Molecular Biology and Biochemistry of Nickel-Containing Hydrogenases

这项研究的长期目标是了解 细菌厌氧生长过程中的氢代谢 根据H2活化的酶促机制， 镍和非血红素铁的作用， 氢化酶，它们的细胞定位机制， 金属氧化还原中心的插入和 厌氧细菌生物能量学中的氢化酶。 氢化酶和H2是一般生物酶， 生物技术的重要性，因为他们密切参与 固氮，从水中光生H2， 用于生产以下物质的复杂微生物发酵 燃料和化学品，以及肠道的正常植物群 高等动物。 拟议研究的具体目标 是：获得镍的X射线晶体结构， 含氢化酶;定义镍的结构- 含有活性中心和三个铁非血红素铁簇; 为了进一步完善涉及的活化和催化循环， H2利用;开发硫酸盐的DNA转移系统 还原细菌;使用该系统进行生物合成 活性氢化酶与重组DNA;以制备修饰的 通过定点诱变和研究， 硫酸盐还原菌的生物化学性质;以及 确定多种氢化酶在选择的 厌氧微生物

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.