THE ACTIVE SITE OF [FE]- AND [NIFE]-HYDROGENASE

这个子项目是利用资源的许多研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， NCRR赠款不直接向子项目或子项目工作人员提供资金。 存在三种遗传学上不同的氢化酶，[Fe]-、[NiFe]-和[FeFe]-氢化酶，其催化分子氢的活化。[Fe]-氢化酶参与氢营养型产甲烷途径，并具有独特的铁-鸟苷酰吡啶醇-辅因子。它的铁离子与两个CO、一个Cys-176-S、一个吡啶醇环的N和一个吡啶醇环上甲酰基-甲基取代基的酰基-C络合。晶体结构，化学分析，红外（IR）和X-射线吸收光谱（XAS）分析这种酶揭示了铁络合物的组成和几何形状。然而，要了解[Fe]-氢化酶催化反应中铁位点的详细化学和电子结构，需要进一步分析。为此目的的一种方法是分析被其独特的特异性抑制剂抑制的[Fe]-氢化酶。我们最近发现[Fe]-氢化酶被异腈类化合物抑制，异腈类化合物不是已知的[NiFe]-和[FeFe]-氢化酶的抑制剂。这些抑制剂的亲和力非常高（Ki < 100 nM）（Shima等人未发表的结果）。紫外-可见光谱分析表明，异氰化物结合到铁网站。Fe K边XAS谱表征了配合物的配位和电子结构。铜离子也可以与[Fe]-氢化酶强烈结合并抑制该酶（Ki < 100 nM）。 [Fe]-氢化酶抑制剂复合物的这些XAS分析将导致理解这些独特的抑制剂的抑制模式。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. There are three phylogenetically different hydrogenases, [Fe]-, [NiFe]- and [FeFe]-hydrogenases, which catalyze activation of molecular hydrogen. [Fe]-hydrogenase is involved in hydrogenotrophic methanogenic pathway and harbors a unique iron-guanylyl pyridinol-cofactor. Its iron ion is complexed with two CO, one Cys-176-S, one N of the pyridinol ring and one acyl-C of the formyl-methyl substituent from the pyridinol ring. Crystal structure, chemical analysis, and infrared (IR)- and X-ray-absorption spectroscopic (XAS) analyses of this enzyme revealed the composition and geometry of the iron complex. However, to understand the detailed chemical- and electronic structures of the iron site in the catalytic reactions of [Fe]-hydrogenase, further analyses are required. One approach to this end is to analyze [Fe]-hydrogenase inhibited by its unique specific inhibitors. We have recently found that [Fe]-hydrogenase is inhibited by isocyanides, which are not known as the inhibitor of [NiFe]- and [FeFe]-hydrogenases. The affinity of these inhibitors is very high (Ki < 100 nM) (Shima et al. unpublished results). UV-Vis spectroscopic analysis indicated that isocyanides bind to the iron site. Fe K-edge XAS will characterize the coordination and electronic structure of the complex. Copper ions can also bind strongly to [Fe]-hydrogenase and inhibits this enzyme (Ki < 100 nM). These XAS analyses of [Fe]-hydrogenase-inhibitor complexes will lead to understanding of the mode of inhibition by these unique inhibitors.