Structural Studies on Fe-only Hydrogenase (CpI) from Clostridium Pasteurianum

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9807821 Peters The ability to oxidize or evolve hydrogen is a feature of a diverse group of microorganisms containing hydrogenases of a variety of molecular and catalytic properties. To date, there is no available structural information for any of the Fe-only hydrogenases. Mechanistic issues concerning the function of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum will be addressed using a multidisciplinary structure/function based approach. This will combine structure determination by x-ray diffraction methods with complimentary biochemical and biophysical studies. The specific objectives of this research concerning the Fe-only hydrogenase (CpI) from Clostridium pasteurianum include: 1) determination of the native three-dimensional structure by x-ray diffraction methods, 2) development of an expression system that will allow introduction of site-specific amino acid substitutions and simplify the method for purification, 3) investigation of oxidation state dependent structural changes, inhibitor binding, and site-specific amino acid substituted proteins combined with complimentary biochemical and biophysical studies. It is anticipated that these studies will reveal the structural architecture of the, as of yet, uncharacterized active site cluster of the Fe-only hydrogenases. Additional experiments will give insight into the mechanism of activity reversible hydrogen oxidation at this site which will lead to a model for catalysis. In addition, the structure of the Fe-only hydrogenase may be quite informative in developing models for the mechanism of Ni independent reversible hydrogen oxidation. Additionally, comparison of the structure of an Fe-only hydrogenase with the structure of the NiFe-hydrogenase from D. gigas as well as the structure of the active site of nitrogenase (which catalyzes proton reduction as part of the mechanism of nitrogen reduction) may bring to light commonalties suggestive of their individual mechanisms.