Sustained photoevolution of molecular hydrogen in a mutant of Synechocystis sp strain PCC 6803 deficient in the type I NADPH-dehydrogenase complex

The interaction between hydrogen metabolism, respiration, and photosynthesis was studied in vivo in whole cells of Synechocystis sp. strain PCC 6803 by continuously monitoring the changes in gas concentrations (H-2, CO2, and O-2) with an online mass spectrometer. The in vivo activity of the bidirectional [NiFe]hydrogenase [H-2:NAD(P) oxidoreductase], encoded by the hoxEFUYH genes, was also measured independently by the proton-deuterium (H-D) exchange reaction in the presence of D-2 This technique allowed us to demonstrate that the hydrogenase was insensitive to light, was reversibly inactivated by 02, and could be quickly reactivated by NADH or NADPH (+H-2). H-2 was evolved by cells incubated anaerobically in the dark, after an adaptation period. This dark H-2 evolution was enhanced by exogenously added glucose and resulted from the oxidation of NAD(P)H produced by fermentation reactions. Upon illumination, a short (less than 30-s) burst of H-2 output was observed, followed by rapid H-2 uptake and a concomitant decrease in CO2 concentration in the cyanobacterial cell suspension. Uptake of both H-2 and CO, was linked to photosynthetic electron transport in the thylakoids. In the ndhB mutant M55, which is defective in the type I NADPH-dehydrogenase complex (NDH-1) and produces only low amounts of O-2 in the light, H-2 uptake was negligible during dark-to-light transitions, allowing several minutes of continuous H-2 production. A sustained rate of photoevolution of H-2 corresponding to 6 mumol H-2 mg of chlorophyll(-1) h(-1) or 2 ml of H-2 liter(-1) h(-1) was observed over a longer time period in the presence of glucose and was slightly enhanced by the addition of the O-2 scavenger glucose oxidase. By the use of the inhibitors DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] and DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), it was shown that two pathways of electron supply for H-2 production operate in M55, namely photolysis of water at the level of photosystem 11 and carbohydrate-mediated reduction of the plastoquinone pool.