The hydrogenases of anaerobic organisms are fascinating metalloenzyumes that incorporate unusual and unexpected coenzymes to catalyze unusual oxidations and reductions. Three types of hydrogenases have been characterized to date from sulfate reducing bacteria: the first from Desulfovibrio gigas contains non heme iron and redox active nickel (NiFe); the second from D. vulgaris contains exclusively non-heme iron (Fe) and the third purified from D. baculaturs contains non heme iron plus equivalent amounts of nickel and selenium (NiFeSe). The immediate objectives are to develop the basic enzymological, biochemical and stuctural information required to understand the biological function of nickel and the roles of sulfur and silenmia in nickel catalysis. Specific aims are 1) to purify and characterize the (NiFeSe) hydrogenase; 2) To evaluate the potential of Se as a probe of the catalytic significance of nickel; 3) to study the activation of the (NiFe) hydrogenase; 4) to probe the catalytic redox cycling of the (NiFe) hydrogenase; 5) to determine the DNA sequences of the hydrogenase genes; 6) to study the mechanisms involved in cellular localization of the (NiFeSe) hydrogenases; and 7) to determine by site specific mutagenesis the amino acid residues responsible for the liganding of the four redox centers of the (NiFe) hydrogenases.
