The long term objectives of this research are to understand hydrogen metabolism during the anaerobic growth of bacteria in terms of the enzymatic mechanisms of H2 activation, the catalytic roles of nickel and non-heme iron, the number and structure of hydrogenases, the mechanisms of their cellular localizations, the insertion of metal redox centers and the roles of hydrogenases in the bioenergetics of anaerobic bacteria. Hydrogenase and H2 are of general biological and biotechnological importance as they are intimately involved in dinitrogen fixation, the photoproduction of H2 from water, complex microbial fermentations useful for the production of fuels and chemicals, and as normal flora of the intestinal tracts of higher animals. The specific aims of the proposed research are: to obtain an x-ray crystallographic structure of the nickel- containing hydrogenases; to define the structure of the nickel- containing active site and the three iron non-heme iron clusters; to further refine the activation and catalytic cycles involved in H2 utilization; to develope a DNA transfer system for the sulfate reducing bacteria; to use this system for the biosynthesis of active hydrogenases with recombinant DNA; to prepare modified hydrogenases by site-directed mutagenesis and study from the sulfate reducing bacteria their biochemical properties; and to establish the roles of multiple hydrogenases in selected anaerobic microorganisms.
