Hydrogenases and H(+)-reduction in primary energy conservation.

Hydrogenases are metalloenzymes subdivided into two classes that contain iron-sulfur clusters and catalyze the reversible oxidation of hydrogen gas (H(2)[Symbol: see text]left arrow over right arrow[Symbol: see text]2H(+)[Symbol: see text]+[Symbol: see text]2e(-)). Two metal atoms are present at their active center: either a Ni and an Fe atom in the [NiFe]hydrogenases, or two Fe atoms in the [FeFe]hydrogenases. They are phylogenetically distinct classes of proteins. The catalytic core of [NiFe]hydrogenases is a heterodimeric protein associated with additional subunits in many of these enzymes. The catalytic core of [FeFe]hydrogenases is a domain of about 350 residues that accommodates the active site (H cluster). Many [FeFe]hydrogenases are monomeric but possess additional domains that contain redox centers, mostly Fe-S clusters. A third class of hydrogenase, characterized by a specific iron-containing cofactor and by the absence of Fe-S cluster, is found in some methanogenic archaea; this Hmd hydrogenase has catalytic properties different from those of [NiFe]- and [FeFe]hydrogenases. The [NiFe]hydrogenases can be subdivided into four subgroups: (1) the H(2) uptake [NiFe]hydrogenases (group 1); (2) the cyanobacterial uptake hydrogenases and the cytoplasmic H(2) sensors (group 2); (3) the bidirectional cytoplasmic hydrogenases able to bind soluble cofactors (group 3); and (4) the membrane-associated, energy-converting, H(2) evolving hydrogenases (group 4). Unlike the [NiFe]hydrogenases, the [FeFe]hydrogenases form a homogeneous group and are primarily involved in H(2) evolution. This review recapitulates the classification of hydrogenases based on phylogenetic analysis and the correlation with hydrogenase function of the different phylogenetic groupings, discusses the possible role of the [FeFe]hydrogenases in the genesis of the eukaryotic cell, and emphasizes the structural and functional relationships of hydrogenase subunits with those of complex I of the respiratory electron transport chain.

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 (H2, CO2, and O2) with an online mass spectrometer. The in vivo activity of the bidirectional [NiFe]hydrogenase [H2:NAD(P) oxidoreductase], encoded by the hoxEFUYH genes, was also measured independently by the proton-deuterium (H-D) exchange reaction in the presence of D2. This technique allowed us to demonstrate that the hydrogenase was insensitive to light, was reversibly inactivated by O2, and could be quickly reactivated by NADH or NADPH (+H2). H2 was evolved by cells incubated anaerobically in the dark, after an adaptation period. This dark H2 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 H2 output was observed, followed by rapid H2 uptake and a concomitant decrease in CO2 concentration in the cyanobacterial cell suspension. Uptake of both H2 and CO2 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 O2 in the light, H2 uptake was negligible during dark-to-light transitions, allowing several minutes of continuous H2 production. A sustained rate of photoevolution of H2 corresponding to 6 micro mol of H2 mg of chlorophyll(-1) h(-1) or 2 ml of H2 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 O2 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 H2 production operate in M55, namely photolysis of water at the level of photosystem II and carbohydrate-mediated reduction of the plastoquinone pool.