I 



PHOTOREDUCTION OF NADP BY ASCORBATE AND HEMATOPORPHYRIN 

 Anthony San Pietro, Leo P. Vernon and Dorothy Limbach 



Some years ago, Krasnovsky demonstrated a ohotochemical reduction of 

 chlorophyll by ascorbic acid in pyridine solution ^^' . It was shown further 

 that chlorophylls in light catalyzed the transfer of electrons f rom ascorbate 

 to a wide variety of compounds including the pyridine nucleotides ^^"^). 

 Whereas most of the reactions studied by the Krasnovsky group have been 

 carried out in non- aqueous media, certain of them did occur in aqueous 

 media ^^K 



Vernon ^6) extended these observations and showed that the photoreduc- 

 tion of pyridine nucleotides in the presence of ascorbate and partially puri- 

 fied photos ynthetic pyridine nucleotide reductase (PPNR) is catalyzed by 

 chlorophyll a, chlorophyll b, hematoporphyrin, protoporphyrin and copro- 

 porphyrin. The significance of this finding is that the reaction occurs in 

 aqueous solution and requires the protein PPNR which functions in the natural 

 photos ynthetic process wherein pyridine nucleotides are reduced (^). 



During the course of the purification of PPNR it was observed that the 

 reduction of pyridine nucleotides by illuminated chloroplasts requires a 

 flavoprotein 's, 9)^ pyridine nucleotide transhydrogenase (transhydrogenase), 

 in addition to the purified PPNR. There was no reduction of pyridine nucleo- 

 tides in the presence of either protein alone; when both proteins were pres- 

 ent, the reduction of pyridine nucleotides was observed. 



In his initial experiments Vernon (6) used a partially purified prepara- 

 tion of PPNR which is known also to contain the transhydrogenase. It was of 

 interest, therefore, to determine whether the hematoporphyrin and ascorbate 

 system also requires both proteins as does the chloroplast system. Con- 

 trary to expectation, it was found that the reduction of pyridine nucleotides 

 by ascorbate and hematoporphyrin requires only the transhydrogenase. 

 Purified PPNR was completely inactive when present either alone or together 

 with the transhydrogenase. Although NADP was used as the electron acceptor 

 in the experiments reported, similar results were obtained with NAD. 



EXPER I MENTAL 



The experimental procedures employed and the description of most of 

 the materials used have been previously described (^» ^> ^'^K In brief, the 

 standard reaction mixture contained 0. 21-0. 27 pmole of hematoporphyrin; 

 144 )jmoles of tris buffer, pH 7. 8; 44 pmoles of sodium ascorbate; 2. 7 to 3. 4 



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