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 unpublished results).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.
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