OXIDATION-REDUCTION REACTIONS OF P700 AND CYTOCHROME f IN FRACTION 1 PARTICLES PREPARED FROM SPINACH CHLOROPLASTS BY FRENCH PRESS TREATMENT

Abstract— –Fraction-1 particles were prepared by passing spinach chloroplasts three times through the French pressure cell and centrifuging in a sucrose gradient. With the electron donor DAD (diaminodurol or 2,3,5,6-tetramethyl-p-phenylenediamine) and ascorbate, a light-induced difference spectrum revealed the oxidation of both cytochrome f and P700 upon illumination of these particles. The oxidation of cytochrome f was completed in less than 0.5 msec. P700 and cytochrome f thus seem to be tightly bound to each other in these particles. Addition of Triton X-100 abolished the fast oxidation of cytochrome f but not that of P700. Artificial electron donors such as DAD, DCIP (2,6-dichlorophenol indophenol), and PMS (N-methylphenazonium methosulfate) were good electron donors for photoreaction 1 in these particles, while neither plastocyanin, Porphyra cytochrome 553, nor Euglena cytochrome-552 reduced P700 efficiently. However, after treatment of fraction 1 particles with Triton X-100 reduced DAD, DCIP and PMS were no longer efficient electron donors, while plastocyanin and the algal cytochromes were highly active in reducing P700. Mammalian cytochrome c was not a good electron donor either before or after Triton treatment. Measurements of the effectiveness of P700 reduction as a function of concentration in Triton-treated particles showed plastocyanin to be about four times more active than Porphyra or Euglena cytochromes which in turn were about fourteen times more active than mammalian cytochrome c. Recent studies by Murata and Brown have shown that plastocyanin is not required for the reduction of NADP in these particles with DCIP and isoascorbate as electron donors. The present investigation and that of Murata and Brown indicate that disintegration of chloroplasts with the French pressure cell and centrifugation in a sucrose gradient is the best method to separate system-1 particles having an electron-transport system in almost the native state as in chloroplasts.