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。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 有三种系统发育不同的氢酶，[Fe]-，[NiFe]-和[FeFe]-氢酶，它们催化分子氢的激活。[Fe]-氢酶参与了氢遗传营养的产甲烷途径，并含有一种独特的铁-鸟苷酸吡啶醇辅因子。它的铁离子与两个CO、一个半胱氨酸-176-S、一个吡啶醇环上的N和一个来自吡啶醇环上的甲酰甲基取代基的酰基-C配位。该酶的晶体结构、化学分析、红外(IR)和X射线吸收光谱(XAS)分析揭示了该铁络合物的组成和几何结构。然而，要了解[Fe]-氢酶催化反应中铁中心的详细化学和电子结构，还需要进一步的分析。为此，一种方法是分析被其独特的特定抑制剂抑制的[Fe]-氢酶。我们最近发现，[Fe]-氢酶被异氰化物抑制，而异氰化物并不是[NiFe]-和[FeFe]-氢酶的抑制剂。这些抑制剂的亲和力非常高(Ki&lt；100 nm)(Shima等人。未发表的结果)。UV-Vis光谱分析表明，异氰化物与铁中心结合。Fe K边XAS将表征该络合物的配位和电子结构。铜离子也可以与[Fe]-氢酶强烈结合并抑制该酶(Ki&lt；100 NM)。这些对[Fe]-氢酶-抑制剂复合体的XAS分析将有助于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 unpublished results).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.