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

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 unpublished results).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)Carbon monoxide as intrinsic ligands to iron in the active site of [Fe]-hydrogenase. In Metal-carbon bonds in enzymes and cofactors, Vol. 6 of Metal Ions in Life Sciences. Some IR experiments suggested that the copper ions bind to the iron complex. One of the candidates of the copper ions binding site might be the Cys176-thiol ligand bound to the iron. Cu K-Edge XAS will reveal the interaction of the copper ions with the thiol or the other part of the iron complex. These XAS analyses of [Fe]-hydrogenase-inhibitor complexes will lead to understanding of the mode of inhibition by these unique inhibitors and also give important information of the characters of