EVOLUTION OF THE CO2 REDUCTION MECHANISM 1693 



carboxylation reaction — pyruvic to oxalacetic acid — was retained. In sup- 

 port of the hypothesis that vinyl phosphate serves as carbon dioxide acceptor 

 in photosynthesis, Calvin quoted the observation that some C(14) can be 

 volatihzed from Chlorella material obtained after about 1 min. of photo- 

 synthesis, in the form of acetaldehyde, by 10 min. hydrolysis with N HCl 

 at 80°C.; the presence of a tagged vinyl ester offered a plausible explana- 

 tion of this observation. 



Later (1950), Calvin and Benson l^ecame doubtful also of the correct- 

 ness of the oxalacetate-malate-fumarate-succinate segment of the original 

 cycle. The absence from the radiograms of active oxalacetic acid could be 

 attributed to its instability, and the appearance of active aspartic acid 

 (derivable from oxalacetic acid by reductive amination) could be consid- 

 ered as indirect evidence of the occurrence of the latter. The scarcity of 

 active succinic acid was, however, difficult to explain. In some experi- 

 ments, glyceric acid, labelled in all three positions, as well as labelled hexose, 

 have been obtained in complete absence of labelled succinic acid. Further- 

 more it was necessary to account for the early appearance of radioactive 

 glycolic acid (fig. 35.14) and glycine (probably derived from glyoxylic 

 acid) . Malonate, which is known to inhibit the succinic acid-fumaric acid 

 conversion, was found not to interfere with photosynthetic carbon dioxide 

 reduction — not only with the production of active sugar but also with the 

 appearance of active glycolic acid and glycine. (Only the formation of ac- 

 tive malate was totally eliminated by malonate, cf. above, section 6.) 

 This proved that a different path, avoiding the malate-succinate-fumarate 

 series, must exist, leading — it was still assumed — from oxalacetate to acetyl 

 phosphate or another C2 compound. 



Calvin and co-workers (1950) discussed the possibility that a C*i compound could 

 serve as precursor of the C*2 acceptor (a hypothesis which is a priori implausible because 

 of the poisonous nature of both formaldehyde and formic acid). Experimentally, only 

 traces of labelled formaldehyde or formic acid were found. Assuming complete isotopic 

 equilibration of these traces with the C*02 used, the quantities of C* found in Ci com- 

 pounds corresponded to 2 X IQ-io mole H2CO, and 12 X IQ-'" mole HCOOH in 1 g. 

 of wet cells. In all likelihood these small quantities were artifacts (cf. Vol. I, chapter 

 lO.C). 



If the C2 acceptor is not formed via a Ci compound, the most likely 

 mechanism of its formation is splitting in two of a C4 compound, as assumed 

 in scheme 9.II. Calvin and co-workers proceeded to discuss the most likely 

 mechanism of this sphtting. 



With succinic and fumaric acids eliminated as possible intermediates 

 between oxalacetic acid and the C2 compound (their appearance in tagged 

 form being now attributed to respiratory CO2 fixation), and with malic acid 

 also excluded as main line intermediate by malonate inhibition experiments, 



