DESULFOVIBRIO HYDROGENASE--MOLECULAR BIOLOGY

拟议研究的具体目标是：1)最终确定 官能化非血红素铁中心(H簇)的结构，即 唯一参与底物(H2)活化和电子 2)分析H簇和两个铁还蛋白的功能。 利用生物物理技术通过特定的探针对星团进行分类；3) 尝试分离氢酶基因Hyd的基因产物 从普通硫弧菌中分离到的Lambda可能是一种新型的 (Fe)脱硫弧菌中的氢酶，并可能解决 (Fe)氢酶的数量和类型；4)建立DNA转移 利用广泛寄主范围的质粒和产生(Fe)的普通假丝酵母系统 氢酶阴性突变体以获得生物合成活性 (Fe)氢酶和5)研究小亚基的作用和 其信号肽在(Fe)周质定位中的作用 氢酶的目标是确定缺乏的大亚基是如何 信号肽在细胞质膜上转运。

The specific aims of the proposed research are: 1) to finalize the structure of the functionalized non-heme iron center (H cluster) that is uniquely involved in both substrate (H2) activation and electron transfer; 2) to analyze the function of the H cluster and two ferredoxin- type clusters by specific probes utilizing biophysical techniques; 3) to attempt to isolate the gene product of the putative hydrogenase gene, hyd lambda from Desulfovibrio vulgaris which is suspected to be a new-type of (Fe) hydrogenase in Desulfovibrio and may resolve the problem of the number and types of (Fe) hydrogenases; 4) to establish a DNA transfer system with D. vulgaris using a wide host range plasmid and generate (Fe) hydrogenase negative mutants in order to obtain biosynthesis of active (Fe) hydrogenase and 5) to investigate the role of the small subunit and its putative signal peptide in the periplasmic localization of the (Fe) hydrogenase with the goal of determining how the large subunit that lacks a signal peptide is translocated across the cytoplasmic membrane.