Molecular Biology and Biochemistry of Nickel-Containing Hydrogenases

含镍氢化酶的分子生物学和生物化学

• 批准号: 8706205
• 负责人: Harry Peck
• 金额: $ 21.5万
• 依托单位: University of Georgia
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-01 至 1991-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8706205&HistoricalAwards=false
• 关键词: Molecular; Biology; Biochemistry; Nickel; Containing

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.

厌氧生物的氢化酶是一种迷人的金属酶，它结合了不寻常的和意想不到的辅酶来催化不寻常的氧化和还原。迄今为止，硫酸盐还原菌已鉴定出三种类型的氢化酶：第一种来自千个脱硫弧菌，含有非血红素铁和氧化还原活性镍（NiFe）；第二种来自D. vulgaris，只含有非血红素铁（Fe），第三种来自D. baculaturs，含有非血红素铁和等量的镍和硒（NiFeSe）。当前的目标是发展基本的酶学、生化和结构信息，以了解镍的生物学功能以及硫和沉默在镍催化中的作用。具体目的是：1)纯化和表征（NiFeSe）氢化酶；2)评价Se作为镍的催化意义探针的潜力；3)研究（NiFe）氢化酶的活化；4)探讨（NiFe）氢化酶的催化氧化还原循环；5)测定氢化酶基因的DNA序列；6)研究（NiFeSe）氢化酶的细胞定位机制；7)通过位点特异性诱变确定负责（NiFe）氢化酶四个氧化还原中心配位的氨基酸残基。