199 



Daniel I. Arnon 



pyruvic dehydrogenase with hydrogenase in the production of Hg 

 from pyruvate. 



Aside from photoproduction of H2, Tagawa and Arnon (9) found 

 that clostridial ferredoxin catalyzed the photoreduction of TPN 

 by spinach chloroplasts without the participation of the "photo- 

 synthetic pyridine nucleotide reductase" (PPNR) of San Pietro and 

 Lang (29), which has become widely accepted as the specific 

 chloroplast enzyme required for the photoreduction of TPN. More- 

 over, in the presence of added bacterial ferredoxin and hydrogen- 

 ase, ' isolated chloroplasts reduced TPN in the dark with hydrogen 

 gas but without PPNR (see Fig. 5 in ref. 9). Thus, the enzymic 

 apparatus of chloroplasts was found to be able to reduce TPN in- 

 dependently of PPNR and light. 



These results indicated that the true pyridine nucleotide 

 reductase enzyme of chloroplasts was not a component of the PPNR 

 preparation but a component of the remaining chloroplast fraction. 

 A re-examination of the TPN-reducing system of chloroplasts, des- 

 cribed in more detail elsewhere (9,30), proved this interpreta- 

 tion to be correct. The TPN reductase proper, which had also 

 diaphorase (3I) and transhydrogenase (32,33) activities, was 

 found to be localized in the flavoprotein fraction of chloro- 

 plasts (9). The flavoprotein reductase, which was also isolated 

 by Gewitz and Voelker (3^) and Davenport (35), was recently crys- 

 tallized (Fig. h) by Shin et al. (36,30)- The enzyme reduces TPN 

 either in the light or in the dark with H2 (plus hydrogenase) and 

 requires in either case the collaboration of an electron carrier: 

 bacterial or chloroplast ferredoxin. 



Chloroplast ferredoxin was isolated and crystallized by meth- 

 ods similar to those used for bacterial ferredoxin (9). Chloro- 

 plast ferredoxin proved to be a non-heme iron protein, localized 

 in chloroplasts, and was similar to Clostridium ferredoxin in 

 having a redox potential (E'q = -^32 mV, pH 7.55) close to that 

 of the hydrogen electrode and in undergoing reversible oxidation- 

 reduction that was measured by spectral changes (9). As shown in 

 Table 3, the protein from spinach chloroplasts had an iron con- 

 tent of 0.89 per cent, which, on the basis of a molecular weight 

 of 13,000, indicates two atoms of iron per mole. 



Spinach ferredoxin proved to be the same substance as PPNR, 

 the methaemoglobin reducing factor of Davenport, Hill and Whatley 

 (37), the TPN-reducing factor of Arnon, Whatley and Allen (38) 

 and the red enzyme of Gewitz and Voelker (3^) [see review (30)]. 

 The presence of iron in PPNR or the red enzyme was recently re- 



