1) 0 100 200 300 400 7.588.599.510 p Hkcat/Km ( M-1 sec-1)

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35 Structure of MnSOD Mutants The human MnSOD mutants E162D and E 162A crystallized in the hexagonal space group P6122 under similar crystallization cond itions (see methods) (Table 3-2). Both mutants contained a homodimer in the asy mmetric unit with the eukaryotic tetramer formed from a crystallographic, symmetrygenerated dimer. The models for the two mutants superimpose well on wild type (rmsd = .2 for E162D and .2 for E162A). The major difference is the truncation of the hydrogen bond interaction between Glu162 and His163 (Figures 3-6 and 3-7). The E162D structure retains an interaction with His163 through a solvent molecule (Figure 3-7) This interaction does not exist in the E612A mutant and therefore a ny stabilizing force that the residue at position 162 would have on His163 or the active site in genera l is missing. The positions of the active-site residues were altered for the two mutants. For E162A, the distances from Asp159 and His74 to manganese were decreased from 2.04 for wild type to 1.61 and from 2.23 to 1.65 respectively. Distances for the re maining coordinating residues were not significantly different. Distan ces were altered by < 10% for the E162D structure. Figure 3-6 Structure of E162D MnSOD (show n in green) superim posed on wild-type human MnSOD (shown in blue) in the region of the mutated residue. Red spheres represent solvent and the ma nganese is shown in purple. The intervening solvent molecule from th e E162D structure is labeled S*. His26 His74 His163 Asp159 Glu162 Mn S1 S* SO4 2-

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36 Table 3-2 Diffraction data and refinement statistics for human E162D and E162A MnSOD. E162D E162A Space group P6122 P6122 Unit-cell parameters () a = 81.16, c = 241.17 a = 81.26, c = 242.28 = 90, = 120 = 90, = 120 Resolution () 20-2.3 20-2.5 No. of unique reflections 19557 16004 Completeness (%) 89.0 (90.3) 92.6 (93.8) aR sym (%) 12.0 (21.0) 14.5 (46.0) {I/ (I)} 18.7 (19.7) 22.0 (7.2) bR factor (%) 20.7 24.0 R free (%) 22.7 25.6 No. of protein atoms 1553 1550 No. of water molecules 51 38 cR.m.s.d. bond length () 0.005 0.006 cR.m.s.d. bond angle () 1.199 1.426 Avg B (main/side/solve nt) 25.2/28.1/34.7 32.6/34.1/34.6 Ramachandran Plot (%) Most favored regions 92.1 91.5 Addn’l allowed regions 6.7 7.3 Gen allowed regions 1.2 1.2 Disallowed regions 0 0 Data collected at room temperature *Data for the highest resolution sh ell are given in parentheses. aRsym = |I-| / I x 100, where I is the intensity of a reflection and is the average intensity. bRfactor = hkl |Fo-KFc| / hkl |Fo| x 100, Rfree is calculated from 5% randomly selected data for cross-validation. cR.m.s.d. = root mean square deviation. Thermal Stability Differential scanning calorimetry was used to determine thermal transition temperatures for E162D and E162A human MnSOD. E162D MnSOD showed two peaks and these melting temperatures were quite si milar to those observed for wild type. The thermal inactivation temperature was 72o C and the unfolding temperature was 88o C.

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37 These values for wild-type human MnSOD were 68o C and 90o C, respectively (Borgstahl et al., 1996). Replacement with alanine destabilized the enzyme. E162A MnSOD exhibited a split peak with values of 74.5o C and 81o C. The calorimetric data for both mutants were the composite of three experi ments averaged and normalized to a non-two state model with two compone nts. Melting temperatures for E162D and E162A were determined at pH 7.8. Figure 3-7 Structure of E162A MnSOD (show n in green) superim posed on wild-type human MnSOD (shown in blue) in the region of the mutated residue. Red spheres represent solvent and the manga nese is shown in purple. In the mutant, a sulfate molecule was found bound near the position of the carboxyl group of Ala162 and in possible interaction with S2. Discussion Structures of E162D and E162A MnSOD In wild-type MnSOD, the side-chain of Glu162 forms a hydrogen bond with the side chain of His163 from the adjacent s ubunit MnSOD (Borgstahl et al., 1992). This interaction is dampened in E162D MnSOD as the carboxyl group is more distant from Y34 H30 SO4 -2 H163 H74 S1 D159 A162 Mn S2

PAGE 48

38 His163 by the length of one methylene gr oup and by the intervention of a solvent molecule bridging Asp162 and His163 (Fi gure 3-6); however, the hydrogen bond to His163 is maintained through the interveni ng water molecule. This interaction is abolished in E162A MnSOD (Figure 3-7). As a result of the altere d interaction between the side chains of residues 162 and 163, the c oordination geometry is changed for E162A, though not so for E162D, presumably because a solvent molecule bridges the gap between Asp162 and His163. Another possible caus e for the altered ligand distances for E162A may be the low metal occupancy (54% ). The positions of other active-site residues His30, Tyr34, Gln143 are una ltered for either mutant. An area of spurious density was obser ved surrounding Asp162 and Ala162 that was attributed to sulfate bindi ng (Figures 3-6 and 3-7). Ali gnment of the sulfate-binding region of E162D and wild-type MnSOD indicat es an opening of the structure. The solvent at position S2 (Figure 3-1) is replaced by sulf ate in the E162D structure. The sulfate-binding region of E162A shows hydrogen bonding of sulfate to th e side chains of His30 and Tyr34. This could indicate a pathwa y for superoxide entry into the region of the active-site cavity. Sulfate wa s not present in measurements of catalysis or of spectral properties of these mutants of MnSOD although formate was used as a hydroxyl scavenger in pulse radiolysis experiments. Differential scanning calorimetry showed that the main unfol ding transition of E162D MnSOD (88o C) is not significantly altered compared with wild-type human MnSOD (90o C), consistent with the retenti on in the mutant of the hydrogen bond between Asp162 and His163 through an interven ing water molecule. In contrast, this transition for E162A is decreased by 15o C, attributed in significan t part to the removal of

PAGE 49

39 a stabilizing interaction with His163 from the adjacent subunit. The reduced stability, however, did not affect tetramerization of E162A. Visible Spectroscopy The visible spectrum of Mn3+SOD is characterized by a broad absorption in the visible region with a maximum at 480 nm (Hearn et al., 1999; Bull and Fee, 1985). The pH profile of this maximum titrates with reported values of pKa of 9.3 (Bull and Fee, 1985) and 9.7 (Malieka l et al., 2002) for E. coli MnSOD, and 9.4 (Guan et al., 1998) and 9.2 (this study) for the human MnSOD. Although there has been some disagreement as to the source of this ionization, a thorough study in E. coli MnSOD assigns this to Tyr34 (Maliekal et al., 2002), with co rroborating evidence from Y34F that Tyr34 is also the source of this ionization in human MnSOD (Guan et al., 1998). NMR studies conducted on E. coli MnSOD showed that a pH-related ch emical shift change corresponding to ionization of the phenolic hydroxyl of Tyr34 exhibited a pKa of 9.5 0.2 (Maliekal et al., 2002). Human Y34F MnSOD exhibits a pKa near 11 significantly different than the visible spectrum of wild-type MnSOD (Hsu et al., 1996; Guan et al., 1998). Ionization of Tyr34 is also most likely the source of the pKa ~ 9.5 in catalysis by human MnSOD (Bull and Fee, 1985; Hearn et al., 2001; Greenleaf et al., 2004). The mutations E162D and E162A affect this critical pKa in the visible spectrum of human MnSOD; however, this e ffect appears to shift the pKa in opposite directions for each mutant (Figure 3-2). The change in this pKa should be explaine d in terms of the effects of these mutations on the ionization of Tyr34. This comment does not preclude changes in the ionization of liga nds of the metal, just that th ese appear to be outside of the pH range of these studies. Th erefore, the changes in the pKa of the visible absorption

PAGE 50

40 are attributed to Tyr34, the side chain of which is located 6.2 from the carboxylate of Glu162 from the adjacent subunit in wild-type MnSOD. The crys tal structures allow us to comment on the mutations at residue 162 on th e ionization of Tyr34. His163 is within 3.3 of S2 in the hydrogen-bond network, allowing indirect interaction between Glu162 and Tyr34 (Hearn et al., 2003; pdb accession #1LUV). The side chain of Asp162 in the E162D mutant maintains an interaction with Tyr34 through a solvent-bridged interaction with His163 while in E162A this interac tion does not exist. A lteration of Glu162 could affect the ionization of Tyr34 through its a ltered or abolished in teraction with His163. Understanding how Glu162 affects the ionizatio n of Tyr34 warrants further study. Catalysis Replacement of the second-shell ligand Glu162 by Ala and Asp resulted in diminished catalysis in human MnSOD (Tab le 3-1). Though not essential for catalysis, mutation of Glu162 resulted in at least a fi ve-fold decrease in rate constants k1-k3 for E162D and a 20-fold decrease for E162A. This is related to the di minished interaction between the Glu162 and His163 and the possible concomitant effects on the properties of the metal and active-site residues such as Tyr34. There is a preced ent for substantial changes in catalysis with mutations at a sec ond-shell ligand and at the dimeric interface. Table 3-3 Maximal values for kcat/Km and k0/ [E] for the catalysis of human wild-type MnSOD and mutants. WT b 800 500 E162Da 290 270 E162Aa 120 190 H30N c 130 2000 Y166F d 95 360 (a) 2mM TAPS pH 7.7, 50mM EDTA, 30 mM fo rmate (see methods for pulse radiolysis) (b) Ramilo et al., 1998 (c) Hearn et al., 2003 (d) Hearn et al., 2001 Kcat/Km k0/[E] ( M-1 sec-1) (sec-1)

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41 The replacement of the second-shell ligand Gln143 with Asn resulted in a 100-fold reduction in catalysis and evidence of an increas e in the redox potential of the active site (Leveque et al., 2000; Hsieh et al., 1998). The mutation Y166F at the dimeric interface of human MnSOD resulted in a 10-fold decrea se in catalysis (H earn et al., 2004). One interesting aspect of the catalysis k1 and k2 for the mutants at residue 162, as well as the step that forms the inhibited complex k3 (Figure 3-4), is that they appear to have pH profiles similar to th e titration of their visible sp ectra. E162D MnSOD showed a pH dependence for rate constants k1-k3 with values of kinetic pKa from 8.0 to 8.7 that roughly matched the pKa of the molar absorptivity (F igures 3-2, 3-4). This pH dependence was also evident in the values for kcat/Km (Figure 3-5). There was no observed pH dependence for the kinetic constants k1-k3 for E162A MnSOD in our pH range of 7.5 – 10.0, roughly consistent with its higher pKa derived from the pH dependence of its visible spectrum. Presum ably, the pH profiles for E162D and E162A are the result of the altered, indirect interaction between Glu162 and Tyr34. E162D MnSOD is the first reported variant of human MnSOD w ith a pH dependence that appears well within the range of practicable kinetic measurements and should be useful for further studies. Comparison with MnSOD from E. coli There have been rather few differences noted in k1 and k2 in catalysis between MnSOD from E. coli and humans; consequently, several differences revealed in this study warrant further attention. The mutant E162A human MnSOD reta ins specificity for manganese and is catalytically active although at about 5% the level of human wild type

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42 (Table 3-1). Additionally, it remains a tetramer in solution. This is in contrast to the equivalent mutation in E. coli E170A, which results in complete loss of catalytic activity, dimer destabilization in solution, and is acco mpanied by a change in specificity to Fe2+/3+ (Whittaker and Whittaker, 1998) It is possi ble that measurement of catalysis for E. coli E170A was not sensitive enough to measure 20-fo ld decreased catalysis. In addition, the presence of a tetrameric interface in the E162A enzyme confers added stability not present in the E. coli E170A, thus the E. coli mutant is both monomeric and dimeric in solution. However, the retention of metal speci ficity is unique to the human E162A. Comparison of the crystal structures of the human and E. coli forms of MnSOD shows nearly superimposable residues for the ligands of the metal and side chains Tyr34 and His30. However, there is a substructure of the active site that is considerably different for these two forms of MnSOD, and th is offers significant cl ues to the different responses of the human and E. coli forms of MnSOD to replacements at residue 162. Specifically, described here are the structural features likely to account for the more extensive changes in E. coli MnSOD compared with human MnSOD upon mutation at 162 and estimate how these structural features might relate to activ ity and ionization of Tyr34. Included in the interactions that form the dimeric interface of MnSOD is a van der Waals interaction betw een Phe66 and Gln119 (3.5 ) in the human enzyme (Quint and Reutzel et al., 2006; pdb # 1ADQ) and between Phe124 and Asn73 (4.0 ) in E. coli (Edwards et al., 1998; pdb # 1VEW) (Figure 38). The orientations of these residues are similar in the E. coli enzyme though they are at a gr eater distance from each other compared to human MnSOD. In addition, Phe124 from the adjacent subunit in E. coli MnSOD interacts with Tyr34 (3.2 ) in E. coli MnSOD. Dimeric destabilization of E.

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43 coli E170A could alter this inte raction with Tyr34 and thus affect ionization of the phenolic hydroxyl. The observation s described here clarify diffe rences in stability and in catalytic activity between th e two enzymes, though they do not sufficiently address the altered metal selectivity exhibited by the two mutants, opening another avenue for future study. Figure 3-8 Structure of E. coli MnSOD (g reen) superimposed on wild-type human MnSOD (blue) in the dimeric interface. Red sphere represents S2 from the hydrogen-bond network. Shown is the in teraction between Phe66 and Gln119 from the adjacent subunit (green). Also shown are Tyr34 and His30. Residues Phe124 and Asn73 (green) are shown for the E. coli enzyme with an interaction between Phe124 and Ty r34 denoted with a dotted line. Product Inhibition Product inhibition is a prominent feature of catalysis by human and E. coli MnSOD (Hearn et al., 2001). The rather significant decreases in k1-k4 describing catalysis and inhibition by E162D and E162A human MnSOD (Table 3-1), are similar to the observed zero-order component of catalys is. The maximal values of k0/[E], the normalized, zeroHis30 Tyr34 Phe66 Gln119 S2 Asn73 Phe124

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44 order rate constant for product inhibi tion, are similar for E162D, E162A, though diminished compared to wild-type MnSOD: 267 s-1, 189 s-1, and 500 s-1, respectively (Table 3-3) (Hsu et al., 1996). For E162A, the value of k0/[E] was reduced to 20 s-1 at pH > 8.5 reflecting the pH dependence of k4. The cause of the similar values of k0/[E] for E162D and E162A is due in significant pa rt to the values of the ratio of k2/k3 in the two mutants (Table 3-1). This is a gating ratio th at determines the extent of reaction that proceeds to catalysis versus inhibition (eq 3-2, 3-3), and the similar gating ratios for E162D and E162A are consistent with simila r extents of product inhibition. The gating for wild-type human MnSOD is 1:1 while it is ~1:2 for both E162D and E162A (Table 31). This gating ratio is 5:1 for E. coli MnSOD (unpublished) (Table 3-1) indicating a less product-inhibited enzyme. This represents another key difference between human and E. coli MnSOD that warrants further study. The side-chain of Glu162 is important fo r dissociation of the product-inhibited complex as evidenced by the similar values for k4 for both E162A and E162D (Table 3-1) The E162A mutant is the only mutant of human MnSOD observed to date that has exhibited a pH related decr ease in the value for k4. This provides another avenue for future investigation since neither the structur e of the inhibited complex nor its mechanism of dissociation is known. The values for k4 do not follow the same pH dependence as k1k3 because protonation of the bound peroxi de is a different process from k1-k3 (Hearn et al., 2001).

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45 CHAPTER 4 STRUCTURE OF NITRATED HUMAN MA NGANESE SUPEROXIDE DISMUTASE Introduction The presence of nitrated prot eins is associated with a number of pathological states (Ischiropoulos and Beckman, 2003; Radi, 2004; Sh ishehbor et al., 2003) and with certain diseases characterized by inflammatory pr ocesses (MacMillan-Crow et al., 2003). Human MnSOD in the presence of peroxyni trite is nitrated at a numbe r of sites, but the observed near complete inhibition of catalysis is asso ciated with the nitra tion of Tyr34 (Yamakura et al., 1998; MacMillan-Crow, Crow a nd Thompson, 1998; MacMillan-Crow and Thompson, 1999). Chapter 3 described the importa nce of the dimeric interfacial residue Glu162 and its role in su pporting an important pKa for catalysis through its interaction with Tyr34. Building on the findings of chapters three, the structure of nitrated human MnSOD was solved, emphasizing the importance of Tyr34 in catalysis and providing a structural explanation for peroxynitrit e-mediated inactivation of MnSOD. The side chain of Tyr34 plays an importan t role in catalysis. The replacement of Tyr34 with Phe causes minor effect s on the catalytic efficiency (kcat/Km) of human and E. coli MnSOD (Guan et al., 1998; Whittaker and Whittaker, 1997); however, it does decrease by 10-fold the value of kcat which determines the maximal velocity of catalysis (Guan et al., 1998). The crystal structure of the mutant human MnSOD with Phe34 is nearly identical to that of wild type, w ith Phe34 closely superimposed on the phenolic side chain of Tyr34 in the wild-type (Gua n et al., 1998). Moreover, the replacement of

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46 Tyr34 with Phe causes no significant change in the redox potential of the human enzyme (Leveque et al., 2001). Nitration of tyrosine occurs through two possible mechanisms shown in figure 4-1. .NO + O2 .ONOO+ H ONOOH [.NO2 + .OH] (1) ONOO+ CO2 ONOOCO2 [.NO2 + CO3 .-] (2) Figure 4-1 Scheme for nitration of tyrosine in the presence of peroxynitrite showing nitration through the pathway of equation 2. Nitration of biomolecules by peroxynitrite is enhanced by the presence of CO2 with the reaction of equation 2 predominating over the reaction of equation 3. Production of carbonate radical promotes tyrosyl form ation and subsequent reaction with .NO2 yields 3nitrotyrosine (Figure 4-1) (B onini et al., 1999). Nitration of tyrosine 34 in MnSOD is associated with abolished activity. The X-ray crystal structure of nitrated wild-type human MnSOD, as well as the unmodified enzyme, were both resolved to 2.4 resolution. Although mass spectrometry detected partial nitration of several tyrosi nes and a tryptophan near the surface of the protein, the crystal structure show s only nitration of Tyr34 in the active site. This nitrated side chain, 3-nitrotyrosin e 34, exhibited only one conformer with the nitro group extending toward the metal-bound hydroxide /water but not forming a hydrogen bond with it. Instead the O1 of the nitro group appears to form a hydrogen bond with the N 2 of residue Gln143. The structure of the nitrat ed enzyme including active-site residues and OH COO-+H3NCO3.+ + .NO2nitrosoperoxycarbonate tyrosyl radical tyrosine 3-nitrotyrosineCH2C.OH COO-+H3N CH2OH COO-+H3N CH2NO2

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47 the phenyl ring of 3-nitrotyros ine 34 are closely superimpos able with the unmodified wild-type MnSOD. The confor mation in the active-site cavity of the nitrated MnSOD strongly suggests inhibition by steric interf erence, by a possible weakening of a hydrogen bond network, and by the electrosta tic effects related to the pr esence of the nitro group and the resulting change of the redox potential. Materials and Methods Preparation of Nitrated Human MnSOD Peroxynitrite was produced by mixing equal volumes (5 ml) of NaNO2 (0.8 M) and acidified H2O2 (0.7 M H2O2 and 0.3 M HCl) in a manually operated dual-syringe mixer and quickly quenched with (3 ml) 3 M Na OH (Crow, Beckman and McCord, 1995). The final solution was purified using a MnO2 gravity column to remove excess H2O2 and a subsequent Chelex 100 gravity column to remove extraneous metal ions. The concentration of peroxynitrite was determin ed by measuring optical density at 302 nm ( 302 = 1670 M-1 cm-1) (Hughes and Nicklin, 1968). The nitration of human MnSOD was carried out by the bolus addition of pe roxynitrite to MnSOD in the presence of CO2/HCO3 -. MnSOD samples were equi librated overnight at 4oC prior to reaction with peroxynitrite. The mixed solution contained MnSOD (50 M), all species of CO2 (25 mM), peroxynitrite (8 mM), and phosphate buffer (20 mM) at pH 7.8 and 25o C. Following reaction with peroxynitrite, sample s of modified enzyme were pooled and concentrated in 20 mM phosphate pH 7.8. Modified enzyme was digested with acetylated trypsin for 3 hours at 37o C. Capillary reverse phase HPLC separation of tryptic fragments was performed on a PepMap C18 column (LC Packings, San Franci sco, CA) in combination with an Ultimate

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48 Capillary HPLC System (LC Packings, San Fran cisco, CA) operated at a flow rate of 200 nL/min. Extent of nitration was measured using a QSTAR XL (LC/MS/MS system, MDS Sciex, Ontario, Canada). To determine the ra tio of nitrated versus unmodified MnSOD, the peak areas for modified and unmodified masses were compared. We determined the following extents of nitration: 75% nitration of Tyr34, 36% ov erall nitration of Tyr9 and Tyr11 (residues 9 and 11 were in the same tryptic fragment), 46% overall nitration of Trp180 and Trp186 (residues 180 and 186 were also in the same tryptic fragment). Crystallization Wild-type and nitrated human MnSOD were buffer exchanged into 20 mM phosphate buffer at pH 7.8 and concentrated (16 mg/ml) using a centricon YM-10 (Amicon). The samples were crystallized using the hanging drop vapor diffusion method (McPherson, 1982). The drops consisted of 5 l of enzyme mixed with 5 l of precipitant solution (3 M ammonium phosphate, 100 mM imidazole, 100 mM malate) and suspended over 1 ml of precip itant solution at 25o C. The crystals grew to full size (0.8 x 0.5 x 0.5 mm) in approximately one week. Data Collection and Processing Both wild-type and nitrated human MnSOD X -ray diffraction data were collected from single crystals, wet mounted in quart z capillaries (Hampton Research), on an RAXIS IV++ image plate (IP) system with Osmic mirrors and a Rigaku HU-H3R CU rotating anode operating at 50 kV and 100 mA (Rigaku/MSC). A 0.3 mm collimator was used with a crystal to IP distance of 220 mm and the 2 angle fixed at 0o. The frames were collected using a 0.3o oscillation angle with an e xposure time of 5 min/frame at room temperature. Both data sets were indexed using DENZO a nd scaled and reduced

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49 with SCALEPACK software (Otwinowski and Minor, 1997). Diffractio n intensities were visible to 2.4 resolution and a total of 200 frames were collected from both wild type and nitrated huma n MnSOD crystals. Structure Determination and Refinement To prevent any model phase bias, the init ial phasing model for both the unmodified and nitrated human MnSOD wa s the structure of the W161A MnSOD mutant (Hearn et al. 2001; pdb accession number 1JA8) from wh ich the nine tyrosines (residues: 9, 11, 34, 45, 165, 166, 169, 176, and 193) and six tryp tophans (residues: 78, 123, 126, 161, 181, and 186) had been replaced by alanines, and the Mn2+ ion and solvent molecules had been removed. The structures were phased a nd refined using the software package CNS (Brunger et al., 1998). Refinement cycling (using rigid body, si mulated annealing (for the first cycle), minimization, and individual B-f actor refinement) was interspersed with rounds of manual model building using the mo lecular graphics program O (Jones et al., 1991). Following the first cycle of refinement, the positions of the manganese ion, nine tyrosines, and six tryptophans were clearly identified and built into Fo-Fc electron density maps for both structures. After the second cy cle of refinement the unambiguous electron density for a nitro group was observed in the vicinity of the C 1 atom of tyrosine 34 for the nitrated human MnSOD and a model fo r a 3-nitrotyrosine residue was built and energy minimized using the PRODRG2 serv er (Schuettelkopf and Aalten, 2004) and placed into the electron density. The mass spectrometric analysis had previously determined the extent of Tyr 34 nitration to be ~75 %. Due to this observation, the atoms comprising the nitro group were refined with an occupancy of 0.75 compared to the rest of the atoms in the protein and solvent. Both structures were furt her refined for several more cycles with some minor manual bui lding, after which solvent molecules were

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50 picked both automatically in CNS (using a 3 sigma cut off) and manually in O by inspection of Fo-Fc electron density maps. The bond geometry of the models was analyzed using the software package PROC HECK (Laskowski et al., 1993). The final, refined models and structure factor files have been deposited with the Protein Data Bank, PDB (accession codes 2ADQ, and 2ADP for the unmodified and nitrated human MnSOD, respectively). Results The structures of the unmodified and th e nitrated human wild-type MnSOD have been solved in the hexagonal space group P6122, with unit cell para meters a = b = 81.3 and c = 242.2 , and refined to 2.4 resolu tion (Table 4-1, Supplementary material 1 and 2). The final refined structure of the unmodified human MnSOD had an Rcryst of 21.7 % (Rfree of 24.0 %) with an aver age B-factor of 26.2 2 ; and the nitrated human MnSOD had an Rcryst of 19.7% (Rfree of 21.8 %) with an average B-factor of 31.2 2 (Table 4-1). Following the second cycle of refinement the position of a single 3nitrotyrosine 34 was built into Fo-Fc and 2 Fo-Fc electron density maps in the nitrated human MnSOD structure (Figure 4-2). The occupancy for the nitro group on 3nityrosine34 was modified to reflect the percent nitration determined by mass spectrometry. There was no unique density su rrounding tyrosines 9 and 11 or tryptophans 181 and 186. A comparison of the wild-type and nitrat ed human MnSOD structures showed no significant side-chain conformational changes or solvent displacement in the active site with the nitration of tyro sine 34 (Figure 4-3). The root-mean-squared difference for all C atoms was 0.13 comparing th e nitrated with unmodified MnSOD. The active-site

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51 manganese of wild-type human MnSOD has been previously reported (Borgstahl et al., 1992) as coordinated by the N 2 of three histidine residues (H26, H74, H163), O 1 of Asp159, and a Mn bound water/hydroxide molecu le arranged in a distorted trigonal bipyramidal geometry. The ni tration of Tyr34 caused no si gnificant change in this geometry or in first shell ligand distances (F igure 4-2). Also of interest was that the overall refined thermal individual atom B va lues for both structures were similar and there was no significant difference for the ma nganese in unmodified and nitrated human MnSOD with values of 16.9 and 21.6 2, respectively. Of note, which may or may not be of significance, was the B values for the Mn bound water/hydroxide molecule which was 11.6 2 for wild-type and nearly twice the value 19.1 2 for nitrated human MnSOD. The significance of this can only be resolv ed with either higher resolution X-ray diffraction data or a neut ron diffraction structure. In comparing the crystal structures of th e nitrated and unmodified wild-type human MnSOD, we observed that the side chain of 3nitrotyrosine 34 was in a single side-chain conformer with the O1 and O2 positi oned 3.6 and 3.8 from the manganese ion. Probably the most significant interaction observed by the ni tration of Tyr34 is the inferred hydrogen bond (3.1 ) between O1 of 3-nitrotyrosine 34 and N 2 of Gln143 (Table 4-2, Figure 4-3). A comparison of the two non-crystallogra phic subunits of the human nitrated MnSOD in the hexagonal space group P6122 (data not shown) showed no differences in orientation of the nitrated Tyr34 residues and they were subs equently averaged in the refinement protocols. There was also no evidence of dityrosine (3,3’-dityrosine) formation in the crystal structure.

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52 Figure 4-2 Structure of the active site of the nitrated human MnSOD. Stereo diagram showing the initial, no model bias (Fo-Fc) and (2Fo-Fc) electron density maps contoured at 3.0 (black) and 1.0 (grey), respectively, into which the 3nitrotyrosine 34 and manganese-bound hydroxide were modeled. The active site manganese (pink) is pentacoordi nate with inner shell ligands His26, His74, His163, Asp159, and the metal-bound solvent molecule. Figure 4-3 The structure of the active-site regi on of nitrated (yellow) superimposed onto unmodified human MnSOD (green). A) The proposed hydrogen bond network (red dashed lines) for unmodified wild type, and B) the proposed hydrogen bond network (blue dashed lines) for n itrated human MnSOD. The proposed hydrogen bond network involves residues Gln143, Tyr34, His30, solvent molecule (W2) and Tyr166. See Table 4-2 for a complete listing of distance geometry.

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53 Table 4-1 X-ray crystallographic structure st atistics of unmodified and nitrated human MnSOD The nitration at Tyr9 and 11 was less ex tensive (36% overall) and was not observed in the crystal structure; mobility of these surface residues may have made the nitration less evident. Nitration at Tr p180 and 186 was also not observed in the crystal structure. Discussion The structure of human MnSOD containing 3-nitrotyrosine at position 34 is welldefined by the 2.4 electron density map (Figur e 4-1) with a three-dimensional structure closely superimposable with the unmodified enzyme (Figure 4-2); specifically, there are Unmodified Nitrated Data Collection Resolution () 20.0-2.4 (2.49-2.4)*20.0-2.4 (2.49-2.4) Space group P6122 P6122 Unit cell () a=b=81.3, c=242.2 a = b =81.3, c =242.2 Molecules/a.s.u. 2 2 aRsym (%) 10.2 (19.1) 10.9 (14.9) Reflections (total/unique) 244,364 / 61,091 231,776 / 60,459 Completeness (%) 95.8 (90.2) 91.3 (80.6) Refinement Non-hydrogen atoms 1629 1605 Water molecules 74 46 Average B factors (2) Protein main-chain 25.1 30 Protein side-chain 27.2 32.3 Solvent molecules 35.9 36.8 Tyrosine 34 20.3 27.6 Rcryst/Rfree (%) 21.7 / 24.0 19.7 / 21.8 R.m.s.d bond lengths () /angles (o) 0.006 / 1.3 0.006 / 1.3 *Data for the highest resolution sh ell are given in parentheses. aRsym = |I-| / I x 100, where I is the intensity of a reflection and is the average intensity. bRcryst = hkl |Fo-KFc| / hkl |Fo| x 100, Rfree is calculated from 5% randomly selected data for cross-validation. cR.m.s.d. = root mean square deviation.

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54 no conformational changes in the active-site cavity. This is the first reported crystal structure of a nitrated MnSOD, to our knowledge. Table 4-2 Distance geometries () in the active-sites of unmodified and nitrated human MnSOD Interaction Wild-typeNitrated Mn2+ aSolvent W12.1 2.1 aSolvent W1 Q143-N 2 3.1 3.1 Q143-N 2 Y34-OH 2.5 3.1 Y34-OH aSolvent W23.1 3.3 aSolvent W1 Y34-O1 --3.5 Q143-N 2 Y34-O1 --3.1 Y34-O1 Mn2+ --3.6 Y34-O2 Mn2+ --3.8 aSolvent W2 H30-N 1 2.9 3.0 H30-N 2 Y166-OH 2.6 2.6 aSee figure 4-2 for position of solvent W1 and W2. The nitrated Tyr34 side chain shows only a single conformer with the nitro group directed toward the metal (Figure 4-1). Th is conformer predominates in large part because of electrostatic gradients within the active-site cavity as discussed below; however, there are probably also steric consid erations that limit the range of side-chain conformations of nitrated Tyr34. For example, the side chain of Phe66 is within 3.6 of that of 3-nitrotyrosine 34 and limits the range of orientations of this nitrated residue. In addition, there is a role for manganese in the reaction of MnS OD with peroxynitrite (Quijano et al., 2001); hence, the orientation of 3-nitrotyrosine in the nitrated enzyme may reflect the reactive site of the side chai n of Tyr34 that is closest to the metal. Although the nitro group of modified Tyr34 is near the manganese bound hydroxyl/water, the distance between oxygen atoms at 3.5 (Table 4-2) is too great to form a hydrogen bond, nor is the nitro group sufficiently close, ~3.7 , to the metal to be considered an inner shell ligand. The nitro group is viewed more accurately as a second-

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55 shell ligand of the manganese, although its presence causes no changes in geometry or distances of the first-shell lig ands. However, the crystal stru cture is consistent with a hydrogen bond between the nitro group at Tyr34 and the N 2 of Gln143, with a distance of 3.1 from the O1 of n itrated-Tyr34. There is stil l evidence for a hydrogen bond between the N 2 of Gln143 and the phenolic OH of nitrated-Tyr34 with a distance of 3.1 (Table 4-2, Figure 4-2). However, this dist ance is considerably lengthened compared with that of the unmodified enzyme and this hydrogen bond involving N 2 of Gln143 may be bifurcated between the phenolic OH a nd the nitro group of 3-nitrotyrosine 34. Nitration of human MnSOD i nhibits catalysis by greater than 90% (Yamakura et al., 1998; MacMillan-Crow et al., 1998). This in hibition is associated with nitration of Tyr34 (Yamakura et al., 1998; MacMillan-Cro w et al., 1998), alt hough there is evidence that nitration of othe r tyrosine residues may also decr ease activity (MacMillan-Crow et al., 1999). We comment then on the likely causes of inhibiti on by MnSOD containing 3nitrotyrosine at position 34. First, it app ears that nitration ha s not affected the stereochemistry of the active site residues; that is, the conformation of Gln143, which forms a hydrogen bond with the aqueous ligand of the metal, is not altered and the sidechain orientation of Tyr34, a lthough nitrated, is not change d. So conformational changes induced by nitration are not pertinent in the inhibiti on. However, the hydrogen bond network involving the side chains of Gln143, Tyr34, His30, and Tyr166 from an adjacent subunit appears to be altered and possibly weak ened at 3-nitrotyrosine (Table 4-2, Figure 4-2), as mentioned above. This network, and pa rticularly Tyr34, has been associated with proton transfer in catalysis by wild-type MnSOD, either proton transfer to product peroxide or to the metal-bound hydroxide (Whittaker and Whittaker, 1997; Silverman

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56 and Nick, 2002; Hunter et al ., 1997; Sorkin, Duong and Miller 1997; Bull and Fee, 1985; Stallings et al., 1991), and alteration of this network in th e nitrated enzyme is consistent with inhibition. Another likely cause of inhibition is simply that the bulk of the nitrated side chain inhibits catalysis, perhaps by its presence in the substrate access channel and/or steric overlap with the enzyme-substrate complex or wi th the transition state. This was the case with H30V MnSOD in which the C of Val 30 is 4.4 from the manganese and either blocks the substrate access channel or has a steric overlap with the developing transition state. The O1 and O2 of the ni tro group of nitrated Tyr34 ar e closer to the metal (near 3.7 ) and also lie along a likely substrate access channel. The substrate access channel in MnSOD and FeSOD is exceedingly narrow a nd there is evidence from dynamics simulations that substrate diffusion to the vicinity of the manganese requires conformational fluctuations along this cha nnel (Sines et al., 1990). A prominent side chain, the motion of which can open this channe l, is Tyr 34 (Sines et al., 1990). Chemical modification by nitration of the phenolic side chain would certainly slow this process. Yet another possibility for inhi bition is the change in pKa of nitrated Tyr, which is expected to be lower by about 2 pKa units compared with unmodified Tyr. We have no measure of the pKa of nitrated Tyr34 in MnSOD, a lthough this value was estimated from spectroscopic data at pKa 7.95 for FeSOD nitrated at Tyr34 (Soulere, 2001). Nitrated tyrosine would have a larger fraction as ty rosinate anion than unmodified, and could decrease catalysis by electrosta tic repulsion of the substrate O as well as have a major effect on the redox potential of the enzyme (Miller et al., 2003). The active-sites of MnSOD and FeSOD are very finely tuned to catalyze both the oxidative and reductive

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57 stages of catalysis (Vance and Miller, 1998) and introduction of a nitro group near the metal is likely to alter this tuning. That is, the nitration of Tyr 34 has almost certainly altered the redox potential. This study provides a connection between the nitration and subse quent inhibition of MnSOD. The location in the active site of the NO2 group of 3-nitrotyrosine 34 gives a basis for understanding the str ong inhibition of this essentia l antioxidant enzyme. It is notable that replacement of Tyr34 with Phe ha s very little effect on catalysis up to low micromolar concentrations of O2 (Guan et al., 1998; Whittaker and Whittaker, 1997) and does not decrease thermal stability or a lter the crystal structur e (Guan et al., 1998). Yet its prominence in the active-site cavity ma kes it a site for nitra tion and inhibition of MnSOD.

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58 CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS Conclusions This dissertation focused on the two stru cturally unique interfaces of human MnSOD. The overall goal was to understand the contributions to stability and catalysis of the two interfaces and to compare the te trameric human MnSOD to the dimeric E. coli MnSOD. The data presented in chapters 2 and 3 of this study suggest that the tetrameric interface provides stability wh ile dimeric interfacial residue s play a greater role in catalysis. The importance of Tyr34 in mainta ining catalysis was explored in chapter 4 with a structure of nitrated human MnSOD. The Tetrameric Interface in Human MnSOD One key characteristic of human Mn SOD that differentiates it from E. coli is the presence of a tetrameric interface. Chapter 2 presented 19F NMR and differential scanning calorimetry studies elucidating the ro les of the two interfaces of human MnSOD and the data suggest a stabilizing role for th e tetrameric interface. Though the tetrameric interface is generally associated with higher organisms, it may in fact be a more primitive structural arrangement. The endosymbiotic theory suggests a b acterial origin for mitochondria; perhaps mitochond ria resulted from phagocytosis by a much larger cell billions of years ago. MnSOD may have orig inally been a tetram er to accommodate a higher average temperature caused by increased atmospheric CO2 levels. As atmospheric CO2 levels waned in the presence of photos ynthesizing cyanobacter ia, prokaryotes and eukaryotes diverged and the mitochondrion be came a selective advantage for eukaryotes.

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59 It is interesting that some heat extremophiles, like T. thermophillus and P. aerophilus utilize tetrameric MnSODs (Wagner et al., 1993; Whittaker and Whittaker, 2000). The Dimeric Interface and Differentia l Roles of Glu162 in Human MnSOD and Glu170 in E. coli MnSOD Considering the importance of the dimeri c interface in stability and catalysis, chapter 3 emphasized the contribution to st ability and catalysis of Glu162 in human MnSOD and Glu170 in E. coli MnSOD. In addition to an observed decrease in catalysis and altered product inhibition, E162D exhib ited a pH dependence for catalysis not observed in the native enzyme. This observed pKa for catalysis may be the result of an altered interaction with Ty r34 through His163 and a bridging solvent molecule. The E162A mutant is less stable, though it is still te trameric in solution and is associated with a significantly diminished catalysis for the enzyme, emphasizing the importance of Glu162 in supporting catalysis and stabilizing the enzyme. In contrast, the equivalent mutation in the E. coli MnSOD, Glu170Ala, exhibits no activ ity, is selective for iron over manganese is a mixture of monomer and dimer in solution. This represents a significant difference between human and E. coli MnSOD, the active sites of which are structurally equivalent. A stabilizing inte raction between Phe66 and Gln119 in the dimeric interface of human MnSOD is absent in the E. coli enzyme, thus the E170A mutant destabilizes to a greater extent than E162A. In addition, an interaction between Ph e124 and Tyr34 exists in the E. coli MnSOD that is not present in th e human enzyme. This could affect ionization of Tyr34 for the E. coli enzyme and perhaps affect its metal sel ectivity, though the latter remains an area for further research.

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60 A Structural Explanation for Abolishe d Catalysis of Nitrated Human MnSOD To add to a growing literature on the reaction of peroxynitrite with MnSOD, chapter 4 presented a structural explanati on for abolished catalys is resulting from nitration of Tyr34. Previous studi es have shown that nitration of Tyr34 is associated with complete catalytic inhibition (MacMillan-C row et al., 1995) (Yamakura et al., 1996). The structure of nitro-MnSOD indi cates that diminished catalys is is the result of steric blockade of the substrate as we ll as possible electrostatic repu lsion of superoxide anion. These findings will help elucidate the role of nitro-MnSOD in certain diseases involving peroxynitrite-mediated nitration of biomolecules. Future Directions The Dimeric Interface of Human MnSOD The peaks corresponding to fluorine labele d tyrosines shown in chapter 2 were significantly broad and four of the nine fluor ine labeled tyrosines were not observed in the fluorine NMR spectrum (F igure 2-2). The peaks for Tyr34, Tyr165, Tyr166 and Tyr176 were not observed and may have been broadened due to their proximity to the paramagnetic manganese of the active site; all four tyrosines that were not observed are within 10 of the active site metal. One way to alleviate this problem would be to construct an apo-fluoro-MnSOD. This w ould involve expression and subsequent chelation of the metal from fluoro-MnSOD. Th e manganese is tightly bound in the active site and chelating the metal would involve denaturation and refolding of the enzyme. Initial attempts to chelate the manganese ha ve resulted in an unfolded enzyme. Further attempts may involve the use of other chel ators and perhaps changes in pH. Expression with a non-paramagnetic metal s ubstitute may also alleviate the issue of excessively broadened lines.

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61 Redox properties of E162 mutants Measurement of redox properties of human MnSOD are difficult due to the size of the active site and require an electron mediator to measur e the redox potential (Leveque et al., 2001). Mediators incl uding ferricyanide and pentacy anoaminoferrate have been used successfully though the effective range of study is limited; to use ferricyanide, the midpoint potential for the enzyme must be below 435 mV. The cause of reduced catalysis in mutants E162A and E162D is not fully understood and measurement of the redox potential of E162D and E162A MnS OD may elucidate these causes. Catalytic Properties of Nitrated MnSOD Indirect studies (including xanthine oxidase assays) have shown that nitration of MnSOD abolishes activity of MnSOD. Dir ect methods like stopped-flow and pulse radiolysis have not been used to measure cata lysis of nitro-MnSOD, in part because it is difficult to achieve 100% nitration of MnSOD. Catalytic studies could provide a more thorough explanation for catalytic decrease. Chapter 4 described the structure of nitrated MnSOD comprised of 74% nitration of Ty34. A pulse radiolysis study would require the purification of 100% nitrated enzyme usi ng affinity chromatography. It would be interesting to determine rate constants k1-k4 for fully nitrated enzyme to see how nitration has altered catalysis of MnS OD. For instance, the enzyme could quickly en ter a product inhibited state from whic h it does not dissociate. Future of Therapeutic Studies The potential for human MnSOD in drug discovery is great and future therapeautic studies will focus on the dimeri c interface. For example, MnSOD mutants that are less product-inhibited, such as H30N are useful as anti -proliferative agents (Davis et al., 2004) and coul d potentially be used as life-saving therapies during

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62 reperfusion injury. In addition, the pres ence of nitrated MnSOD will serve as a biomarker for chronic diseases such as allograf t rejection and could pot entially be used as markers for cancer.

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68 Miller, A. F. (2004) Superoxide dismutases: acti ve sites that save, but a protein that kills, Curr. Opin. Chem. Biol. 8 162-8. Miller, A.-F., Padmakumar, K., Sorkin, D. L., Karapetian, A., Vance, C. K. (2003) Proton-coupled electron tran sfer in Fe-superoxide di smutase and Mn-superoxide dismutase. J. Inorg. Biochem. 93 71-83. Mizuno, K., Whittaker, M. M., Bachinger, H. P., Whittaker, J. W. (2004) Calorimetric studies on the tight binding metal intera ctions of Escherichia coli manganese superoxide dismutase, J. Biol. Chem. 279 27339-44. Otwinowski, Z. a. M., W. (1997) Processi ng of X-ray diffraction data collected in Oscillation mode, Methods Enzymol. 276 307-326. Purrello, M, Di Pietro, C, Ragusa, M, Pulv irenti, A, Giugno, R, Pi etro, VD, Emmanuele, G, Travali, S, Scalia, M., Ferro, A. (2005) In vitro and in silico cloning of Xenopus laevis SOD2 cDNA and its phylogenetic analysis DNA Cell Bio. 24 111-116. Quijano, C., Hernandez-Saavedra, D., Castro, L., McCord, J. M., Freeman, B.A., Radi, R. (2001) Reaction of peroxynitrite with Mn-superoxide dismutase. Role of the metal center in decomposition kinetics and nitration. J. Biol. Chem. 276 1163111638. Quint, P., Reutzel, R., Mikulski, R., McKenna R., Silverman, D. (2005) Crystal structure of nitrated human manganese superoxide dismutase: mechanism of inactivation, Free Radic. Biol. Med. 40 453-458. Quint, P.S., Ayala, I., Busby, S. A., Chalmers, M. J., Griffin, P. R., Rocca, J., Nick, H. S., Silverman, D. N., (in press) Structural mobility in human manganese superoxide dismutase, Biochemsitry Rabini, J., Nielson, S. O. (1969) Absorp tion specrum and decay kinetics of O2 .and HO2 in aqueous solutions by pulse radiolysis, J. Phys. Chem 73 3736-3744. Radi, R. (2004) Nitric oxide, oxidant s, and protein tyrosine nitration. Proc. Nat. Acad. Sci. USA 101 4003-4008. Radi, R., Cassina, A., Hodara, R., Quijano, C., Castro, L. (2002) Pe roxynitrite reactions and formation in mitochondria. Free Rad. Biol. and Med. 33 1451-1464. Ren, X., Bhatt, D., Perry, J. J. P., Tainer, J. A., Cabelli, D. E., and Silverman, D. N. (in press) Kinetic and structural charac terization of human MnSOD containing 3fluorotyrosine. J. Molec. Structure Santos, C. X., Bonini, M. G., and Augusto, O. (2000) Role of the ca rbonate radical anion in tyrosine nitration and hydroxylation by peroxynitrite, Arch. Biochem. Biophys. 377 146-52.

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69 Schuettelkopf, A. W., van Aalten, D. M. F. (2004) PRODRG a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallographica D60 13551363. Schwarz, H. (1981) Free radicals genera ted by radiolysis of aqueous solutions, Journal of Chemical Education 58 101-105. Shishehbor, M. H., Aviles, R. J., Brennan, M. L., Fu, X., Goormastic, M., Pearce, G. L., Gokce, N., Keaney, J. F. Jr, Penn, M. S., Sprecher, D. L., Vita, J. A., Hazen, S. L. (2003) Association of nitr otyrosine levels with car diovascular disease and modulation by statin therapy. JAMA 289 1675-80. Silverman, D.N. and Nick, H. S. (2002) Ca talytic pathway of manganese superoxide dismutase by direct observation of superoxide. Methods Enzymol. 349 61-74. Sines, J., Allison, S.A., Wierzbicki, A. and McCammon, J.A.. (1990) Brownian Dynamics Simulation of the Superoxide-S uperoxide Dismutase Reaction: Iron and Manganese Enzymes. J. Phys. Chem 94 959-961. Slykhouse, T. O., and Fee, J. A. (1976) P hysical and chemical studies on bacterial superoxide dismutases. Purification and some anion binding properties of the ironcontaining protein of Escherichia coli B, J. Biol. Chem. 251 5472-5477. Sorkin, D. L., Duong, D. K., Miller A. F. ( 1997) Mutation of tyrosine 34 to phenylalanine eliminates the active site pK of reduced iron-containing supe roxide dismutase. Biochemistry 36 8202-9208. Soulere, L., Claparols, C., Perie, J., Hoffm ann, P. (2001) Peroxynitr ite-induced nitration of tyrosine-34 does not inhibit Escher ichia coli iron superoxide dismutase. Biochem. J. 360 563-567. Stallings W. C., Metzger, A. L., Pattridge K. A., Fee J. A., Ludwig M. L. (1991) Structure-function relationships in iron and manganese superoxide dismutases. Free Radical Res. Commun. 12-13 259-268. Tu, C., Quint, P., and Silverman, D. N. ( 2005) Exchange of (18)O in the reaction of peroxynitrite with CO(2), Free Radic. Biol. Med. 38 93-97. Vance, C. K., Miller, A. F. (1998) Spectrosc opic comparisons of the pH dependencies of Fe-substituted (Mn)superoxide dismut ase and Fe-superoxide dismutase. Biochemistry 37 5518-5527. Wagner, U. G., Pattridge, K. A., Ludwig, M. L., Stallings, W. C., Werber, M. M., Oefner, C., Frolow, F., and Sussman, J. L. (1993) Comparison of the crystal structures of genetically engineered human manganes e superoxide dismutase and manganese superoxide dismutase from Thermus ther mophilus: differences in dimer-dimer interaction, Protein Sci. 2 814-825.

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70 Weisiger, R. A., and Fridovich, I. (1973) M itochondrial superoxide simutase. Site of synthesis and intramito chondrial localization, J. Biol. Chem. 248 4793-4796. Whittaker, M. M. and Whittaker J. W. (1997) Mutagenesis of a proton linkage pathway in Escherichia coli manganese superoxide dismutase. Biochemistry 36 8923-8931. Whittaker, M. M. and Whittaker, J. W. (1998) A glutamate bridge is essential for dimer stability and metal selectivity in manganese superoxide dismutase. J. Biol. Chem. 273 22188-22193. Whittaker, M. M., and Whittaker, J. W. (2000) Recombinant superoxide dismutase from a hyperthermophilic archaeon, Pyrobaculum aerophilium, J. Biol. Inorg. Chem. 5 402-408. Wintjens, R., Noel, C. May, A. C. W., Gerbod, D., Dufernez, F., Caparon, M., Viscogliosi, E. and Rooman, M. (2004) Sp ecificity and phenetic relationships of Feand Mn-containing superoxide dismut ases on the basis of structure and sequence. J. Biol. Chem. 279 9248-9254. Yamakura, F., Taka, H., Fujimura, T., Mu rayama, K. (1998) Inactivation of human manganese-superoxide dismutase by peroxyni trite is caused by exclusive nitration of tyrosine 34 to 3-nitrotyrosine. J. Biol. Chem. 273 14085-14089.

PAGE 81

71 BIOGRAPHICAL SKETCH In 1995, Patrick Quint received his high school diploma from Rocky Mountain High School in Ft. Collins, CO, and then moved to St. Paul, MN, where he attended Macalester College. Upon receip t of his BA in biology, he worked at 3M for two years before attending graduate school at University of Florida. After successfully defending his dissertation, he will receive a post-doctoral appointment in the lab of Dr. Bob Bergen at the proteomics division at Ma yo Clinic in Rochester, MN.


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KINETIC AND STRUCTURAL EFFECTS OF INTERFACIAL INTERRUPTION AND
PROTEIN NITRATION IN HUMAN MANGANESE SUPEROXIDE DISMUTASE















By

PATRICK QUINT


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


2006


























Copyright 2006


by

PATRICK QUINT















ACKNOWLEDGMENTS

Sincere thanks go to my parents, brothers and sisters, and all those with whom I

have shared the joy and frustration of this work. However, this dissertation would not

have been possible without the patient encouragement of my wife Ingvild, the impish

smile of my son Anders or the expert mentorship of Drs. Silverman and McKenna.















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iii

LIST O F TA BLE S ....................... ........................... ........... .. ............ vi

L IST O F FIG U R E S .... ...... ...................... ........................ .. ....... .............. vii

ABSTRACT ........ .............. ............. ...... ...................... ix

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

Superoxide Form ation and R eactions............................................... ..................
Classes of Superoxide D ism utase.......................................................... ............... 2
Hum an M anganese Superoxide D ism utase ........................................ .....................2
Active Site and Hydrogen Bond Network................... .......... ................ ...2
Manganese Superoxide Dismutase and its Reaction with Superoxide................3
The Subunit Interfaces of Human Manganese Superoxide Dismutase ...............4
P eroxy nitrite .................................................. .......................... 4
T yrosine N itration ................................................................. ............ .. 5
R e se arch G o als ...................................... ............... ........... ....... ...............6
Interfacial Mobility at the Dimeric and Tetrameric Interfaces of Human
M anganese Superoxide Dismutase ....................................... ............... 7
Replacement of a Key Dimeric Interfacial Residue...........................................7
Structure of Nitrated Human M nSOD .............................................. ............... 8

2 STRUCTURAL MOBILITY IN HUMAN MANGANESE SUPEROXIDE
D ISM U TA SE ........................................... ............................ .. 9

Intro du action ..................................................................................................... .... .. 9
M materials and M ethods ................................................................................... 11
Labeling with Fluorotyrosine, Expression in E. coli, and Purification ...............11
Site-Directed Mutagenesis of Fluorine Manganese Superoxide Dismutase .......12
19F Nuclear Magnetic Resonance Spectroscopy .........................................13
Differential Scanning Calorim etry ............................ ................................... 14
R e su lts ......... .. ... ... .......................................... 14
Assignment of Fluorine Resonances .................. ...................................14
Therm al Stability .................... ............................................... 17
Temperature Dependence of Fluorine Resonances ...................... ........ 17









D iscu ssio n ...................................... ................................................. 19

3 ROLE OF A GLUTAMATE BRIDGE SPANNING THE DIMERIC
INTERFACE OF HUMAN MANGANESE SUPEROXIDE DISMUTASE ............24

Introduction ................. ....... .......................................................... 24
M materials and M methods ....................................................................... ..................2 5
R e su lts ................................................... ......................... ................2 9
D isc u ssio n ................................................... ................... ................ 3 7

4 STRUCTURE OF NITRATED HUMAN MANGANESE SUPEROXIDE
D ISM U T A SE ....................................................... ................ 4 5

In tro du ctio n ...................................... ................................................ 4 5
M materials and M methods .................................................................... .....................47
Preparation of Nitrated Human MnSOD..........................................................47
C ry stalliz atio n ............................ ........... ...... ................ ................ 4 8
Data Collection and Processing......................... .............. ............... 48
Structure Determination and Refinement............................ ................49
R e su lts ............... ......................................... ............................... 5 0
Discussion .............. ............ .... ...... ........ ......................... 53

5 CONCLUSIONS AND FUTURE DIRECTIONS .....................................................58

C o n c lu sio n s .......................... ......... ................................ ................ 5 8
The Tetrameric Interface in Human M nSOD ....................... ................... ......58
The Dimeric Interface and Differential Roles of Glu162 in Human MnSOD
and Glul70 in E. coli MnSOD...................................... ...............59
A Structural Explanation for Abolished Catalysis of Nitrated Human MnSOD.60
F utu re D direction s ................................................................................................. 60
The Dimeric Interface of Human MnSOD ...................................................60
Redox properties of E162 m utants ........................................... ............... 61
Catalytic Properties of Nitrated M nSOD .................................... ............... 61
Future of Therapeutic Studies ........................................ ........................ 61

L IST O F R E FE R E N C E S .......... ........... ........................................................ ........... 63

B IO G R A PH IC A L SK E T C H ..................................................................... ..................71















LIST OF TABLES

Tablege

3-1 Maximal rate constants for the catalysis and inhibition of human wild type
M nSO D and m utants ........................ .. ........................ .... .... ........... 33

3-2 Diffraction data and refinement statistics for human E162D and E162A
M n SO D ............................................................................36

3-3 Maximal values for kcat/Km and kO/[E] for the catalysis of human wild-type
M n SO D and m utants ........................................................................ ..................40

4-1 X-ray crystallographic structure statistics of unmodified and nitrated human
M n S O D .................................................................................. 5 3

4-2 Distance geometries (A) in the active-sites of unmodified and nitrated human
M n S O D .................................................................................. 54















LIST OF FIGURES


Figure page

1-1 Hydrogen-bond network of human MnSOD. Shown in cyan are intervening
residues from the adjacent, non-crystallographic subunit .......................................3

1-2 Tetrameric human MnSOD. The dimeric interface is shown in green and blue
and the tetrameric interface is shown in blue and orange. ........................................6

2-1 Structure of human MnSOD showing the dimeric and tetrameric interfaces. .........10

2-2 19F NMR spectrum (470 MHz) of wild-type human MnSOD in which all nine
tyrosines were replaced with 3-fluorotyrosine .................................. ............... 16

2-3 Stacked fluorine NMR spectra for wild-type human MnSOD in which tyrosines
9, 11, 34, 45, 166, 176 and 193 have been mutated to phenlylalanine. .................16

2-4 The temperature dependence of five 19F chemical shifts of wild-type human
MnSOD in which all tyrosine residues are replaced with 3-fluorotyrosine.............18

2-5 The temperature dependence of 19F linewidths at half height for wild-type
human MnSOD in which all tyrosine residues are replaced with 3-
flu orotyrosine.. ......................................................................... 18

2-6 H/D exchange map for human M nSOD....................................... ............... 20

3-1 Active-site structure including the hydrogen bond network for human wild-type
M nSOD (Borgstahl et al., 1992). ........................................ ......................... 25

3-2 pH profile for molar absorptivity at 480 nm for human wild-type Mn3+SOD,
162D Mn3+SOD and E162A Mn3+SOD................ .. .......... ..... ...... ......... 30

3-3 Change in absorbance at 420 nm over a millisecond timescale after generation of
5.6 tM superoxide.................. ............................ .. ......32

3-4 pH profile for ki (A), k2 (e), and (m) k3 in catalysis of the disproportionation of
superoxide by E 162D M nSO D .. ......................... ............................................... 34

3-5 pH profile for Kcat/Km for E162D MnSOD............. ......................................... 34

3-6 Structure of E162D MnSOD (shown in green) superimposed on wild-type
human MnSOD (shown in blue) in the region of the mutated residue. ...................35









3-7 Structure ofE162A MnSOD (shown in green) superimposed on wild-type
human MnSOD (shown in blue) in the region of the mutated residue ..................37

3-8 Structure ofE. coli MnSOD (green) superimposed on wild-type human MnSOD
(blue) in the dim eric interface.. ........................... ............................................... 43

4-1 Scheme for nitration of tyrosine in the presence of peroxynitrite showing
nitration through the pathway of equation 2. ................................ ..................46

4-2 Structure of the active site of the nitrated human MnSOD. ...................................52

4-3 The structure of the active-site region of nitrated (yellow) superimposed onto
unm odified human M nSOD (green).. ........................................... ...............52











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

KINETIC AND STRUCTURAL EFFECTS OF INTERFACIAL INTERRUPTION AND
PROTEIN NITRATION IN HUMAN MANGANESE SUPEROXIDE DISMUTASE

By

Patrick Quint

August 2006

Chair: David Silverman
Major Department: Biochemistry and Molecular Biology

This dissertation investigated interfacial attributes of human manganese superoxide

dismutase (MnSOD), a homotetramer with two structurally unique interfaces. A

conserved dimeric interface is formed from the junction of two subunits and the

tetrameric interface is formed from the interaction of two dimer pairs. Using 19F NMR,

observation of chemical shift and linewidth changes for fluorine-labeled tyrosines in the

dimeric and tetrameric interfaces of human MnSOD (Tyr169 and Tyr45 respectively)

indicated greater rigidity in the dimeric interface compared to the tetrameric interface.

Replacement of the dimeric, interfacial residue Glu162 with aspartate or alanine did not

significantly alter the crystal structure of human MnSOD, though an interaction with a

histidine ligand of the active site manganese was truncated. This interaction was

mediated in E162D by an intervening solvent molecule. Catalytic activity for E162D and

E162A was 5-20% that of wild-type enzyme and differential scanning calorimetry

indicated a role for E162 in thermal stability. Nitration of Tyr34, a dimeric, interfacial

residue involved in a hydrogen-bond network emanating from the active-site manganese,

abolishes activity in human MnSOD. In vitro nitration of human MnSOD yielded 74%









nitration of Tyr34 with moderate nitration of other aromatic residues. A 2.4 A structure of

nitrated MnSOD aligned well with wild type though an NO2 group covalently linked to

Tyr34 is observed. The structure of nitro-MnSOD indicates that alteration of the

hydrogen-bond network as well as steric blockade and electrostatic repulsion of substrate

all account for the loss in catalytic activity associated with nitration. Taken together,

these findings provide a role for the tetrameric interface in stability and the dimeric

interface in catalysis. In addition, this dissertation provides a structural explanation for

abolished catalytic activity associated with nitration of Tyr34 in MnSOD. Future

therapeutic studies on this enzyme will involve the study of residues that form the

dimeric interface of MnSOD.














CHAPTER 1
INTRODUCTION

Superoxide Formation and Reactions

Superoxide is an oxygen radical formed from the reaction of free electrons with 02

to form 02 -. There are several enzymatic sources of superoxide including xanthine

oxidase, NADPH-oxidase within phagocytes, and other oxidases and an important

endogenous source of superoxide are the mitochondria. Leaks in the electron transport

chain allow for the addition of a single electron to 02 to form 02' (Kalra et al. 1994;

Haliwell, 1995). Though superoxide is itself toxic to the cell, its primary mode of damage

to cellular structures occurs through the Haber-Weiss reaction to form the highly unstable

hydroxyl radical (Haber and Weiss, 1934). In addition, superoxide reacts with nitric

oxide to form the nitrating agent peroxynitrite, which dissociates to form hydroxyl radical

and nitrite radical (Beckman et al., 1990; Beckman et al., 1992). Superoxide and its

breakdown products can cause damage to several biomolecules including lipids, DNA

and proteins and can affect their normal function. Its reaction with proteins, lipids and

DNA has implicated superoxide in several pathological states including reperfusion

injury (Becker, 2004), degenerative diseases like amyotrophic lateral sclerosis and

muscular dystrophy and has been implicated in damage induced aging of cells (Harman,

1956). To avoid the deleterious effects of the superoxide anion and hydroxyl radical, cells

have evolved a mechanism to scavenge and catalyze the disproportionation of

superoxide.









Classes of Superoxide Dismutase

Organisms that thrive in aerobic environments utilize superoxide dismutase (SOD)

to scavenge and detoxify superoxide radicals. There are four isoforms of SOD that utilize

for catalysis the metals copper and zinc, manganese, iron, and nickel. A copper

containing protein with dismutase activity was first discovered in 1968 by McCord and

Fridovich and a year later identified as a copper-containing SOD (McCord and Fridovich,

1968; McCord and Fridovich, 1969). The same group reported the discovery of a

structurally unique SOD in E. coli that utilized manganese in its active site (Keele,

McCord and Fridovich, 1970). It was later discovered that both Cu/ZnSOD and MnSOD

are utilized by eukaryotes, though their localizations are different; Cu/ZnSOD is localized

primarily in the cytosol while MnSOD is localized exclusively within the inner-

mitochondrial matrix (Weisinger and Fridovich, 1973). Its localization in the

mitochondrial matrix suggests a role for MnSOD in the protection of mitochondrial

DNA, lipids and proteins. Eukaryotic organisms also express an extracellular SOD which

utilizes copper and zinc for catalysis (Marklund, 1982). Like eukaryotic MnSOD, it is

tetrameric in solution. Two other classes of SOD also exist: FeSOD, which is structurally

similar to MnSOD with an identical active-site structure (Slykhouse and Fee, 1976) and

the structurally unique NiSOD (Chodhury et al,. 1999; Barondeau et al., 2004).

Human Manganese Superoxide Dismutase

Active Site and Hydrogen Bond Network

Human manganese superoxide dismutase (MnSOD) is a homotetramer of 22 kDa

subunits that catalyzes the disproportionation of superoxide into 02 and H202 (Fridovich

et al., 1989). Localized in the mitochondria, MnSOD is an enzyme with kcat/Km = 8 x 108

M-1 sec-1 (Hsu et al., 1996). Structural studies done by Borgstahl et al. in 1992 reveal a









trigonal, bipyramidal geometry about the active site manganese composed of three

histidines (His26, 74 and 163), one aspartate (Asp159) and one


Trp123


Asp159


His26


Gin143


Tyrl66
(adjacent subunit)


Glu162
(adjacent subunit)


Figure 1-1 Hydrogen-bond network of human MnSOD. Shown in cyan are intervening
residues from the adjacent, non-crystallographic subunit.

solvent molecule (Figure 1-1). A hydrogen bond network extends from the active site

metal to the coordinating solvent molecule, through Gln143 to Tyr34, through a solvent

to His30, and finally to Tyr166 from the adjacent subunit (Figure 1-1).

Manganese Superoxide Dismutase and its Reaction with Superoxide

Human MnSOD catalyzes the rapid disproportionation of superoxide through a

two-step reaction in which the active-site manganese is first reduced and then oxidized

with formation of products 02 and H202 (see equation 1 and 2).


Mn(III)(OH)SOD + 02O- + H+

Mn(II)(H20)SOD + 02 -+ H+

Mn(II)(H20)SOD + 02"

Mn(III)(X)SOD + H+ + H20


kl Mn(II)(H20)SOD + 02

k2 1 Mn(III)(OH)SOD + H202

k3 Mn(III)(X)SOD

k4 Mn(III)(OH)SOD + H202









The scheme shown above indicates that the coordinated solvent of MnSOD is

protonated upon reduction (Eq. 1) (Miller et al., 2003) and that one proton is donated

from the coordinating solvent to form product H202 (Eq. 2). Catalysis is further

complicated by the reversible formation and dissociation of a product inhibited complex,

shown in equations 3 and 4 (McAdam et al., 1977; Bull et al., 1991). It is speculated that

inhibition of the enzyme occurs through the oxidative addition of 02- to Mn(II), shown in

equation 3, forming a peroxo-bound complex of Mn(III) with the reverse of this reaction,

shown in equation 4, yielding active enzyme (Bull et al., 1991).

The Subunit Interfaces of Human Manganese Superoxide Dismutase

Human MnSOD contains two interfaces, the dimeric interface, conserved between

prokaryotes and eukaryotes, and the tetrameric interface generally associated with the

eukaryotic enzyme (Figure 1-2) (Borgstahl et al., 1992). The dimeric interface is

composed of several residues that participate in hydrogen bond interactions and the

location of the hydrogen bond network (Fig 1-1 and Fig 1-2). The tetrameric interface is

formed from the dimerization of dimers in the eukaryotic enzyme, creating a novel four-

helix bundle, first described by Borgstahl et al. in 1992. The role of the tetrameric

interface is unknown, though it likely plays a role in stabilizing the enzyme, thus

providing an evolutionary advantage. The tetrameric interface does not appear to

participate in catalysis. The scheme shown above indicates that the coordinating solvent

of MnSOD is protonated upon reduction (Eq. 1) (Miller et al., 2003) and that one proton

is donated from the coordinating solvent to form product H202 (Eq. 2).

Peroxynitrite

An important mechanism for catalytic inhibition is through the reaction of MnSOD

with peroxynitrite. Peroxynitrite is a nitrating agent formed from the diffusion-controlled









reaction of superoxide with nitric oxide (Beckman et al., 1990). Upon protonation,

peroxynitrite dissociates to form nitrate (70%) and hydroxyl and nitrite radicals (30%)

with a first order rate constant of 0.17 sec-1 (Beckman et al., 1990). The reaction of

hydroxyl radicals with biomolecules results in the one-electron oxidation of lipids,

proteins and DNA. However, hydroxyl radical formation is likely to occur at acidic pH

when peroxynitrite is in its protonated form, but at physiological pH its reaction with CO2

becomes important. At pH 7.8, peroxynitrite reacts with CO2 to form

nitrosoperoxycarbonate and out competes the spontaneous dissociation of peroxynitrous

acid (Bonini et al., 1999). The short-lived nitrosoperoxycarbonate intermediate

dissociates into nitrate (-65%) and carbonate radicals (-35%) which mediate the

formation of 3-nitrotyrosine.

Tyrosine Nitration

Electron paramagnetic resonance studies have shown that tyrosine nitration by

peroxynitrite is increased in the presence of CO2, and membrane inlet mass spectrometry

studies have shown that CO2 catalyzes the isomerization of peroxynitrite into nitrate

through a nitrosoperoxycarbonate intermediate (Santos et al., 2000; Tu et al., 2004). The

predominant pathway for tyrosine nitration is through the reaction of carbonate radical

with tyrosine to form tyrosyl radical. Though hydroxyl radical is capable of forming

tyrosyl radical, it is a more promiscuous oxidative agent than CO3-. It has been shown

previously that catalytic inhibition of MnSOD results primarily from the nitration of

Tyr34 (MacMillan-Crow et al., 1996; Yamakura et al., 2001). The mechanism for this

substantial decrease has been elucidated by a 2.4 A structure of nitrated human MnSOD

indicating that steric hindrance or bulk blockade of the substrate access channel may be









responsible for the change in activity (Quint and Reutzel et al., 2005). These results are

discussed in chapter 4.


Dimeric Interface











Tetrameric
Interface








Figure 1-2 Tetrameric human MnSOD. The dimeric interface is shown in green and blue
and the tetrameric interface is shown in blue and orange. Magenta spheres are
active site manganese.

Research Goals

The data and conclusions presented in this thesis focus on the role of the subunit

interfaces in human MnSOD and the structural effects of nitration of Tyr34. Techniques

utilized include 19F NMR, x-ray crystallography, differential scanning calorimetry, mass

spectrometry, site-directed mutagenesis and pulse radiolysis. The first aim was to define

the contribution of the two subunit interfaces to enzymatic stability. Building on the

conclusions of the first study, the involvement of the dimeric interface in stability and

catalysis was probed through replacement of Glul62. Finally, a structure of nitrated

human MnSOD was determined.









Interfacial Mobility at the Dimeric and Tetrameric Interfaces of Human Manganese
Superoxide Dismutase

The tetrameric interface is generally associated with eukaryotic enzymes. The

advantage of a tetrameric structure over a dimeric structure in human MnSOD is not yet

understood. The goal of this study was to understand differences in stability exhibited by

the two structurally unique interfaces of human MnSOD. Using fluorine labeled Tyrl69

as a reporter for the dimeric interface and fluorine labeled Tyr45 as a reporter for the

tetrameric interface, 19F NMR was utilized to probe the conformational mobility of the

two interfaces. The data indicate that the dimeric interface is significantly less

conformationally mobile than the tetrameric interface, suggesting a role for the tetrameric

interface in thermal stability. This study will provide a better understanding of the role of

both interfaces in stability and may aid in our understanding of the evolutionary role of

the tetrameric interface.

Replacement of a Key Dimeric Interfacial Residue

Following the thermostability work of the previous study, an important dimeric

interfacial residue, Glu162, was replaced with aspartate and alanine. Glu162 interacts

through a hydrogen bond with Glu162 and His163 of the adjacent subunit (Fig 1-1). The

goals of this study were twofold: determine the role of Glu162 in structure and catalysis

and compare the human E162A to the equivalent mutation in E. coli (Whittaker and

Whittaker, 1998). The Glul70A mutant in E. coli is less stable, catalytically inactive and

exhibits an altered metal specificity. Replacement of Glu162 in the human enzyme

resulted in diminished catalysis and a higher degree of product inhibition than wild-type

enzyme. A crystal structure of both E162A and E162D indicated an abolished interaction

between the side chains of residues 162 and 163 though an intervening solvent molecule









bridged the interaction between Asp162 and His163 in the E162D mutant. Both mutants

were tetrameric in solution and metal specificity was not altered for either mutant.

Structure of Nitrated Human MnSOD

The third goal was to establish a structural explanation for the decrease in catalysis

observed when human MnSOD is nitrated at position 34. Previous studies have shown

that nitration of Tyr34 in human MnSOD is associated with abolished catalysis, and

recently this lab published the structure of nitrated MnSOD (Quint and Reutzel et al.,

2005). Chapter 4 describes the first structure of a nitrated MnSOD. A 2.4 A crystal

structure of nitrated human MnSOD indicates exclusive nitration of Tyr34 though mass

spectrometry indicates nitration of other positions. The orientation of the NO2 group on

3-nitrotyrosine-34 suggests that steric blockade and potentially electronic repulsion of

substrate could both cause catalytic inhibition.














CHAPTER 2
STRUCTURAL MOBILITY IN HUMAN MANGANESE SUPEROXIDE
DISMUTASE

Introduction

The presence of a tetrameric interface in human MnSOD suggests enhanced

stability; the E. coli dimeric MnSOD has a melting temperature of 76C compared to

90C for human, tetrameric MnSOD. A polymorphism in the tetrameric interface of

human MnSOD, I58T, is associated with 50% decreased activity compared to wild type

MnSOD and its melting temperature is decreased to 760 C (Borgstahl et al., 1996). In

addition, the heat extremophile Thermus thermophilus utilizes a tetrameric MnSOD

(Wagner et al., 1993). This also suggests a role for the tetrameric interface in thermal

stability.

The properties of the dimeric and tetrameric interfaces in human MnSOD have

been investigated using 19F NMR. Human MnSOD has been prepared with all nine

tyrosine residues of each subunit replaced by 3-fluorotyrosine (abbreviated Fluoro-

MnSOD) (Fig 2-1). The use of 19F labels allows the observation of specific, well-resolved

NMR signals of labeled-tyrosine residues at the dimeric and tetrameric interfaces. The

NMR frequency of 19F resonances are nearly as high as 1H, thus producing about the

same signal-to-noise as 1H. Moreover, they have a much larger chemical shift range than

1H, making them considerably more sensitive to local electronic environment. The

replacement of hydrogen by fluorine in 3-fluorotyrosine is a minor steric change since the

van der Waals radius of a fluorine is just 0.15 A larger than the hydrogen it replaces









(Bondi, 1993), and the C-F moiety is a rather weak hydrogen bond acceptor (Jeffrey,

1997).
Tyr9/11
Tyr169 Tyrl76 Tyr9/1




.. Tyr193




STyr34
.- ,
Tyr45







Tyr165


Tyr166




Figure 2-1 Structure of human MnSOD showing the dimeric and tetrameric interfaces.
Also shown are the positions of five tyrosine residues that give sharp 19F
resonances when labeled with fluorine. Tyr45 is located at the tetrameric and
Tyrl69 at the dimeric interfaces.

The replacement of all tyrosines with 3-fluorotyrosine in human MnSOD has no

observed effect on the structure of the enzyme as determined by X-ray crystallography at

1.5 A resolution (Ayala et al., 2005); the fluorinated and unfluorinated structures are

closely superimposable with the root-mean-square deviation for 198 a-carbon atoms at

0.3 A (Ayala et al., 2005). We point out that the crystal structure of Fluoro-MnSOD

showed a single side chain rotamer for each of the nine 3-fluorotyrosines (Ayala et al.,









2005). The catalytic activity of Fluoro-MnSOD was lower than that of MnSOD by a

factor of 25 (Ren et al., 2005). This decrease could not be attributed to a single 3-

fluorotyrosine residue, and was not primarily due to 3-fluorotyrosine at residue 34, which

is in the active site.

The 19F NMR show that Tyrl69 at the dimeric interface of human MnSOD (Figure

2-1), has significantly less conformational freedom or mobility than does Tyr45 at the

tetrameric interface. Consistent with these results, differential scanning calorimetry of

human MnSOD showed that replacement by site-specific mutagenesis of Tyrl69 at the

dimeric interface decreased thermal stability and replacement of Tyr45 at the tetrameric

interface did not. These results are discussed in terms of catalysis and stability of

MnSOD.

Materials and Methods

Labeling with Fluorotyrosine, Expression in E. coli, and Purification

E. coli that express wild-type and the site-directed mutants of human MnSOD were

grown for 17 hours at 370C in 50 mL of minimal media. The minimal medium (M9),

which consisted of 0.06 M phosphate buffer at pH 8.2, 8.6 mM NaC1, and 0.02 M NH4Cl,

was sterilized by autoclaving. The overnight culture was supplemented with 0.1 mM

CaC12, 1 mM MgSO4, 11 mM glucose, 1 [tg/mL of thiamine, 0.2 mg/mL of amino acids

(except the aromatic amino acids), 1 mM tryptophan, 1 mM phenylalanine, and

ampicillin. The overnight growth was then transferred to 7.5 L of minimal media and

supplemented in the same manner as the overnight culture plus the addition of MnSO4 to

18 aM. The cells were allowed to grow for approximately 5 hours until an OD595 of 0.3-

0.4 was reached. At this point, the cells were induced with 0.3 mM IPTG and

supplemented with 1 mM 3-fluorotyrosine (or unlabeled L-tyrosine as a control) and were









allowed to grow for an additional 4 hours. Due to the low solubility of L-tyrosine and its

fluorinated analog in water, these compounds were added as solids to the growing media.

The cells were placed at 40C overnight and harvested the next day by centrifugation. The

resulting pellet was frozen at -700C overnight and the pellet was then lysed the following

day.

Depending on the particular sample preparation, the amount of 3-fluorotyrosine

incorporated into MnSOD was 67% to 76%, as determined by amino acid analysis

composition (Protein Chemistry Laboratory, Texas A&M University, College Station,

TX) and corroborated by hybrid LCQ-ToF (QSTAR) mass spectrometry (ICBR,

University of Florida, Gainesville, FL).

Site-Directed Mutagenesis of Fluorine Manganese Superoxide Dismutase

Site-directed mutants of human Fluoro-MnSOD were constructed for the purpose

of assigning 19F spectra of enzyme containing 3-fluorotyrosine. Each tyrosine of the

enzyme was replaced individually by phenylalanine. An exception was the double mutant

Y9F-Y1 IF; since these residues are near in sequence and tertiary structure we replaced

these together. These mutants were generated with the Stratagene QuikChange Site-

Directed Mutagenesis Kit (La Jolla, CA) in a Perkin Elmer GeneAmp PCR System 2400

(Foster City, CA). The plasmid of wild type MnSOD contained in the pTrc99A vector

was used as the template. PCR was performed using specific oligonucleotides (Sigma-

Genosys, The Woodlands, TX) containing the desired mutations as primers. The PCR

products were digested with the restriction enzyme Dpn I and transformed into

supercompetent XL-1 cells for selection. The plasmid containing the mutation of interest

was isolated using the plasmid mini prep kit from Qiagen and the mutation was

corroborated by DNA sequencing of the entire coding region (ICBR, University of









Florida, Gainesville, FL). The plasmid containing the desired mutation was then

transformed into QC774 cells from E. coli. This particular strain lacks the genes that

encode for endogenous FeSOD (SodB-) and MnSOD (SodA-).

Amide Hydrogen/Deuterium Exchange Kinetics.

We employed amide H/D exchange mass spectrometry to examine backbone

dynamics for human MnSOD (not fluorinated). By measuring the rate of amide H/D

exchange over defined regions of human MnSOD, we were able to develop a

comprehensive map of backbone dynamics that was complementary to the NMR studies.

On-exchange experiments of amide backbone hydrogens with deuterium were performed

in triplicate and involved exposing native human MnSOD to solvent 80% D20 for 0, 1,

15, 300, 900, 1800, 3600, and 12000 s prior to quenching of amide hydrogen exchange

by rapidly lowering the solution pH and temperature. After quenching, the protein was

digested by exposure to pepsin, and the resultant peptide pool was examined by LC-MS.

The uptake of deuterium for MnSOD pepsin-derived peptides was determined by

measuring the increase in number-average m/z values of the ion isotopic distributions for

each peptide from an on-exchange time point (deuterated peptide) when compared to the

same peptide from t = 0 (nondeuterated peptide). The percent deuterium incorporation

was determined for each peptide by dividing the measured number of deuterium atoms

incorporated by the calculated number of exchangeable amide hydrogen atoms for that

peptide

19F Nuclear Magnetic Resonance Spectroscopy

The 19F NMR spectra of fluorinated samples were recorded on a Bruker Avance

500 MHz spectrometer. A H 5mm TXI probe tuned for 19F at 470 MHz was employed.

We were not able to use a fluorine specific probe; the probe used displayed a very broad









background 19F resonance upon which the peaks of our enzyme were superimposed. This

arrangement precluded measurements of T2 and we report linewidths instead. Due to this

broad fluorine background, a T2 filter was utilized. This allows for the broader signals of

the spectrum to decay before the start of the data collection. The enzyme concentrations

were 0.5 mM in phosphate buffer at pH 7.8, unless otherwise specified, and 10% (by

volume) D20 for an internal lock. Chemical shifts were referenced to the internal

standard trifluoroacetate (TFA) at 0 ppm; high-field or shielded values with respect to

TFA are taken as negative. Temperature was varied from 17 C to 620 C by the flow of

heated nitrogen gas. Spectra were acquired by averaging 4000 scans with a scan rate of

8000 per hour.

Differential Scanning Calorimetry

Proteins were prepared in potassium phosphate buffer (20 mM, pH 7.8) at a

concentration of 1 mg/ml. A solution of 20 mM potassium phosphate (pH 7.8) was used

as a buffer reference. Both the sample and reference were degassed for 10 minutes before

scanning from 250C to 1200C at a rate of 10C per minute (Microcal VP-DSC). A buffer

blank was subtracted from the final protein scan and a cubic baseline was fit to the

profile. Changes in heat capacity (ACp) for the unfolding peaks were corrected by fitting

a non-two state model with a single component. Baseline correction and peak fitting were

performed using Origin (Microcal Software, Northampton, MA).

Results

Assignment of Fluorine Resonances

There are nine 3-fluorotyrosine residues in each subunit of the tetramer in human

wild-type Fluoro-MnSOD; five appear as distinct major peaks spanning about 8 ppm in

the 19F NMR spectrum (Figure 2-2). The integrated intensity of each individual peak was









approximately the same and represents one fluorine atom each. The assignment of the

peaks in Figure 2 was achieved by measuring the 19F NMR spectra of individual site-

specific mutants in which each tyrosine was replaced by phenylalanine (Figure 2-2).

Since residues 9 and 11 are near each other in sequence, we saved effort by preparing the

double mutant; hence, the NMR resonance assignments are not yet verified. However, in

the crystal structure ofFluoro-MnSOD (Ayala et al., 2005; PDB # 1XDC) the side chain

of Fluor-Tyr9 is buried with the phenolic hydroxyl hydrogen bonded to the backbone

carbonyl of residue 78 and in near van der Waals contact with Pro8, suggesting the

downfield shifted 19F resonance of residue 9 with respect to Fluoro-Tyrl 1. Tyrl 1 is more

exposed to the solvent than Tyr9. Thus we assign the more downfield resonance at -57.6

ppm to Tyr9 and the resonance at -60.1 ppm to Tyrl 1 (Figure 2-3). For reference, the 19F

resonance for monomeric tyrosine (pH 7.8, 25 C) is -61.4 ppm, and the chemical shift of

the single large resonance of collapsed Fluoro-MnSOD collapses at 620 C is -62.3 ppm.

Of the nine 3-fluorotyrosine residues of each monomer, four are not observed in the

19F NMR spectrum under the conditions of Figure 2-2. These are residues 34, 165, 166,

and 176, the side chains of which are located at distances less than 9 A from the

manganese (manganese to hydroxyl distance). All of the observed resonances, residues 9,

11, 45, 169, and 193, are located at distances greater than 13 A from the manganese.

Thus it is a reasonable suggestion that the four residues of 3-fluorotyrosine not observed

are broadened by the paramagnetic manganese, in addition to broadening by the overall

slow motion of the homotetramer. At the pH 7.8 of these studies it is not expected that

any of the tyrosine residues are ionized, consistent with literature on structure (Borgstahl

et al., 1996) and catalysis (Hsu et al., 1996).








1 193





169







-62 -4 -56 -58 -60 -2 -64 ppm
Figure 2-2 19F NMR spectrum (470 MHz) of wild-type human MnSOD in which all nine
tyrosines were replaced with 3-fluorotyrosine. Residue assignments were
made by replacement of individual 3-fluorotyrosine residues with Phe and are
written above the peaks. Chemical shifts referenced to TFA as internal
standard.






Tyr176

STyr9/11

Tyr45


Tyrl93
Tyr166


Tyr34

WT


-124 -126 -128 -130 -132 -134 -136 -138 -140 -142 ppm

Figure 2-3 Stacked fluorine NMR spectra for wild-type human MnSOD in which
tyrosines 9, 11, 34, 45, 166, 176 and 193 have been mutated to phenlylalanine.










Thermal Stability

Differential scanning calorimetry was used to determine changes in thermal

stability for two unfluorinated mutants of human MnSOD at positions 45 and 169

measuring the main unfolding transition of the enzyme. The mutant with Tyr45 replaced

by Ala exhibited an unfolding temperature of 94.3 C compared to 90.70 C for the wild

type MnSOD (Hsu et al., 1996; Greenleaf et al., 2004)(standard deviation estimated at 0.3

C). The unfolding temperature for the mutant with Tyrl69 replaced with Ala was

decreased to 86.50 C. These site-specific mutants, one with Tyr45 replaced by Ala and the

second with Tyrl69 also replaced by Ala, showed no significant change in catalytic

decay of superoxide measured by pulse radiolysis at Brookhaven National Lab (data not

shown). Analysis by native polyacrylamide gel electrophoresis indicated that both

mutants, Y45A and Y169A, remained tetrameric in solution.

Temperature Dependence of Fluorine Resonances

The change as temperature was increased from 17 to 570 C in the chemical shifts

of each of the five assigned resonances was uniform with no significant changes in slope

for individual peaks over this temperature range (Figure 2-3). The two residues that had

the largest downfield 19F chemical shift, Fluoro-Tyr45 and Fluoro-Tyr9, also showed the

largest changes in chemical shift (Figure 2-3) and in linewidth at half height (Figure 2-4)

as temperature increased. The remaining assigned residues Fluoro-Tyrl 1, 169, and 193

showed smaller changes in chemical shifts and in linewidths with changes in temperature

over the range of temperatures in Figures 2-4 and 2-5. It is notable that Fluoro-Tyrl69,

iun the dimeric interface, showed almost no change in linewidth with temperature

(Figure 2-5).












Tyr45


L. -0.8

S-0.6

-0.4

-0.2

0


17 22 27 32 37 42 47 52 57 62


Temperature ( C)

Figure 2-4 The temperature dependence of five 19F chemical shifts of wild-type human
MnSOD in which all tyrosine residues are replaced with 3-fluorotyrosine.
Values are normalized to show a single chemical shift at 17o C in order to
compare trends. Conditions are as described in Figure 2-2. (*) Tyr45; (m)
Tyr9; (A)Tyrl69; (e) Tyrl93; (X)Tyrll.


0.75


0.65


E 0.55


S0.45


- 0.35
.-


Tyr45


Tyrl 1


0.25


0.15
12 17 22 27 32 37 42 47 52 57 62


Temperature (oC)


Figure 2-5 The temperature dependence of 19F linewidths at half height for wild-type
human MnSOD in which all tyrosine residues are replaced with 3-
fluorotyrosine. Conditions are as described in Figure 2-2. (*) Tyr45; (m)
Tyr9; (A)Tyrl69; (e) Tyrl93; (X)Tyrll.









To corroborate these findings, hydrogen/deuterium exchange studies were

performed. Using this approach, amide hydrogen exchange kinetics of 29 peptides

(comprising approximately 78% of the human MnSOD protein) were determined (Figure

2-6). For each peptide, the percentage of deuterium uptake versus time for the seven on-

exchange time intervals was plotted with error bars (plots not shown) representing the

mean standard deviation of the deuterium incorporation percentages determined from

triplicate experiments. The rate of deuterium incorporation varied in different regions of

the protein (Figure 2-6). Peptides corresponding to regions 25-40, 58-77, and 94-113

displayed significant protection from amide H/D exchange as demonstrated by very low

levels of deuterium incorporation with their maximum levels being below 35% (percent

of the maximum on-exchange possible corrected for percent deuterium exposure and

back exchange) at the longest on-exchange time point. Peptides corresponding to regions

1-20, 78-96, 114-135, and 155-173 showed moderate protection from amide H/D

exchange with levels of deuterium incorporation between 50% and 60% at the longest

on-exchange time point.

Discussion

Emphasized here are the properties of Fluoro-Tyr45 and 169, which are located in

the tetrameric and dimeric interfaces, respectively (Figure 2-1). The 19F resonance of

Fluoro-Tyr45 showed a large increase in chemical shift as temperature increased (Figure

2-4), moving toward the position of the 19F peak for denatured enzyme, and showed a

large decrease in linewidth as temperature increased (Figure 2-5). These features

characterize a region at the tetrameric interface with increased conformational and

dynamic mobility as temperature increases. Based on previous studies of the motions of










KS LPD LPYDYCALEPMINAOIMQLMMKIMMAAVNMNLNVTEElKYOEA L/C

1ApVTA I- 9IA PA CF FT I 5 Ai I Kl I R



1!* lea wa 125 lie le 19 5as
TGL t PLLGI DVWEHArYY"LOVYKINJRPDDY LKAl I WNTJV I NWE' VTE RPYMA.C K K
rtn


K.1
----------------' -- -----'---'------ ---'--- ----- ----------------









derived fagment of MnSOD detected by LC-MS and monitored during on-
observed 9F spectrum suggest that such a mechanism is predominant.
P'I I l l ssnt
1. 0 2%O 4 ar H.!T ae. N1 1141 35 1 UITS


Figure 2-6 H/D exchange map for human MnSOD. Each block represents a pepsin-
derived fragment of MnSOD detected by LC-MS and monitored during on-
exchange time periods to determine the degree of deuterium incorporation.
The gradations within each block represent the seven on-exchange time
periods used with the shortest period being on top. The deuteration level, as a
percentage of the theoretical maximum, for each peptide at each time period is
color-coded.

fluorotyrosine rings in proteins (Hull and Sykes, 1975; Hull and Sykes, 1975B), we

anticipate that the dominant spin-lattice relaxation mechanism is dipolar with a

contribution from chemical shift anisotropy, and that the decrease in linewidths observed

in 19F resonances can be largely attributed to motional narrowing. We point out that the

crystal structure shows just one rotamer for the side-chains of each 3-fluorotyrosine

residue in Fluoro-MnSOD (Ayala et al., 2005); although we cannot exclude a

contribution of chemical exchange to line broadening, neither the crystal structure nor the

observed 19F spectrum suggest that such a mechanism is predominant.









The 19F resonance of Fluoro-Tyrl69 is notable because it does not change

appreciably in linewidth over the temperature range from 17 to 57 C (Figure 2-5). The

other 19F resonances have linewidths that decrease with increasing temperature,

suggesting increasing motional processes; Fluoro-Tyrl69 does not show a detectably

higher degree of motional freedom as temperature increases. Also, Fluoro-Tyrl69 shows

a chemical shift change as temperature increases that is modest compared with that of

Fluoro-Tyr45 (Figure 2-4). The linewidth data especially indicate that the environment of

this side chain is rather stable with little change in mobility over the temperature range

studied. Residue 169 is located at the dimeric interface and its side chain appears in near

van der Waals contact with the hydrocarbon side chain of Gln168. Considering residue

169 as a reporter for the dimeric interface, these data indicate stability and lower

motional freedom for the region of Fluoro-Tyrl69.

Of the remaining observed 19F resonances, those of residues 11 and 193 had

chemical shifts closest to monomeric 3-fluorotyrosine or to partially denatured Fluoro-

MnSOD. With increasing temperature, their chemical shifts moved toward that of the

denatured enzyme and their linewidths narrowed somewhat (Figures 2-4, 2-5). These

features are consistent with the partially constrained positions of Tyrl 1 and 193 in the

structure. Fluoro-Tyr9 was different in showing a rather substantial temperature effect in

its 19F linewidth (Figure 2-5), although showing a rather modest temperature effect on 19F

chemical shift (Figure 2-4).

Deutrium exchange studies on native MnSOD corroborate the findings of fluorine

NMR. The most rapidly exchanging MnSOD peptides, or regions of MnSOD that

demonstrate little or no protection to amide H/D exchange, were in the 40-58 region that









showed 96% deuterium incorporation at the 3600 s time point. The exchange kinetics for

a region of a protein is dependent in part on the extent of localized hydrogen bonding as

amide hydrogens are protected from exchange while involved in hydrogen bonding. For

an amide hydrogen involved in a hydrogen bond to become exchange competent,

localized unfolding must occur to break the hydrogen bond and allow exchange with

solvent protons or deuterons. Therefore, slowly exchanging regions of a protein are

considered less dynamic in part due to significant hydrogen bonding. Taken together, the

data indicate that the regions corresponding to the tetrameric domain (40-58) and the

dimeric domain (159-174) display different amide H/D exchange kinetics with the

tetrameric domain affording rapid exchange and the dimeric domain affording moderate

protection from amide H/D exchange (Figure 2-6)

The thermal unfolding data appear consistent with these conclusions. Specifically,

differential scanning calorimetry showed that replacement of Tyr169 with Ala

destabilized human MnSOD with the major unfolding transition decreased about 4 C

compared with wild type, while the replacement of Tyr45 did not destabilize but actually

enhanced stability somewhat. The observation that the melting temperature for Y169A is

decreased unlike the Y45A mutant suggests that the dimeric interface is stabilized by

specific residue interactions whereas the tetrameric interface is stabilized by several non-

specific interactions.

Evolution clearly shows a dimeric MnSOD of primitive species, with tetrameric

MnSOD a different development (Purrelo et al., 2005). In fact, crossing the dimeric

interface are residues such as Glu162 and Tyrl66 that extend into the active site of the

adjacent subunit and are significant contributors to catalysis (Whittaker and Whittaker,






23


1998; Hearn et al., 2004). In human MnSOD, the reporter residue at the dimeric interface

Tyrl69 showed less conformational mobility and greater contribution to stability than

Tyr45 at the tetrameric interface. This may in part reflect the observation that residues at

the dimeric interface are closer to the active site, likely play a greater role in supporting

catalysis, and hence require a greater degree of conformational stiffness than residues at

the tetrameric interface.














CHAPTER 3
ROLE OF A GLUTAMATE BRIDGE SPANNING THE DIMERIC INTERFACE OF
HUMAN MANGANESE SUPEROXIDE DISMUTASE

Introduction

The previous chapter elucidated the roles of the tetrameric and dimeric interfaces

and their contribution to enzyme stability. Recognizing the role of the dimeric interface in

catalysis, this chapter focuses on a mutation in the dimeric interface and the subsequent

effects on catalysis and stability. This study reports the role of Glu162 in human MnSOD,

the side chain carboxyl of which forms a hydrogen bond with the imidazole side chain of

Hisl63 of the adjacent subunit, a ligand of the metal (Figure 3-1). Using both X-ray

crystallography and pulse radiolysis, reported here are the effects of mutation of Glul62

to aspartate and alanine. The X-ray data for E162D and E162A reveal no significant

structural changes compared with wild type other than the lost interaction with Hisl63,

and in the case of E162D an intervening water molecule maintains a hydrogen-bond link

between Aspl62 and Hisl63. However, mutation of Glul62 introduces a pH dependence

in catalysis and in the visible absorption spectrum of Mn3+SOD near pKa 8.5 not

observed in human wild-type MnSOD. Mutation of Glul62 to either aspartate or alanine

results in a greater degree of product inhibition compared to wild-type MnSOD.

Differential scanning calorimetry indicates that the hydrogen bond between Glu162 and

His163 contributes to the stability of MnSOD. These data emphasize the role of the

dimeric interface of human MnSOD in catalysis and thermal stability.











Trp123
Asp159

His74

Mn
His26 ,s
His163
Tyrl 66
GIn143 S1 (adjacent subunit)





Tyr34 S2 Glu162
(adjacent subunit)
Figure 3-1 Active-site structure including the hydrogen bond network for human wild-
type MnSOD (Borgstahl et al., 1992). The manganese is designated as a
purple sphere and solvent molecules as red spheres. Dotted lines indicate a
hydrogen-bonded network emanating from the metal-bound solvent molecule
to Tyr166 from an adjacent subunit.

Materials and Methods

Site Directed Mutagenesis

Mutants were generated with the Stratagene QuikChange Site-Directed

Mutagenesis Kit (La Jolla, CA) in a Perkin Elmer GeneAmp PCR System 2400 (Foster

City, CA). The plasmid of wild type MnSOD contained in the pTrc99A vector was used

as the template. PCR was performed using specific oligonucleotides (Sigma-Genosys,

The Woodlands, TX) containing the desired mutations as primers. The PCR products

were digested with the restriction enzyme Dpn I and transformed into supercompetent

XL-1 cells for selection. The plasmid containing the mutation of interest was isolated

using the plasmid mini prep kit from Qiagen and the mutation was corroborated by

DNA sequencing of the entire coding region (ICBR, University of Florida, Gainesville,









FL). The plasmid containing the desired mutation was then transformed into QC774 cells

from E. coli. This particular strain lacks the genes that encode for endogenous FeSOD

(SodB-) and MnSOD (SodA-).

Expression and Purification of Human MnSOD

The pTrc99A plasmid containing the mutant MnSOD template was transformed

into SodA-/SodB- E. coli QC774 cells. Cells were grown in luria broth media

supplemented with 6 mM MnCl2 and ampicillin for selection. Cultures were grown to 0.8

absorbance units and then induced with IPTG. Cells were centrifuged and then lysed. The

lysate was heat treated at 600 C for 15 minutes to select for MnSOD, which is

thermostable to 700 C. Following heat treatment, the lysate was spun and the supernatant

was dialysed against three exchanges of 20 mM Tris pH 8.2 and 50 iM EDTA. The

dialysate was purified using a Q-sepharose anion exchange column (Pharmacia). Protein

concentrations were determined by UV spectrometry using a Beckman Coulter DU 800

spectrometer at 250 C and pH 7.8 (8280 = 40,500 M-1 cm-1) (Greenleaf et al., 2004).

Manganese and iron content for each enzyme sample were determined using flame

atomic absorption spectroscopy (flame AA), and division by the total protein

concentration gives metal occupancy for the enzyme.

Visible Absorption

The visible spectrum for human MnSOD shows a broad absorption with a

maximum at 480 nm (8480 = 610 M-1 cm1) (Hsu et al., 1996). Profiles for pH dependence

were determined by measuring the absorption at 482 nm at pH varying from 6.5 to 11.5.

Enzyme samples were diluted 1:1 (-500 tM enzyme) in a buffer containing 200 mM









MES and 200 mM TAPS and pH was adjusted using 5 M KOH. The pH was measured

using a Fisher Accumet 610 pH meter with a Coming semi-micro combo electrode.

Pulse Radiolysis

Pulse radiolysis experiments were performed at Brookhaven National Lab using a 2

MeV van de Graff generator to produce superoxide directly in solution. Superoxide

radicals were formed by exposing aqueous, air-saturated solutions to a high-dose electron

pulse according to methods described by Schwarz (Schwarz, 1981). Up to 45 [M

superoxide was produced in solution. Enzyme solutions contained 2 mM buffer

containing either MOPS pH 6.5-8.0, TAPS 8.0-9.0, or CAPS 9.0-10.0 depending on the

desired pH, 50 [M EDTA, and 30 mM format to scavenge hydroxyl radicals. The

reactions were monitored spectrophotometrically using a Cary 210 spectrophotometer at

25 C by following changes in the absorbance of superoxide (8260 = 2000 M-1 cm1)

(Rabini and Nielson, 1969) or by following enzyme absorbance at 420 nm or 480 nm

(Cabelli et al., 1999).

Manganese and Iron Content Determination

Flame atomic absorption spectroscopy was used to determine total metal content in

all enzyme solutions. A Perkin Elemer 308 Flame Atomic Absorption Spectrometer was

utilized to determine manganese concentration. A multi-ion lamp with a 3-slit burner was

used and absorption was measured at 279 nm. Manganese occupancies were determined

by dividing the total metal concentration by the enzyme concentration to yield a

percentage of total active sites containing manganese. Typical occupancies for metal in

the active site range from 70% to 90%. Iron content was measured by ABC Research

Corp (Gainesville, FL) and was determined to account for less than 2% of the total metal









in solution E162D and E162A MnSOD. The manganese concentration was used as the

active enzyme concentration for all pulse radiolysis measurements.

Crystallography

Hexagonal crystals were grown from a solution of 3 M ammonium sulfate

containing 100 mM imidazole and 100 mM malate at pH 7.8-8.2 using the vapor

diffusion method. Crystals approximately 0.2 x 0.2 x 0.3 mm grew within one week and

were magenta in color. Diffraction data were collected from single crystals wet mounted

in quartz capillaries (Hampton Research) on an R-AXIS IV++ image plate (IP) system

with Osmic mirrors and a Rigaku HU-H3R CU rotating anode operating at 50 kV and

100 mA (Rigaku/MSC). Diffraction data was collected at room temperature. A 0.3 mm

collimator was used with a crystal to IP distance of 220 mm and the 20 angle fixed at 0.

The frames were collected using a 0.3O oscillation angle with an exposure time of 5

min/frame at room temperature. Both data sets were indexed using DENZO and scaled

and reduced with SCALEPACK software (Otwinowski, 1997). Diffraction intensities

were visible to 2.3 A resolution for E162D and 2.5 A for E162A. Diffraction and

refinement statistics are given in Table 3-2.

To prevent model bias, human MnSOD mutants were phased using the human,

wild-type MnSOD structure (Quint and Reutzel et al., 2006; PDB accession 2ADQ) from

which the residue at position 162 was replaced with an alanine and the active-site

manganese had been removed. The structures were phased and refined using the software

package CNS (Brunger et al., 1998). Refinement cycling (using rigid body, simulated

annealing for the first cycle, minimization, and individual B-factor refinement) was done









in conjunction with rounds of manual model building using the program COOT for

molecular modeling (Emsley and Cowtan, 1998).

Differential Scanning Calorimetry

Enzyme samples were buffered in 20 mM potassium phosphate pH 7.8 at a

concentration of 1 mg/ml. A solution of 20 mM potassium phosphate pH 7.8 was used as

a buffer reference and was subtracted from the protein scan prior to baseline correction

and model fitting. Both the sample and reference were degassed for 10 minutes before

scanning from 250 C to 110 C at a rate of 1 C per minute (Microcal VP-DSC). A buffer

blank was subtracted from the final protein scan and a cubic baseline was fit to the

profile. Changes in heat capacity (ACp) for the thermal unfolding peaks were corrected

by fitting a reversible, non-two state model with two components. Baseline correction

and peak fitting were performed using the program Origin (Microcal Software,

Northampton, MA).

Results

Visible Spectrometry

Atomic absorption spectroscopy for the mutants of MnSOD studied here showed

that the metal occupancy for E162D was 88% Mn with <1% Fe, and the metal occupancy

for E162A was 54% Mn and <1% Fe. Both mutants were tetramers as determined by

non-denaturing PAGE. Human wild type Mn3+SOD as well as E162D and E162A

Mn3+SOD exhibit a characteristic visible absorbance with a maximum at 480 nM (8480 =

610 M-1 cm-1) corresponding to Mn3+ in the active site (Hsu et al., 1996). The pH profile

for molar absorptivity for wild-type human Mn3+SOD fits a single ionization with a pKa

of 9.2 + 0.1 (Figure 3-2) (Hsu et al., 1996; Greenleaf et al., 2004). Mutation to aspartate









resulted in a diminished pKa (pKa = 8.7 + 0.2) whereas replacement with alanine

increased the pKa(pKa = 10.1 + 0.1) (Figure 3-2).


600 _


500


0400


300
6 8 10 12
pH

Figure 3-2 pH profile for molar absorptivity at 480 nm for (o) human wild-type
Mn3+SOD, (*) E162D Mn3+SOD and (A) E162A Mn3+SOD. Data were fit to
a single ionization with values of pKa 8.7 0.2, 9.2 0.1, and 10.1 0.1 for
E162D, wild type, and E162A Mn3+SOD respectively. Solutions contained
200 mM MES and TAPS at 250 C and pH was adjusted using 5 M KOH.

Catalysis

MnSOD catalyzes the disproportionation of superoxide through a two-step process

in which the active-site metal cycles between the Mn3+ and Mn2+ states concomitant with

oxidation and reduction of superoxide (shown in eqs. 3-1 and 3-2) (Hearn et al. 2001;

McAdam et al., 1977; Cabelli et al., 1999).


Mn3+(OH)SOD + 02 + H+

Mn2+(H20)SOD + 02' + H+

Mn2+(H20)SOD + 02O

Mn3+(O22-)SOD + H+ + H20


ki

k2

k3

k4


Mn2+(H20)SOD + 02

Mn3+(OH)SOD + H202

Mn3+(022-)SOD

Mn3+(OH)SOD + H202


(3-1)

(3-2)

(3-3)

(3-4)









The scheme shown here reflects the observation that the solvent ligand of

Mn3+(OH)SOD takes up a proton upon reduction of the metal as shown in equation 3-1.

Equations 3-3 and 3-4 represent the formation and dissociation of a product inhibited

complex characterized by zero-order catalysis (Hearn et al., 2001; McAdam et al., 1977).

Estimation of the rate constants for eqs 3-1 through 3-4 were carried out by

measuring the rate of change of absorbance of 02' and of enzyme species after the

generation of 02 by pulse radiolysis (Hearn et al., 2001; Cabelli et al., 1999). Decrease

in absorption at 260 nm (E260 = 2000) (Rabini and Nielson, 1969) corresponds to the

disappearance of superoxide catalyzed by wild type, E162D, and E162A MnSOD, which

is characterized by a first-order phase of catalysis that is relatively uninhibited followed

by a zero-order, product inhibited phase (Hsu et el., 1996; Bull, Yoshida and Fee, 1991).

When enzyme and superoxide are similar in concentration, the progress curve for

superoxide decay is dominated by the reaction in eq 3-1, and a rate constant for kl can be

determined from a first-order fit to the decay. Another method for determining ki,

described by Hearn et al., 1999 (Borgstahl et al., 1996), involves measuring changes in

absorption at 480 nm under single turnover conditions when there is a molar excess of

enzyme. Decrease in absorption at 480 nm results from the conversion of Mn3+SOD to

Mn2+SOD shown in eq 3-1. This is a complementary method for determining kl and

values for the two methods are in agreement. After addition of a molar equivalent of

H202 to reduce the active-site manganese to Mn2+, observation of the increase in

absorption at 480 nm yields an estimate for k2 at earlier time points and k4 at the later part

of the curve. The product-inhibited complex has a characteristic absorption at 420 nm

(Bull, Yoshida and Fee, 1991) and measuring absorption increase at 420 nm after









reduction of the enzyme with H202 allows for an estimate of the rate constant k3 (Hearn

et al., 2001). Figure 3-3 shows typical data generated for the calculation of k3.


4









El1


0 *
0.00 0.10 0.20 0.30

Time (msec)


Figure 3-3 Change in absorbance at 420 nm over a millisecond timescale after generation
of 5.6 [M superoxide by pulse radiolysis in a solution containing 120 [tM
E162D MnSOD buffered by 2 mM TAPS at pH 8.35 with 50 tM EDTA and
30 mM sodium format at 25 C. Prior to pulsing, the sample was reduced
with 300 tM H202. Increase in absorbance at 420 nm was fit to a first-order
process giving the rate constant 14.4 ms1.

The values of kl-k4 describing catalysis by human wild-type MnSOD are

independent of pH in the range of pH 7 to 9.5 with a slight decrease above pH 9.5

(Greenleaf et al., 2004; Hearn et al., 2001). Values of the rate constants ki-k3 for catalysis

by E162D MnSOD were pH dependent (Figure 3-4) and could be fit to a single ionization

with maxima given in Table 3-1 and values of pKa in the range of 8.0 to 8.7 given in the

legend to Figure 3-4. The rate constant k4 describing the dissociation of the product-

inhibited complex was independent of pH though it was diminished three-fold compared









to wild-type enzyme. E162A MnSOD exhibited no pH dependence for kl-k3 with values

given in Table 3-1 concomitant with oxidation and reduction of superoxide (shown in

eqs. 3-1 and 3-2) (Hearn et al., 2001; Cabelli et al., 1999; McAdam et al., 1977).

However, above pH 8.0, the rate constant k4 was decreased 10-fold, from 30 3 sec-1 to 3

1 sec1.

Table 3-1 Maximal rate constants for the catalysis and inhibition of human wild type
MnSOD and mutants.

ki k2 k3 k4
(LaM-1 sec-1) (LaM-1 sec-1) (LaM-1 sec-1) (sec-1)


WTb 1500 1100 1100 120
E162Da 355 33 133 16 215 20 40 1
E162Aa 63 4 50 4 87 3 30 3
H30Nc 210 400 680 480
Y166Fd 0.2 0.2 0.2 270
E. coli 1000 800 150 60

(a) 2mM TAPS pH 7.7, 50mM EDTA, 30 mM format (see methods for pulse radiolysis)
(b) Ramilo et al., 1998
(c) Heam et al., 2003
(d) Heam et al., 2001
(e) unpublished

The maximal value for the steady-state rate constant kcat/Km for E162D was 290

[tM-1 sec-1 compared to 800 [tM-1 sec-1 for wild-type enzyme, and decreased in a pH

dependent manner with a pKa of 8.8 (Figure 3-5). The maximal value for kcat/Km for

E162A was diminished 8-fold (120 [tM-1 sec-1) independently of pH (Table 3-3). The rate

constant ko/[E] describes the product-inhibited, zero-order region of catalysis. The

maximal value of ko/[E] for E162D MnSOD was 270 sec-1 and was mostly pH

independent, though there was a decrease at higher pH (190 sec-1 at pH 8.3) (Table 3-3).

The E162A mutant exhibited a more extensive decrease with pH. The value for ko/[E] at

pH 7.7 was 190 sec-1 while at pH 8.4 it decreased to 18 sec-1. For comparison, the value










of ko/[E] is near 500 s-1 and is pH independent (Hearn et al., 2001; Hsu et al., 1996;

Greenleaf et al., 2004) (Table 3-3).

500

400


8 300

:L 200

100 r

0 -
7.5 8 8.5 9 9.5 10
pH


Figure 3-4 pH profile for kl (A), k2 (e), and (m) k3 in catalysis of the disproportionation
of superoxide by E162D MnSOD. Solutions contained 120 [iM enzyme, 2
mM buffer (see methods), 50 |tM EDTA and 30 mM sodium format at 250 C.
A single ionization was fit to the data with values for pKa of 8.7 + 0.2 for kl
and 8.1 + 0.1 and 8.0 0.1 for k2 and k3 respectively.


400


g 300-


I 200
E
100
o
0
7.5 8 8.5 9 9.5 10

pH


Figure 3-5 pH profile for Kcat/Km for E162D MnSOD. Solutions contained 2mM MOPS
(pH 6.5-8.0), TAPS (8.0-9.0), or CAPS (9.0-10.0) 50 [tM EDTA and 30 mM
sodium format at 250 C The line represents a theoretical curve for a single
ionization with value for pKa of 8.8.









Structure of MnSOD Mutants

The human MnSOD mutants E162D and E162A crystallized in the hexagonal

space group P6122 under similar crystallization conditions (see methods) (Table 3-2).

Both mutants contained a homodimer in the asymmetric unit with the eukaryotic tetramer

formed from a crystallographic, symmetry-generated dimer. The models for the two

mutants superimpose well on wild type (rmsd = .2 A for E162D and .2 A for E162A).

The major difference is the truncation of the hydrogen bond interaction between Glu162

and Hisl63 (Figures 3-6 and 3-7). The E162D structure retains an interaction with

His163 through a solvent molecule (Figure 3-7). This interaction does not exist in the

E612A mutant and therefore any stabilizing force that the residue at position 162 would

have on His163 or the active site in general is missing. The positions of the active-site

residues were altered for the two mutants. For E162A, the distances from Asp159 and

His74 to manganese were decreased from 2.04 A for wild type to 1.61 A and from 2.23 A

to 1.65 A respectively. Distances for the remaining coordinating residues were not

significantly different. Distances were altered by < 10% for the E162D structure.



S042-
Glu162 42
His163 His26
S s Hil s His74

S,
Asp159


Figure 3-6 Structure of E162D MnSOD (shown in green) superimposed on wild-type
human MnSOD (shown in blue) in the region of the mutated residue. Red
spheres represent solvent and the manganese is shown in purple. The
intervening solvent molecule from the E162D structure is labeled S*.









Table 3-2 Diffraction data and refinement statistics for human E162D and E162A
MnSOD.
E162D E162A

Space group P6122 P6122
Unit-cell parameters (A) a = 81.16, c = 241.17 a = 81.26, c = 242.28
a = 90, y = 120 a = 90, y = 1200

Resolution (A) 20-2.3 20-2.5
No. of unique reflections 19557 16004
Completeness (%) 89.0 (90.3) 92.6 (93.8)
aR sym (%) 12.0(21.0) 14.5(46.0)
{ I/C (I)} 18.7 (19.7) 22.0(7.2)
bR factor (%) 20.7 24.0
Rfree (%) 22.7 25.6
No. of protein atoms 1553 1550
No. of water molecules 51 38
cR.m.s.d. bond length (A) 0.005 0.006

cR.m.s.d. bond angle (0) 1.199 1.426

Avg B (main/side/solvent) 25.2/28.1/34.7 32.6/34.1/34.6

Ramachandran Plot (%)
Most favored regions 92.1 91.5
Addn'l allowed regions 6.7 7.3
Gen allowed regions 1.2 1.2
Disallowed regions 0 0

Data collected at room temperature
*Data for the highest resolution shell are given in parentheses.
aRsym = |I-1 / E Ix 100, where I is the intensity of a reflection and is the
average intensity.
bRfactor = hkl |Fo-KFc / Ihkl IFol x 100, Rfree is calculated from 5% randomly selected data
for cross-validation.
cR.m.s.d. = root mean square deviation.

Thermal Stability

Differential scanning calorimetry was used to determine thermal transition

temperatures for E162D and E162A human MnSOD. E162D MnSOD showed two peaks

and these melting temperatures were quite similar to those observed for wild type. The

thermal inactivation temperature was 72 C and the unfolding temperature was 880 C.









These values for wild-type human MnSOD were 68 C and 900 C, respectively (Borgstahl

et al., 1996). Replacement with alanine destabilized the enzyme. E162A MnSOD

exhibited a split peak with values of 74.50 C and 81 C. The calorimetric data for both

mutants were the composite of three experiments averaged and normalized to a non-two

state model with two components. Melting temperatures for E162D and E162A were

determined at pH 7.8.

D159

H74


A162 H163


H30


S04-2 s:Y
Y34




Figure 3-7 Structure of E162A MnSOD (shown in green) superimposed on wild-type
human MnSOD (shown in blue) in the region of the mutated residue. Red
spheres represent solvent and the manganese is shown in purple. In the
mutant, a sulfate molecule was found bound near the position of the carboxyl
group of Ala162 and in possible interaction with S2.

Discussion

Structures of E162D and E162A MnSOD

In wild-type MnSOD, the side-chain of Glu162 forms a hydrogen bond with the

side chain ofHisl63 from the adjacent subunit MnSOD (Borgstahl et al., 1992). This

interaction is dampened in E162D MnSOD as the carboxyl group is more distant from









His163 by the length of one methylene group and by the intervention of a solvent

molecule bridging Asp162 and His163 (Figure 3-6); however, the hydrogen bond to

His163 is maintained through the intervening water molecule. This interaction is

abolished in E162A MnSOD (Figure 3-7). As a result of the altered interaction between

the side chains of residues 162 and 163, the coordination geometry is changed for E162A,

though not so for E162D, presumably because a solvent molecule bridges the gap

between Asp162 and Hisl63. Another possible cause for the altered ligand distances for

E162A may be the low metal occupancy (54%). The positions of other active-site

residues His30, Tyr34, Gln143 are unaltered for either mutant.

An area of spurious density was observed surrounding Asp162 and Ala162 that was

attributed to sulfate binding (Figures 3-6 and 3-7). Alignment of the sulfate-binding

region of E162D and wild-type MnSOD indicates an opening of the structure. The

solvent at position S2 (Figure 3-1) is replaced by sulfate in the E162D structure. The

sulfate-binding region of E162A shows hydrogen bonding of sulfate to the side chains of

His30 and Tyr34. This could indicate a pathway for superoxide entry into the region of

the active-site cavity. Sulfate was not present in measurements of catalysis or of spectral

properties of these mutants of MnSOD although format was used as a hydroxyl

scavenger in pulse radiolysis experiments.

Differential scanning calorimetry showed that the main unfolding transition of

E162D MnSOD (88 C) is not significantly altered compared with wild-type human

MnSOD (90 C), consistent with the retention in the mutant of the hydrogen bond

between Asp162 and His163 through an intervening water molecule. In contrast, this

transition for E162A is decreased by 150 C, attributed in significant part to the removal of









a stabilizing interaction with His 163 from the adjacent subunit. The reduced stability,

however, did not affect tetramerization of E162A.

Visible Spectroscopy

The visible spectrum of Mn3+SOD is characterized by a broad absorption in the

visible region with a maximum at 480 nm (Hearn et al., 1999; Bull and Fee, 1985). The

pH profile of this maximum titrates with reported values of pKa of 9.3 (Bull and Fee,

1985) and 9.7 (Maliekal et al., 2002) forE. coli MnSOD, and 9.4 (Guan et al., 1998) and

9.2 (this study) for the human MnSOD. Although there has been some disagreement as to

the source of this ionization, a thorough study in E. coli MnSOD assigns this to Tyr34

(Maliekal et al., 2002), with corroborating evidence from Y34F that Tyr34 is also the

source of this ionization in human MnSOD (Guan et al., 1998). NMR studies conducted

on E. coli MnSOD showed that a pH-related chemical shift change corresponding to

ionization of the phenolic hydroxyl of Tyr34 exhibited a pKa of 9.5 + 0.2 (Maliekal et al.,

2002). Human Y34F MnSOD exhibits a pKa near 11 significantly different than the

visible spectrum of wild-type MnSOD (Hsu et al., 1996; Guan et al., 1998). Ionization of

Tyr34 is also most likely the source of the pKa~ 9.5 in catalysis by human MnSOD (Bull

and Fee, 1985; Hearn et al., 2001; Greenleaf et al., 2004).

The mutations E162D and E162A affect this critical pKa in the visible spectrum of

human MnSOD; however, this effect appears to shift the pKa in opposite directions for

each mutant (Figure 3-2). The change in this pKa should be explained in terms of the

effects of these mutations on the ionization of Tyr34. This comment does not preclude

changes in the ionization of ligands of the metal, just that these appear to be outside of

the pH range of these studies. Therefore, the changes in the pKa of the visible absorption









are attributed to Tyr34, the side chain of which is located 6.2 A from the carboxylate of

Glu162 from the adjacent subunit in wild-type MnSOD. The crystal structures allow us to

comment on the mutations at residue 162 on the ionization of Tyr34. Hisl63 is within

3.3 A of S2 in the hydrogen-bond network, allowing indirect interaction between Glu162

and Tyr34 (Hearn et al., 2003; pdb accession #1LUV). The side chain of Aspl62 in the

E162D mutant maintains an interaction with Tyr34 through a solvent-bridged interaction

with His163 while in E162A this interaction does not exist. Alteration of Glu162 could

affect the ionization of Tyr34 through its altered or abolished interaction with His163.

Understanding how Glu162 affects the ionization of Tyr34 warrants further study.

Catalysis

Replacement of the second-shell ligand Glu162 by Ala and Asp resulted in

diminished catalysis in human MnSOD (Table 3-1). Though not essential for catalysis,

mutation of Glu162 resulted in at least a five-fold decrease in rate constants kl-k3 for

E162D and a 20-fold decrease for E162A. This is related to the diminished interaction

between the Glu162 and His163 and the possible concomitant effects on the properties of

the metal and active-site residues such as Tyr34. There is a precedent for substantial

changes in catalysis with mutations at a second-shell ligand and at the dimeric interface.

Table 3-3 Maximal values for kcat/Km and kO/[E] for the catalysis of human wild-type
MnSOD and mutants.

Kcat/Km ko/[E]
([tM-1 sec-1) (sec-1)
WTb 800 500
E162Da 290 270
E162Aa 120 190
H30N c 130 2000
Y166F d 95 360

(a) 2mM TAPS pH 7.7, 50mM EDTA, 30 mM format (see methods for pulse radiolysis)
(b) Ramilo et al., 1998
(c) Hearn et al., 2003
(d) Hearn et al., 2001











The replacement of the second-shell ligand Gln143 with Asn resulted in a 100-fold

reduction in catalysis and evidence of an increase in the redox potential of the active site

(Leveque et al., 2000; Hsieh et al., 1998). The mutation Y166F at the dimeric interface of

human MnSOD resulted in a 10-fold decrease in catalysis (Hearn et al., 2004).

One interesting aspect of the catalysis kl and k2 for the mutants at residue 162, as

well as the step that forms the inhibited complex k3 (Figure 3-4), is that they appear to

have pH profiles similar to the titration of their visible spectra. E162D MnSOD showed a

pH dependence for rate constants ki-k3 with values of kinetic pKa from 8.0 to 8.7 that

roughly matched the pKa of the molar absorptivity (Figures 3-2, 3-4). This pH

dependence was also evident in the values for kcat/Km (Figure 3-5). There was no

observed pH dependence for the kinetic constants kl-k3 for E162A MnSOD in our pH

range of 7.5 10.0, roughly consistent with its higher pKa derived from the pH

dependence of its visible spectrum. Presumably, the pH profiles for E162D and E162A

are the result of the altered, indirect interaction between Glu162 and Tyr34. E162D

MnSOD is the first reported variant of human MnSOD with a pH dependence that

appears well within the range of practicable kinetic measurements and should be useful

for further studies.

Comparison with MnSOD from E. coli

There have been rather few differences noted in kl and k2 in catalysis between

MnSOD from E. coli and humans; consequently, several differences revealed in this

study warrant further attention. The mutant E162A human MnSOD retains specificity for

manganese and is catalytically active although at about 5% the level of human wild type









(Table 3-1). Additionally, it remains a tetramer in solution. This is in contrast to the

equivalent mutation in E. coli, E170A, which results in complete loss of catalytic activity,

dimer destabilization in solution, and is accompanied by a change in specificity to Fe2+/3+

(Whittaker and Whittaker, 1998) It is possible that measurement of catalysis for E. coli

E170A was not sensitive enough to measure 20-fold decreased catalysis. In addition, the

presence of a tetrameric interface in the E162A enzyme confers added stability not

present in the E. coli E170A, thus the E. coli mutant is both monomeric and dimeric in

solution. However, the retention of metal specificity is unique to the human E162A.

Comparison of the crystal structures of the human and E. coli forms of MnSOD

shows nearly superimposable residues for the ligands of the metal and side chains Tyr34

and His30. However, there is a substructure of the active site that is considerably

different for these two forms of MnSOD, and this offers significant clues to the different

responses of the human and E. coli forms of MnSOD to replacements at residue 162.

Specifically, described here are the structural features likely to account for the more

extensive changes in E. coli MnSOD compared with human MnSOD upon mutation at

162 and estimate how these structural features might relate to activity and ionization of

Tyr34. Included in the interactions that form the dimeric interface of MnSOD is a van der

Waals interaction between Phe66 and Glnl 19 (3.5 A) in the human enzyme (Quint and

Reutzel et al., 2006; pdb # 1ADQ) and between Phe124 and Asn73 (4.0 A) in E. coli

(Edwards et al., 1998; pdb # 1VEW) (Figure 3-8). The orientations of these residues are

similar in the E. coli enzyme though they are at a greater distance from each other

compared to human MnSOD. In addition, Phe124 from the adjacent subunit in E. coli

MnSOD interacts with Tyr34 (3.2 A) in E. coli MnSOD. Dimeric destabilization of E.








coli E170A could alter this interaction with Tyr34 and thus affect ionization of the

phenolic hydroxyl. The observations described here clarify differences in stability and in

catalytic activity between the two enzymes, though they do not sufficiently address the

altered metal selectivity exhibited by the two mutants, opening another avenue for future

study.






s2
.f-r th.. djacent. s.. ..... ... His30: Residue..s
"-., ~: ,, 52 ^ ""^ '" ,,. ,














Figure 3-8 Structure of E. coli MnSOD (green) superimposed on wild-type human




interaction between Phel24 and Tyr34 denoted with a dotted line.
Ph66- -
Gin11 1 n .












Product Inhibition

Product inhibition is a prominent feature of catalysis by human and E. coli MnSOD

(Hearn et al., 2001). The rather significant decreases in ki-k4 describing catalysis and

inhibition by E162D and E162A human MnSOD (Table 3-1), are similar to the observed

zero-order component of catalysis. The maximal values of ko/[E], the normalized, zero-









order rate constant for product inhibition, are similar for E162D, E162A, though

diminished compared to wild-type MnSOD: 267 s-1, 189 s-, and 500 s-1, respectively

(Table 3-3) (Hsu et al., 1996). For E162A, the value of ko/[E] was reduced to 20 s-1 at pH

> 8.5 reflecting the pH dependence of k4. The cause of the similar values of ko/[E] for

E162D and E162A is due in significant part to the values of the ratio of k2/k3 in the two

mutants (Table 3-1). This is a gating ratio that determines the extent of reaction that

proceeds to catalysis versus inhibition (eq 3-2, 3-3), and the similar gating ratios for

E162D and E162A are consistent with similar extents of product inhibition. The gating

for wild-type human MnSOD is 1:1 while it is -1:2 for both E162D and E162A (Table 3-

1). This gating ratio is 5:1 forE. coli MnSOD (unpublished) (Table 3-1) indicating a less

product-inhibited enzyme. This represents another key difference between human and E.

coli MnSOD that warrants further study.

The side-chain of Glu162 is important for dissociation of the product-inhibited

complex as evidenced by the similar values for k4 for both E162A and E162D (Table 3-1)

The E162A mutant is the only mutant of human MnSOD observed to date that has

exhibited a pH related decrease in the value for k4. This provides another avenue for

future investigation since neither the structure of the inhibited complex nor its mechanism

of dissociation is known. The values for k4 do not follow the same pH dependence as ki-

k3 because protonation of the bound peroxide is a different process from ki-k3 (Hearn et

al., 2001).














CHAPTER 4
STRUCTURE OF NITRATED HUMAN MANGANESE SUPEROXIDE DISMUTASE

Introduction

The presence of nitrated proteins is associated with a number of pathological states

(Ischiropoulos and Beckman, 2003; Radi, 2004; Shishehbor et al., 2003) and with certain

diseases characterized by inflammatory processes (MacMillan-Crow et al., 2003). Human

MnSOD in the presence of peroxynitrite is nitrated at a number of sites, but the observed

near complete inhibition of catalysis is associated with the nitration of Tyr34 (Yamakura

et al., 1998; MacMillan-Crow, Crow and Thompson, 1998; MacMillan-Crow and

Thompson, 1999). Chapter 3 described the importance of the dimeric interfacial residue

Glu162 and its role in supporting an important pKa for catalysis through its interaction

with Tyr34. Building on the findings of chapters three, the structure of nitrated human

MnSOD was solved, emphasizing the importance of Tyr34 in catalysis and providing a

structural explanation for peroxynitrite-mediated inactivation of MnSOD.

The side chain of Tyr34 plays an important role in catalysis. The replacement of

Tyr34 with Phe causes minor effects on the catalytic efficiency (kat/Km) of human and E.

coli MnSOD (Guan et al., 1998; Whittaker and Whittaker, 1997); however, it does

decrease by 10-fold the value of kcat which determines the maximal velocity of catalysis

(Guan et al., 1998). The crystal structure of the mutant human MnSOD with Phe34 is

nearly identical to that of wild type, with Phe34 closely superimposed on the phenolic

side chain of Tyr34 in the wild-type (Guan et al., 1998). Moreover, the replacement of









Tyr34 with Phe causes no significant change in the redox potential of the human enzyme

(Leveque et al., 2001).

Nitration of tyrosine occurs through two possible mechanisms shown in figure 4-1.

NO + 02' N ONOO- + H t ONOOH t [NO2 + OH] (1)
ONOO- + CO2 = ONOOCO2 = [NO2 + CO3s] (2)
nitrosoperoxycarbonate
OH OH OH


ccO. + "NO2

CH2 CH2 CH2

+H3N COG +H3N COG H3N COOC
tyrosine tyrosyl radical 3-nitrotyrosine

Figure 4-1 Scheme for nitration of tyrosine in the presence of peroxynitrite showing
nitration through the pathway of equation 2.

Nitration of biomolecules by peroxynitrite is enhanced by the presence of CO2 with the

reaction of equation 2 predominating over the reaction of equation 3. Production of

carbonate radical promotes tyrosyl formation and subsequent reaction with NO2 yields 3-

nitrotyrosine (Figure 4-1) (Bonini et al., 1999). Nitration oftyrosine 34 in MnSOD is

associated with abolished activity.

The X-ray crystal structure of nitrated wild-type human MnSOD, as well as the

unmodified enzyme, were both resolved to 2.4 A resolution. Although mass spectrometry

detected partial nitration of several tyrosines and a tryptophan near the surface of the

protein, the crystal structure shows only nitration of Tyr34 in the active site. This nitrated

side chain, 3-nitrotyrosine 34, exhibited only one conformer with the nitro group

extending toward the metal-bound hydroxide/water but not forming a hydrogen bond

with it. Instead the 01 of the nitro group appears to form a hydrogen bond with the Ne2

of residue Gln143. The structure of the nitrated enzyme including active-site residues and









the phenyl ring of 3-nitrotyrosine 34 are closely superimposable with the unmodified

wild-type MnSOD. The conformation in the active-site cavity of the nitrated MnSOD

strongly suggests inhibition by steric interference, by a possible weakening of a hydrogen

bond network, and by the electrostatic effects related to the presence of the nitro group

and the resulting change of the redox potential.

Materials and Methods

Preparation of Nitrated Human MnSOD

Peroxynitrite was produced by mixing equal volumes (5 ml) of NaNO2 (0.8 M) and

acidified H202 (0.7 M H202 and 0.3 M HC1) in a manually operated dual-syringe mixer

and quickly quenched with (3 ml) 3 M NaOH (Crow, Beckman and McCord, 1995). The

final solution was purified using a MnO2 gravity column to remove excess H202 and a

subsequent Chelex 100 gravity column to remove extraneous metal ions. The

concentration of peroxynitrite was determined by measuring optical density at 302 nm

(g302 = 1670 M-1 cm-1) (Hughes and Nicklin, 1968). The nitration of human MnSOD was

carried out by the bolus addition of peroxynitrite to MnSOD in the presence of

C02/HC03-. MnSOD samples were equilibrated overnight at 40C prior to reaction with

peroxynitrite. The mixed solution contained MnSOD (50 piM), all species of CO2 (25

mM), peroxynitrite (8 mM), and phosphate buffer (20 mM) at pH 7.8 and 25 C.

Following reaction with peroxynitrite, samples of modified enzyme were pooled and

concentrated in 20 mM phosphate pH 7.8.

Modified enzyme was digested with acetylated trypsin for 3 hours at 370 C.

Capillary reverse phase HPLC separation of tryptic fragments was performed on a

PepMap C18 column (LC Packings, San Francisco, CA) in combination with an Ultimate









Capillary HPLC System (LC Packings, San Francisco, CA) operated at a flow rate of 200

nL/min. Extent of nitration was measured using a QSTAR XL (LC/MS/MS system, MDS

Sciex, Ontario, Canada). To determine the ratio of nitrated versus unmodified MnSOD,

the peak areas for modified and unmodified masses were compared. We determined the

following extents of nitration: 75% nitration of Tyr34, 36% overall nitration of Tyr9 and

Tyrl 1 (residues 9 and 11 were in the same tryptic fragment), 46% overall nitration of

Trpl80 and Trp186 (residues 180 and 186 were also in the same tryptic fragment).

Crystallization

Wild-type and nitrated human MnSOD were buffer exchanged into 20 mM

phosphate buffer at pH 7.8 and concentrated (16 mg/ml) using a centricon YM-10

(Amicon). The samples were crystallized using the hanging drop vapor diffusion method

(McPherson, 1982). The drops consisted of 5 [tl of enzyme mixed with 5 dtl of precipitant

solution (3 M ammonium phosphate, 100 mM imidazole, 100 mM malate) and suspended

over 1 ml of precipitant solution at 250 C. The crystals grew to full size (0.8 x 0.5 x 0.5

mm) in approximately one week.

Data Collection and Processing

Both wild-type and nitrated human MnSOD X-ray diffraction data were collected

from single crystals, wet mounted in quartz capillaries (Hampton Research), on an R-

AXIS IV++ image plate (IP) system with Osmic mirrors and a Rigaku HU-H3R CU

rotating anode operating at 50 kV and 100 mA (Rigaku/MSC). A 0.3 mm collimator was

used with a crystal to IP distance of 220 mm and the 20 angle fixed at 0. The frames

were collected using a 0.3 oscillation angle with an exposure time of 5 min/frame at

room temperature. Both data sets were indexed using DENZO and scaled and reduced









with SCALEPACK software (Otwinowski and Minor, 1997). Diffraction intensities were

visible to 2.4 A resolution and a total of 200 frames were collected from both wild type

and nitrated human MnSOD crystals.

Structure Determination and Refinement

To prevent any model phase bias, the initial phasing model for both the unmodified

and nitrated human MnSOD was the structure of the W161A MnSOD mutant (Hearn et

al. 2001; pdb accession number 1JA8) from which the nine tyrosines (residues: 9, 11, 34,

45, 165, 166, 169, 176, and 193) and six tryptophans (residues: 78, 123, 126, 161, 181,

and 186) had been replaced by alanines, and the Mn2+ ion and solvent molecules had

been removed. The structures were phased and refined using the software package CNS

(Brunger et al., 1998). Refinement cycling (using rigid body, simulated annealing (for the

first cycle), minimization, and individual B-factor refinement) was interspersed with

rounds of manual model building using the molecular graphics program O (Jones et al.,

1991). Following the first cycle of refinement, the positions of the manganese ion, nine

tyrosines, and six tryptophans were clearly identified and built into Fo-Fo electron density

maps for both structures. After the second cycle of refinement the unambiguous electron

density for a nitro group was observed in the vicinity of the Cel atom of tyrosine 34 for

the nitrated human MnSOD and a model for a 3-nitrotyrosine residue was built and

energy minimized using the PRODRG2 server (Schuettelkopf and Aalten, 2004) and

placed into the electron density. The mass spectrometric analysis had previously

determined the extent of Tyr 34 nitration to be -75 %. Due to this observation, the atoms

comprising the nitro group were refined with an occupancy of 0.75 compared to the rest

of the atoms in the protein and solvent. Both structures were further refined for several

more cycles with some minor manual building, after which solvent molecules were









picked both automatically in CNS (using a 3 sigma cut off) and manually in O by

inspection of Fo-Fo electron density maps. The bond geometry of the models was

analyzed using the software package PROCHECK (Laskowski et al., 1993). The final,

refined models and structure factor files have been deposited with the Protein Data Bank,

PDB (accession codes 2ADQ, and 2ADP for the unmodified and nitrated human

MnSOD, respectively).

Results

The structures of the unmodified and the nitrated human wild-type MnSOD have

been solved in the hexagonal space group P6122, with unit cell parameters a = b = 81.3

and c = 242.2 A, and refined to 2.4 A resolution (Table 4-1, Supplementary material 1

and 2). The final refined structure of the unmodified human MnSOD had an Rryst of 21.7

% (Rfree of 24.0 %) with an average B-factor of 26.2 A2 ; and the nitrated human

MnSOD had an Rcryst f 19.7% (Rfree of 21.8 %) with an average B-factor of 31.2 A2

(Table 4-1). Following the second cycle of refinement the position of a single 3-

nitrotyrosine 34 was built into Fo-Fo and 2 Fo-Fo electron density maps in the nitrated

human MnSOD structure (Figure 4-2). The occupancy for the nitro group on 3-

nityrosine34 was modified to reflect the percent nitration determined by mass

spectrometry. There was no unique density surrounding tyrosines 9 and 11 or tryptophans

181 and 186.

A comparison of the wild-type and nitrated human MnSOD structures showed no

significant side-chain conformational changes or solvent displacement in the active site

with the nitration of tyrosine 34 (Figure 4-3). The root-mean-squared difference for all Ca

atoms was 0.13 A comparing the nitrated with unmodified MnSOD. The active-site









manganese of wild-type human MnSOD has been previously reported (Borgstahl et al.,

1992) as coordinated by the Ne2 of three histidine residues (H26, H74, H163), 061 of

Asp 159, and a Mn bound water/hydroxide molecule arranged in a distorted trigonal

bipyramidal geometry. The nitration of Tyr34 caused no significant change in this

geometry or in first shell ligand distances (Figure 4-2). Also of interest was that the

overall refined thermal individual atom B values for both structures were similar and

there was no significant difference for the manganese in unmodified and nitrated human

MnSOD with values of 16.9 and 21.6 A2, respectively. Of note, which may or may not

be of significance, was the B values for the Mn bound water/hydroxide molecule which

was 11.6 A2 for wild-type and nearly twice the value 19.1 A2 for nitrated human MnSOD.

The significance of this can only be resolved with either higher resolution X-ray

diffraction data or a neutron diffraction structure.

In comparing the crystal structures of the nitrated and unmodified wild-type human

MnSOD, we observed that the side chain of 3-nitrotyrosine 34 was in a single side-chain

conformer with the 01 and 02 positioned 3.6 and 3.8 A from the manganese ion.

Probably the most significant interaction observed by the nitration of Tyr34 is the

inferred hydrogen bond (3.1 A) between 01 of 3-nitrotyrosine 34 and NE2 of Gln143

(Table 4-2, Figure 4-3).

A comparison of the two non-crystallographic subunits of the human nitrated

MnSOD in the hexagonal space group P6122 (data not shown) showed no differences in

orientation of the nitrated Tyr34 residues and they were subsequently averaged in the

refinement protocols. There was also no evidence of dityrosine (3,3'-dityrosine)

formation in the crystal structure.
























Figure 4-2 Structure of the active site of the nitrated human MnSOD. Stereo diagram
showing the initial, no model bias (Fo-Fo) and (2Fo-Fc) electron density maps
contoured at 3.0 a (black) and 1.0 a (grey), respectively, into which the 3-
nitrotyrosine 34 and manganese-bound hydroxide were modeled. The active
site manganese (pink) is pentacoordinate with inner shell ligands His26,
His74, Hisl63, Asp159, and the metal-bound solvent molecule.


Figure 4-3 The structure of the active-site region of nitrated (yellow) superimposed onto
unmodified human MnSOD (green). A) The proposed hydrogen bond network
(red dashed lines) for unmodified wild type, and B) the proposed hydrogen
bond network (blue dashed lines) for nitrated human MnSOD. The proposed
hydrogen bond network involves residues Gln143, Tyr34, His30, solvent
molecule (W2) and Tyrl66. See Table 4-2 for a complete listing of distance
geometry.


A) solvent(W)

6/' C H26
_143 Mn



Y34 H30 -
solvent (W2) Y166' %


(symmetry related subunit)









Table 4-1 X-ray crystallographic structure statistics of unmodified and nitrated human
MnSOD

S Unmodified Nitrated


Data Collection
Resolution (A)
Space group
Unit cell (A)
Molecules/a.s.u.
aRsym (%)
Reflections (total/unique)
Completeness (%)
Refinement
Non-hydrogen atoms
Water molecules
Average B factors (A2)
Protein main-chain
Protein side-chain
Solvent molecules
Tyrosine 34
Rcryst/Rfree (%)
R.m.s.d bond lengths (A)
/angles (o)


20.0-2.4 (2.49-2.4)*
P6122
a=b=81.3, c=242.2
2
10.2(19.1)
244,364 / 61,091
95.8 (90.2)

1629
74

25.1
27.2
35.9
20.3
21.7 / 24.0

0.006/ 1.3


20.0-2.4 (2.49-2.4)
P6122
a=b=81.3, c=242.2
2
10.9(14.9)
231,776 / 60,459
91.3(80.6)

1605
46

30
32.3
36.8
27.6
19.7/21.8

0.006/ 1.3


*Data for the highest resolution shell are given in parentheses.
aRsym = |I-| / I x 100, where I is the intensity of a reflection and is the
average intensity.
bRcryst = Zhkl Fo-KFC / hkl |Fol x 100, Rfree is calculated from 5% randomly selected
data for cross-validation.
cR.m.s.d. = root mean square deviation.

The nitration at Tyr9 and 11 was less extensive (36% overall) and was not observed

in the crystal structure; mobility of these surface residues may have made the nitration

less evident. Nitration at Trpl80 and 186 was also not observed in the crystal structure.

Discussion

The structure of human MnSOD containing 3-nitrotyrosine at position 34 is well-

defined by the 2.4 A electron density map (Figure 4-1) with a three-dimensional structure

closely superimposable with the unmodified enzyme (Figure 4-2); specifically, there are









no conformational changes in the active-site cavity. This is the first reported crystal

structure of a nitrated MnSOD, to our knowledge.

Table 4-2 Distance geometries (A) in the active-sites of unmodified and nitrated
human MnSOD
Interaction Wild-type Nitrated
Mn2 aSolvent Wl 2.1 2.1
aSolvent Wl Q143-Ne2 3.1 3.1
Q143-Ne2 Y34-OH 2.5 3.1
Y34-OH aSolvent W2 3.1 3.3
aSolvent Wl Y34-01 --- 3.5
Q143-Ne2 Y34-01 --- 3.1
Y34-01 Mn2+ --- 3.6
Y34-02 Mn2 --- 3.8
aSolvent W2 H30-N61 2.9 3.0
H30-Ne2 Y166-OH 2.6 2.6
aSee figure 4-2 for position of solvent W1 and W2.


The nitrated Tyr34 side chain shows only a single conformer with the nitro group

directed toward the metal (Figure 4-1). This conformer predominates in large part

because of electrostatic gradients within the active-site cavity as discussed below;

however, there are probably also steric considerations that limit the range of side-chain

conformations of nitrated Tyr34. For example, the side chain of Phe66 is within 3.6 A of

that of 3-nitrotyrosine 34 and limits the range of orientations of this nitrated residue. In

addition, there is a role for manganese in the reaction of MnSOD with peroxynitrite

(Quijano et al., 2001); hence, the orientation of 3-nitrotyrosine in the nitrated enzyme

may reflect the reactive site of the side chain of Tyr34 that is closest to the metal.

Although the nitro group of modified Tyr34 is near the manganese bound

hydroxyl/water, the distance between oxygen atoms at 3.5 A (Table 4-2) is too great to

form a hydrogen bond, nor is the nitro group sufficiently close, -3.7 A, to the metal to be

considered an inner shell ligand. The nitro group is viewed more accurately as a second-









shell ligand of the manganese, although its presence causes no changes in geometry or

distances of the first-shell ligands. However, the crystal structure is consistent with a

hydrogen bond between the nitro group at Tyr34 and the Ne2 of Gln143, with a distance

of 3.1 A from the 01 of nitrated-Tyr34. There is still evidence for a hydrogen bond

between the Ne2 of Gln143 and the phenolic OH of nitrated-Tyr34 with a distance of 3.1

A (Table 4-2, Figure 4-2). However, this distance is considerably lengthened compared

with that of the unmodified enzyme and this hydrogen bond involving Ne2 of Gln143

may be bifurcated between the phenolic OH and the nitro group of 3-nitrotyrosine 34.

Nitration of human MnSOD inhibits catalysis by greater than 90% (Yamakura et

al., 1998; MacMillan-Crow et al., 1998). This inhibition is associated with nitration of

Tyr34 (Yamakura et al., 1998; MacMillan-Crow et al., 1998), although there is evidence

that nitration of other tyrosine residues may also decrease activity (MacMillan-Crow et

al., 1999). We comment then on the likely causes of inhibition by MnSOD containing 3-

nitrotyrosine at position 34. First, it appears that nitration has not affected the

stereochemistry of the active site residues; that is, the conformation of Gln143, which

forms a hydrogen bond with the aqueous ligand of the metal, is not altered and the side-

chain orientation of Tyr34, although nitrated, is not changed. So conformational changes

induced by nitration are not pertinent in the inhibition. However, the hydrogen bond

network involving the side chains of Gln143, Tyr34, His30, and Tyrl66 from an adjacent

subunit appears to be altered and possibly weakened at 3-nitrotyrosine (Table 4-2, Figure

4-2), as mentioned above. This network, and particularly Tyr34, has been associated with

proton transfer in catalysis by wild-type MnSOD, either proton transfer to product

peroxide or to the metal-bound hydroxide (Whittaker and Whittaker, 1997; Silverman









and Nick, 2002; Hunter et al., 1997; Sorkin, Duong and Miller, 1997; Bull and Fee, 1985;

Stallings et al., 1991), and alteration of this network in the nitrated enzyme is consistent

with inhibition.

Another likely cause of inhibition is simply that the bulk of the nitrated side chain

inhibits catalysis, perhaps by its presence in the substrate access channel and/or steric

overlap with the enzyme-substrate complex or with the transition state. This was the case

with H30V MnSOD in which the C of Val30 is 4.4 A from the manganese and either

blocks the substrate access channel or has a steric overlap with the developing transition

state. The 01 and 02 of the nitro group of nitrated Tyr34 are closer to the metal (near 3.7

A) and also lie along a likely substrate access channel. The substrate access channel in

MnSOD and FeSOD is exceedingly narrow and there is evidence from dynamics

simulations that substrate diffusion to the vicinity of the manganese requires

conformational fluctuations along this channel (Sines et al., 1990). A prominent side

chain, the motion of which can open this channel, is Tyr 34 (Sines et al., 1990). Chemical

modification by nitration of the phenolic side chain would certainly slow this process.

Yet another possibility for inhibition is the change in pKa of nitrated Tyr, which is

expected to be lower by about 2 pKa units compared with unmodified Tyr. We have no

measure of the pKa of nitrated Tyr34 in MnSOD, although this value was estimated from

spectroscopic data at pKa 7.95 for FeSOD nitrated at Tyr34 (Soulere, 2001). Nitrated

tyrosine would have a larger fraction as tyrosinate anion than unmodified, and could

decrease catalysis by electrostatic repulsion of the substrate O'- as well as have a major

effect on the redox potential of the enzyme (Miller et al., 2003). The active-sites of

MnSOD and FeSOD are very finely tuned to catalyze both the oxidative and reductive









stages of catalysis (Vance and Miller, 1998), and introduction of a nitro group near the

metal is likely to alter this tuning. That is, the nitration of Tyr 34 has almost certainly

altered the redox potential.

This study provides a connection between the nitration and subsequent inhibition of

MnSOD. The location in the active site of the NO2 group of 3-nitrotyrosine 34 gives a

basis for understanding the strong inhibition of this essential antioxidant enzyme. It is

notable that replacement of Tyr34 with Phe has very little effect on catalysis up to low

micromolar concentrations of 02 (Guan et al., 1998; Whittaker and Whittaker, 1997)

and does not decrease thermal stability or alter the crystal structure (Guan et al., 1998).

Yet its prominence in the active-site cavity makes it a site for nitration and inhibition of

MnSOD.














CHAPTER 5
CONCLUSIONS AND FUTURE DIRECTIONS

Conclusions

This dissertation focused on the two structurally unique interfaces of human

MnSOD. The overall goal was to understand the contributions to stability and catalysis of

the two interfaces and to compare the tetrameric human MnSOD to the dimeric E. coli

MnSOD. The data presented in chapters 2 and 3 of this study suggest that the tetrameric

interface provides stability while dimeric interfacial residues play a greater role in

catalysis. The importance of Tyr34 in maintaining catalysis was explored in chapter 4

with a structure of nitrated human MnSOD.

The Tetrameric Interface in Human MnSOD

One key characteristic of human MnSOD that differentiates it from E. coli is the

presence of a tetrameric interface. Chapter 2 presented 19F NMR and differential

scanning calorimetry studies elucidating the roles of the two interfaces of human MnSOD

and the data suggest a stabilizing role for the tetrameric interface. Though the tetrameric

interface is generally associated with higher organisms, it may in fact be a more primitive

structural arrangement. The endosymbiotic theory suggests a bacterial origin for

mitochondria; perhaps mitochondria resulted from phagocytosis by a much larger cell

billions of years ago. MnSOD may have originally been a tetramer to accommodate a

higher average temperature caused by increased atmospheric CO2 levels. As atmospheric

CO2 levels waned in the presence of photosynthesizing cyanobacteria, prokaryotes and

eukaryotes diverged and the mitochondrion became a selective advantage for eukaryotes.









It is interesting that some heat extremophiles, like T thermophillus and P. aerophilus

utilize tetrameric MnSODs (Wagner et al., 1993; Whittaker and Whittaker, 2000).

The Dimeric Interface and Differential Roles of Glu162 in Human MnSOD and
Glul70 in E. coli MnSOD

Considering the importance of the dimeric interface in stability and catalysis,

chapter 3 emphasized the contribution to stability and catalysis of Glu162 in human

MnSOD and Glul70 in E. coli MnSOD. In addition to an observed decrease in catalysis

and altered product inhibition, E162D exhibited a pH dependence for catalysis not

observed in the native enzyme. This observed pKa for catalysis may be the result of an

altered interaction with Tyr34 through His163 and a bridging solvent molecule. The

E162A mutant is less stable, though it is still tetrameric in solution and is associated with

a significantly diminished catalysis for the enzyme, emphasizing the importance of

Glu162 in supporting catalysis and stabilizing the enzyme. In contrast, the equivalent

mutation in the E. coli MnSOD, Glul70Ala, exhibits no activity, is selective for iron over

manganese is a mixture of monomer and dimer in solution. This represents a significant

difference between human and E. coli MnSOD, the active sites of which are structurally

equivalent. A stabilizing interaction between Phe66 and Gln 119 in the dimeric interface

of human MnSOD is absent in the E. coli enzyme, thus the E170A mutant destabilizes to

a greater extent than E162A. In addition, an interaction between Phe124 and Tyr34 exists

in the E. coli MnSOD that is not present in the human enzyme. This could affect

ionization of Tyr34 for the E. coli enzyme and perhaps affect its metal selectivity, though

the latter remains an area for further research.









A Structural Explanation for Abolished Catalysis of Nitrated Human MnSOD

To add to a growing literature on the reaction of peroxynitrite with MnSOD,

chapter 4 presented a structural explanation for abolished catalysis resulting from

nitration of Tyr34. Previous studies have shown that nitration of Tyr34 is associated with

complete catalytic inhibition (MacMillan-Crow et al., 1995) (Yamakura et al., 1996). The

structure of nitro-MnSOD indicates that diminished catalysis is the result of steric

blockade of the substrate as well as possible electrostatic repulsion of superoxide anion.

These findings will help elucidate the role of nitro-MnSOD in certain diseases involving

peroxynitrite-mediated nitration of biomolecules.

Future Directions

The Dimeric Interface of Human MnSOD

The peaks corresponding to fluorine labeled tyrosines shown in chapter 2 were

significantly broad and four of the nine fluorine labeled tyrosines were not observed in

the fluorine NMR spectrum (Figure 2-2). The peaks for Tyr34, Tyrl65, Tyrl66 and

Tyrl76 were not observed and may have been broadened due to their proximity to the

paramagnetic manganese of the active site; all four tyrosines that were not observed are

within 10 A of the active site metal. One way to alleviate this problem would be to

construct an apo-fluoro-MnSOD. This would involve expression and subsequent

chelation of the metal from fluoro-MnSOD. The manganese is tightly bound in the active

site and chelating the metal would involve denaturation and refolding of the enzyme.

Initial attempts to chelate the manganese have resulted in an unfolded enzyme. Further

attempts may involve the use of other chelators and perhaps changes in pH. Expression

with a non-paramagnetic metal substitute may also alleviate the issue of excessively

broadened lines.









Redox properties of E162 mutants

Measurement of redox properties of human MnSOD are difficult due to the size of

the active site and require an electron mediator to measure the redox potential (Leveque

et al., 2001). Mediators including ferricyanide and pentacyanoaminoferrate have been

used successfully though the effective range of study is limited; to use ferricyanide, the

midpoint potential for the enzyme must be below 435 mV. The cause of reduced catalysis

in mutants E162A and E162D is not fully understood and measurement of the redox

potential of E162D and E162A MnSOD may elucidate these causes.

Catalytic Properties of Nitrated MnSOD

Indirect studies (including xanthine oxidase assays) have shown that nitration of

MnSOD abolishes activity of MnSOD. Direct methods like stopped-flow and pulse

radiolysis have not been used to measure catalysis of nitro-MnSOD, in part because it is

difficult to achieve 100% nitration of MnSOD. Catalytic studies could provide a more

thorough explanation for catalytic decrease. Chapter 4 described the structure of nitrated

MnSOD comprised of 74% nitration of Ty34. A pulse radiolysis study would require the

purification of 100% nitrated enzyme using affinity chromatography. It would be

interesting to determine rate constants kl-k4 for fully nitrated enzyme to see how nitration

has altered catalysis of MnSOD. For instance, the enzyme could quickly enter a product

inhibited state from which it does not dissociate.

Future of Therapeutic Studies

The potential for human MnSOD in drug discovery is great and future

therapeutic studies will focus on the dimeric interface. For example, MnSOD mutants

that are less product-inhibited, such as H30N, are useful as anti-proliferative agents

(Davis et al., 2004) and could potentially be used as life-saving therapies during






62


reperfusion injury. In addition, the presence of nitrated MnSOD will serve as a

biomarker for chronic diseases such as allograft rejection and could potentially be used as

markers for cancer.















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BIOGRAPHICAL SKETCH

In 1995, Patrick Quint received his high school diploma from Rocky Mountain

High School in Ft. Collins, CO, and then moved to St. Paul, MN, where he attended

Macalester College. Upon receipt of his BA in biology, he worked at 3M for two years

before attending graduate school at University of Florida. After successfully defending

his dissertation, he will receive a post-doctoral appointment in the lab of Dr. Bob Bergen

at the proteomics division at Mayo Clinic in Rochester, MN.