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The first generation of biochemical studies of complex, iron-sulfur-cluster-containing [FeFe]-hydrogenases and Mo-nitrogenase were carried out on enzymes purified from Clostridium pasteurianum (strain W5). These Previous studies suggested that two distinct [FeFe]-hydrogenases are expressed differentially under nitrogen-fixing and non-nitrogen-fixing conditions. As a result, the first characterized [FeFe]-hydrogenase (CpI) is presumed to have a primary role in central metabolism, recycling reduced electron carriers that accumulate during fermentation via proton reduction. A role for capturing reducing equivalents released as hydrogen during nitrogen fixation has been proposed for the second hydrogenase, CpII. Biochemical characterization of CpI and CpII indicated CpI has extremely high hydrogen production activity in comparison to CpII, while CpII has elevated hydrogen oxidation activity in comparison to CpI when assayed under the same conditions. This suggests that these enzymes have evolved a catalytic bias to support their respective physiological functions. Using the published genome of C. pasteurianum (strain W5) hydrogenase sequences were identified, including the already known [NiFe]-hydrogenase, CpI, and CpII sequences, and a third hydrogenase, CpIII was identified in the genome as well.We identified four hydrogenases: three [FeFe]-hydrogenases, one of which had not previously been known, and one [NiFe]-hydrogenase in the published genome of C. pasteurianum (strain W5). Quantitative real-time PCR experiments were performed in order to analyze transcript abundance of the hydrogenases under diazotrophic and non diazatrophic growthconditions. show There is a markedly reduced levels of CpI gene expression together with concomitant increases in CpII gene expression under nitrogen-fixing conditions. Structure-based analyses of the CpI and CpII sequences reveals variations in their catalytic sites that may contribute to their alternative physiological roles. This work demonstrates that the physiological roles of CpI and CpII are to evolve and to consume hydrogen, respectively, in concurrence with their catalytic activities in vitro, with CpII capturing excess reducing equivalents under nitrogen fixation conditions. Comparison of the primary sequences of CpI and CpII and their homologs provides an initial basis for identifying key structural determinants that modulate hydrogen production and hydrogen oxidation activities.

The production of H2 for use as a renewable energy carrier using solar energy and electrons derived from water is widely regarded as one of the most environmentally benign and sustainable energy solutions. Several water-oxidizing, phototrophic algae and cyanobacteria have the remarkable ability to use low potential electrons from the photosynthetic electron transport chain, or from sugar oxidation, to reduce protons to H2. major research efforts are aimed at developing a more informed understanding of the physiological parameters dictating H2 production in water-oxidizing, phototrophic microorganisms, with the ultimate goal of improving H2 yields. Currently, the yields of H2 production are far below those required for the economically-viable production of H2 and substantial improvements are required to generate the quantities of H2 necessary to replace a meaningful portion of our current energy portfolio. Nevertheless, recent biological H2 production research efforts are rapidly elucidating (a) the metabolic pathways that supply reductant to H2-producing enzymes, (b) the metabolic and mechanistic requirements for maturation of the metallo-enzyme centers required in H2-producing enzymes, (c) novel hydrogenase enzymes, and (d) the genetic techniques required for manipulating metabolism in H2-producing organisms.