The project goals are to develop molecular level understanding of the hydrogenase metalloenzymes, which effect the interconversion of H2 and H+. Now is the perfect time to attack this problem from the modeling perspective because the active sites have recently been elucidated by X-ray crystallography on four enzymes, two [Fe]- and two [NiFe]-hydrogenases. The structural similarities of these enzymes and their extraordinary features (with respect to other metal sites in biology) strongly suggests that mechanistic insights obtained for one metallo hydrogenase will be applicable to the others. Thus the study here is broadly applicable to the entire (metallo) hydrogenase problem. A specific impetus for this project is the pair of recent (1999, and late 1998) crystallographic results on the two Fe- hydrogenases. These reports describe a surprisingly simple core structure that is amenable to chemical synthesis from organometallic precursors, an area of specific expertise to the PI. Our plans for preparing active site models are supported by compelling preliminary evidence that the Fe2(SR)2(CO)2(CN)2 core of the [Fe]-hydrogenase can be efficiently synthesized. Crystallographic analysis of our model confirms that it displays many of the structural characteristics seen in the two most recent protein crystallography reports. Furthermore, the synthetic analogue reduces protons to dihydrogen. In other words, we begin the project with a model that has fidelity with nature in terms of both structure and function. It is therefore quite likely that we will be able to obtain considerable molecular detail about how nature processes hydrogen. The first part of the project involves the preparation of accurate structural models for the Fe-hydrogenase. This preparative effort is coupled to spectroscopic characterization as well as mechanistic studies on the hydrogenase activity of the models. The synthetic effort entails both stepwise methods and multicomponent assembly processes starting from the most primitive precursor reagents. These models are subjected to protonation studies to give H2. The second part of the project is broadly aimed at gaining an understanding of the electronic and structural factors that determine the function of nonheme Fe centers to bind and activate H2. These studies probe libraries of thiolato, amino, and cyano ligation to afford Fe centers capable of activating H2, with connections to the binding and activation of RCN, N2, and CO. Initial studies are already promising with respect to LFe(CN)3-, LFe(CN)2(CO), LFe(SR)(CO)2+, and LFe(SR)2(CO) in conjunction with diversity in L as well. These studies will define how nature uses Fe-S and Fe-Ni-S ensembles to activate weakly basic small molecule substrates.
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