DIFFERENT OXIDANTS 1577 



(e. g., by a "transphosphorylation" reaction with adenosine triphosphate, 

 ATP). Reduction then leads to the degradation of a "high energy" car- 

 boxyl phosphate to a "low energy" carbonyl phosphate, and the energy 

 required for reduction is thus decreased by the energy difference between 

 the two types of phosphate esters (about 10 kcal, corresponding to a shift 

 of about 0.22 volt downward on the redox potential scale). We will return 

 to these relationships below. 



Attempts to use pyridine nucleotides as ultinaate Hill oxidants ^^■ere 

 unsuccessful (Holt and French 1948; Mehler 1951). 



Mehler found no reduction of DPN also when the dye dichlorophenol-indophenol 

 (which reacts rapidly with DPNH.) was supplied as intermediate, although the dye was 

 reduced in light practically completely. (Considering the wide difference between the 

 normal potentials of DPN and the dye, this is not astonishing, cf. considerations on p. 

 1522). Similar results were obtained by Wessels (1954) when lie tried to use quinone, or 

 dichlorophenol-indophenol, as intermediate catalyst to reduce oxidized glutathione, de- 

 hydroascorbic acid, dehydroxyphenylalanine, riljoflavin or DPN. 



The reason for the negative result of attempts to reduce pyridine nucleo- 

 tides in the Hill reaction could be a rapid back reaction of reduced pyridine 

 nucleotides with intermediates in the oxidation of water (or with the final 

 oxidation product, molecular oxygen). The surmise that a small amount 

 of DPNH2 or TPNH2 is present in the photostationary state was confirmed 

 by "trapping" these compounds with one of the enzymatic systems which 

 use them for the reduction of pyruvic acid or other respiration intermedi- 

 ates. This proof was given independently by Vishniac in Ochoa's labora- 

 tory in New York, by Tolmach in Franck and Gaffron's laboratory in 

 Chicago and by Arnon in Berkeley. 



Vishniac (see Ochoa 1950; Vishniac 1951; Ochoa and Vishniac 1951; 

 Vishniac and Ochoa 1951) based his experiments on previous findings con- 

 cerning reversible oxidative decarboxylation of malic acid (or c?-isocitric 

 acid) to pyruvic acid (or ketoglutaric acid) in the presence of "malic en- 

 zyme" (or isocitric dehydrogenase and aconitase), with TPN as specific 

 hydrogen acceptor. These reactions normally proceed in the direction of 

 decarboxylation and oxidation, but they can be reversed by the supply of 

 excess TPNH2 and CO2, e. g. : 



malic 

 enzvme 



(35.34) CO2 + CH3COCOOH + TPNH2 . " ^ COOHCH2CHOHCOOH + TPN 



pyruvate malate 



It was surmised by Ochoa, Veiga Solles and Ortiz (1950) that illuminated 

 chloroplasts may be capable of converting TPN to TPNH2 and thus driving 

 reaction (35.34) from left to right. The first proof of this was obtained by 

 Vishniac and Ochoa, who showed that if pyruvate and bicarbonate are 

 added to a chloroplast suspension in the presence of TPN, malic enzyme 



