Ascorbate as a Substrate for Photoproduction of Hydrogen by Photosystem I of Chloroplasts 1

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The photoproduction of hydrogen by 2-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-inhibited chloroplasts from ascorbate under anaerobic conditions was studied in the pH range 5.0 to 7.5 using methyl viologen (MV), N,N,N′,N′-tetramethyl-P-phenylenediamine (TMPD), and excess hydrogenase from Desulfovibrio desulfuricans. (a) At neutral and basic pHs, the photoreduction of MV, which reacted back with photoxidized ascorbate (dehydroascorbate [DHASC]), and the rates of H2 photoproduction were very low. The slow H2 photoproduction was explained by the reversible reduction of MV by the photoproduced H2 (catalyzed by hydrogenase) and its reoxidation by DHASC resulting in H2 uptake. (b) At pH 5.2, relatively high initial rates of H2 photoproduction were obtained, which were comparable to the rates of O2 consumption at pH 5.2 by photosystem I (catalyzed by photoreduced MV). However, accumulation of photoreduced MV under anaerobic conditions was not detected. In the presence of high concentrations of protons, the H2 uptake by DHASC was very slow because the equilibrium concentration of H2-reduced MV was very small, thus allowing H2 evolution mediated by photoreduced MV to compete with the back reaction with DHASC. (c) The continuous accumulation of DHASC, which was generated together with H2, gradually slowed the H2 evolution until it stopped after about 3 hours. At high concentrations, DHASC was able to compete with the coupling of photoreduced MV to hydrogenase and H2 evolution. (d) Dithiothreitol (DTT) reduced the DHASC and consequently competed with the back reaction of the photoreduced and H2-reduced MV with DHASC. DTT thus prolonged the time period of H2 photoproduction from ascorbate and abolished the dependence of its rate on pH in the range of 5.2 to 7.5 (e) A study of H2 uptake by chemically oxidized ascorbate (in the dark) showed that MV and hydrogenase were both required to catalyze electron transfer from H2 to DHASC. TMPD prevented this H2 consumption by DHASC (in a chloroplast reaction mixture containing MV and hydrogenase). Illumination restored the H2 uptake presumably by generating reduced MV which activated the hydrogenase.

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