i^'|V10lX-'0^7 7tyi^//iAppc'/o^i MDDC - 1027 UNITED STATES ATOMIC ENERGY COMMISSION THE DARK REDUCTIONS OF PHOTOSYNTHESIS By A. Benson M. Calvin University of California This document consists of 2 pages. Date of Manuscript: May 27, 1947 Date Declassified: June 12, 19,7 \ This document is issued for official use. Its issuance does not constitute authority to declassify copies or versions of the ■ same or similar content and title and by the same author(s). Technical Information Division, Oak Ridge Directed Operations Oak Ridge, Tennessee THE DARK REDUCTIONS OF PHOTOSYNTHESIS By A. Benson and M. Calvin Although green plants have been shcwn^ to fix CO, in the dark, the conditions influencing that fixation and the compounds formed were unknown. We are investigating these variables. The conditions of the experiments were as follows: A sample of actively growing algae was split into two parts (approximately 1 cc algae/l5 cc suspension). One part (I) was kept in the dark exposed to 4 per cent COj in N^ for about 8 hours. The other part (II) was exposed to the light of a 150 watt tungsten lamp (.7 g cal/cm^/min) for one hour during which time it was kept free of COj by constant flushing with Nj. The two samples were then evacuated, kept In the dark, and simultaneously exposed to the same gas containing C^Oj in Nj for a period of 5 minutes. At the end of this period, the algae were killed by an acetic acid-HCl mixture and the remaining active C^Oj 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 COj by chlorella. I n Pretreatment CO, in the dark Light in the absence of COj Total (relative units) 1 5.5 Succinic acid* 70^ 6% Fumaric acid 3% ■ 1% Malic acid > — 6% Cationic substancest (not extractable by ether from pH 1 — 15% 30% probably amino acids) Anionic substancest (not extractable by ether from pH 1) 9% 10% Neutral (sugars) < .1% 1.5% Unidentified 2% 6% (extractable by ether from pH 6) Unidentified — 25% (extractable 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. MDDC - 1027 [ 1 2] MDDC - 1027 From these results alone, it Is clear that the reduction of COj 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 1 of the table.^ 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 COj to sugar. Using some of the reactior.s already established in animal tissue and bacteria,^ it is possible to account for the above results as well as the observed distribution of radiocarbon in sugar produced by a shori photosynthesis.' Starting with either acetate or pyruvate, the numbers over each carbon + [2H] Oxalacetate ^ ^ +C0 321 ATP Tricarboxylic • CHjCO" IHw] * ^^» ^^ ^^^ * Phospo-enol pyruvate acid cycle I ^ [-4H] -2CO2 f[2H] -* -O2C -CEj -CHj -COj" f[2H] "0,C-CH = CH-CO;«- ^22 ^ -H^O + 00, ■OjC-CHj-COCO," f[2H] "OjC -CH2-CHOH-CO2" (sugars, starch) atom Indicate which carbon atoms are labeled each time arovind cycle A. The reducing power, of course, (indicated as [2H]) is ultimately derived from the light reaction and some of it might well be reduced coenzymes I or 11. 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 leaves^ 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. REFERENCES 1. Ruben, Kamen, and Hassid, J. Am. Chem. Soc. 61: 661 (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). UNIVERSITY OF Fl ORIDA 3 1262 08909 7520 \