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

9807821 Peters氧化或演化氢的能力是含有多种分子和催化性质的氢化酶的多种微生物的特征。到目前为止，还没有任何一种纯铁氢化酶的结构信息。关于巴氏梭菌中仅铁氢化酶（CpI）功能的机制问题将采用基于多学科结构/功能的方法来解决。这将结合x射线衍射方法的结构测定与互补的生化和生物物理研究。本研究对巴氏梭菌纯铁氢化酶（CpI）的具体研究目标包括：1)通过x射线衍射方法确定天然三维结构；2)开发一种表达系统，允许引入位点特异性氨基酸取代并简化纯化方法；3)研究氧化态依赖的结构变化、抑制剂结合和位点特异性氨基酸取代蛋白，结合互补的生化和生物物理研究。预计这些研究将揭示到目前为止尚未表征的纯铁氢化酶活性位点簇的结构结构。进一步的实验将深入了解该位点活性可逆氢氧化的机理，从而建立催化模型。此外，纯铁氢化酶的结构可能为建立不依赖镍的可逆氢氧化机理模型提供重要信息。此外，将纯铁氢化酶的结构与D. gigas的nfe氢化酶的结构以及氮酶活性位点（催化质子还原作为氮还原机制的一部分）的结构进行比较，可能会揭示出它们各自机制的共性。

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.