Molecular Biology and Biochemistry of Nickel-Containing Hydrogenases

厌氧生物的氢化酶很吸引人 金属酶，包括不寻常的和意想不到的 辅酶催化不寻常的氧化和还原。 迄今为止，已经鉴定了三种类型的氢化酶 硫酸盐还原菌：第一个来自脱硫弧菌 gigas含有非血红素铁和氧化还原活性镍（NiFe）; 第二个是D。普通型只含有非血红素 铁（Fe）和从D. baculaturs包含 非血红素铁加等量的镍和硒 （NiFeSe）。 近期目标是发展基本的 所需的酶学、生物化学和结构信息 了解镍的生物学功能及其作用 硫和silenmia在镍催化。 具体目标 1）（NiFeSe）氢化酶的纯化和表征; 2)为了评价硒作为探针的潜力， 镍的催化作用; 3）研究了镍的活化作用 （NiFe）氢化酶的催化氧化还原反应 （NiFe）氢化酶的循环; 5）确定DNA 氢化酶基因的序列; 6）研究氢化酶基因， 参与（NiFeSe）细胞定位的机制 氢化酶;和7）通过位点特异性测定 诱变负责突变的氨基酸残基。 （NiFe）的四个氧化还原中心的配位 氢化酶

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.