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Articles

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

Department of Plant Sciences, King's College, London SE24 9JF, United Kingdom

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.