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UNITED STATES ATOMIC ENERGY COMMISSION
THE DARK REDUCTIONS OF PHOTOSYNTHESIS
University of California
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Date of Manuscript: May 27, 1947
Date Declassified: June 12, 19-.7
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trI" ; .'" .p D M PK .DUCTIQNS OF PHQTOYNTHESIS
.,*, '.: :!i. ... .,. By A, Benson and M. Calvtn
Although green plants have been shown' to fix CO, in the dark, the conditions influencing that
fiati~oh and the compound formed re unknown. We are investigating these variables.
\dw-,, .The renditionslof the experiments were as follo h: A sample of actively growing algae was split
into two parts (approximately 1 cc algae/15 cc suspension). One part (I) was kept in the dark exposed
to 4 per cent CO, in N, for about 8 hours. The other part (II) was exposed to the light of a 150 watt
r.tungstellamp (.Tg cal/cmn/min) for one hour during which time it was kept free of CO, by constant
flushing with N,. The two samples were then evacuated, kept in the dark, and simultaneously exposed
to the same gas containing CU40 in N, for a period of 5 minutes. At the end of this period, the algae
weil killed by an acetic acid-HCI mixture and the remaining active CO, pumped off.
'The total non-volatile radiocarbon content of the two samples was then measured and its chem-
ical distribution determined. The preliminary results are given in the following table.
Dark fixation of CO, by chlorella.
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.PetreaSment CO, in the dark Light in the absence
S: .. .. of Cp,
T. otal (relative units) 1 5.5
Succinic acid* 70% 6%
i P 'larie acid 3% 1%
Malic acid -- 6%
-: '.:. Catloale substances
(not extractable by ether from pH 1- 15% 30%
probably amino acids)
(not extractable by ether from pH 1) 9% 10%
Neutral (sugars) < .1% 1.5%
Unidentified 2% 6%
extractablee by ether from pH 6)
extractablee by ether from pH 1)
The succinic acid was isolated without carrier and identified by extraction
coefficient, equiv. wt, C and H analysis, m.p., and X-ray powder pattern.
t Absorbed by Duolite ion exchange resins C-3 and A-3, respectively.
2 MDDC 1027
From these results alone, it is clear that the reduction of CO to sugars and the intermediates
in that reduction does not involve the primary photochemical step itself. This is further substan-
tiated by the appearance of an appreciable fraction (up to 15 per cent) of the radiocarbon in the meth-
ylene groups of the succinic acid isolated from sample I of the table.2
It is thus confirmed that the photochemical process establishes a reservoir, small to be sure,
of reducing power which can subsequently carry out all of the reduction steps necessary to bring
CO, to sugar.
Using some of the reactions already established li animal tissue and bacterias it id'posaib to
account for the above results as well as the observed distribution of radiocarbon in sugar produed
by a short photosynthesis.4 Starting with either acetate or pyruvate, the numbers over each carbon
Oxalacetate 3 2 +C2 a I ATP".
Tricarboxylie xCHC --- +[2H] CH, CO CQ-- hospo-ea olp ylrte
acid cycle .. ,
B +[2J i-C Cr .- I r
[-4H] -2CO, .....+[IM,
I 2 2 I I I
-oC-C- -CH,-Co; A -OC-CcH-CO C-. (sugar, starch)
H,0 i i
-OC-CH= CH-CO, ,C-CH,-CHOH-COj-
atom Indicate which carbon atoms-are labeled each time around cycle A. The' reducing power, of
course, (indicated as [2HJ) is ultimately derived from the light reaction and some of it might well be
reduced coenzymes I or II. The high-energy phosphate required in these reductions is not explicitly
shown in the chart. All or part of it could easily be derived from the combustion of part of the
acetate through cycle B.
It should be mentioned that this scheme cannot be a simple reversal of the respiratory system of
reactions since CO, derived from respiration of barley leaves5 containing freshly photosynthesized
radioactive sugars has a lower specific activity (per mg C) than the sugar itself. If respiration in-
volves some of the same intermediates as those shown in the chart, the respiratory system must be
physically separated from the photosynthetic system.
1. Ruben, Kamen, and Hassid, J. Am. Chem. Soc. 61: 861 (1939).
2. Benson, Bassham, and Calvin, unpublished.
3. Wood, Physiol. Rev. 26: 198 (1946).
4. Aronoff, Barker, and Calvin, J. Biol. Chem. 664, No. 2: 459-60 (1947).
5. Aronoff, Benson, Hassid, and Calvin, Science 105: 664-665 (1947).
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