Chlamydomonas reinhardtii cultures, deprived of inorganic sulfur, undergo dramatic changes during adaptation to the nutrient stress [Biotechnol. Bioeng. 78 (2002) 731]. When the capacity for Photosystem II (PSII) O(2) evolution decreases below that of respiration, the culture becomes anaerobic [Plant Physiol. 122 (2000) 127]. We demonstrate that (a) the photochemical activity of PSII, monitored by in situ fluorescence, also decreases slowly during the aerobic period; (b) at the exact time of anaerobiosis, the remaining PSII activity is rapidly down regulated; and (c) electron transfer from PSII to PSI abruptly decreases at that point. Shortly thereafter, the PSII photochemical activity is partially restored, and H(2) production starts. Hydrogen production, which lasts for 3-4 days, is catalyzed by an anaerobically induced, reversible hydrogenase. While most of the reductants used directly for H(2) gas photoproduction come from water, the remaining electrons must come from endogenous substrate degradation through the NAD(P)H plastoquinone (PQ) oxido-reductase pathway. We propose that the induced hydrogenase activity provides a sink for electrons in the absence of other alternative pathways, and its operation allows the partial oxidation of intermediate photosynthetic carriers, including the PQ pool, between PSII and PSI. We conclude that the reduced state of this pool, which controls PSII photochemical activity, is one of the main factors regulating H(2) production under sulfur-deprived conditions. Residual O(2) evolved under these conditions is probably consumed mostly by the aerobic oxidation of storage products linked to mitochondrial respiratory processes involving both the cytochrome oxidase and the alternative oxidase. These functions maintain the intracellular anaerobic conditions required to keep the hydrogenase enzyme in the active, induced form.
This report elucidates the distinctions of redox properties between two uptake hydrogenases in Escherichia coli. Hydrogen uptake in the presence of mediators with different redox potential was studied in cell-free extracts of E. coli mutants HDK103 and HDK203 synthesizing hydrogenase 2 or hydrogenase 1, respectively. Both hydrogenases mediated H(2) uptake in the presence of high-potential acceptors (ferricyanide and phenazine methosulfate). H(2) uptake in the presence of low-potential acceptors (methyl and benzyl viologen) was mediated mainly by hydrogenase 2. To explore the dependence of hydrogen consumption on redox potential of media in cell-free extracts, a chamber with hydrogen and redox ( E(h)) electrodes was used. The mutants HDK103 and HDK203 exhibited significant distinctions in their redox behavior. During the redox titration, maximal hydrogenase 2 activity was observed at the E(h) below -80 mV. Hydrogenase 1 had maximum activity in the E(h) range from +30 mV to +110 mV. Unlike hydrogenase 2, the activated hydrogenase 1 retained activity after a fast shift of redox potential up to +500 mV by ferricyanide titration and was more tolerant to O(2). Thus, two hydrogenases in E. coli are complementary in their redox properties, hydrogenase 1 functioning at higher redox potentials and/or at higher O(2) concentrations than hydrogenase 2.
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