Structural and Biochemical Investigations on Fe-Only Hydrogenases

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The main objective of this projectl is to investigate the structural and catalyticproperties of Fe-Only hydrogenases. Hydrogenases catalyze reversible hydrogenoxidation and function physiologically to either recycle the reduced electron carriers thataccumulate during a fermentative metabolism or couple the oxidation of hydrogen to thegeneration of reduced electron carriers that can be utilized in energy yielding processes.The metal-containing hydrogenases are classified according to their metal content andinclude those that contain both Ni and Fe (NiFe-hydrogenases) and those that contain justFe (Fe-Only hydrogenases). These enzymes are of paramount importance in themetabolism of numerous microorganisms. In addition, since hydrogen is being exploredas a future alternative fuel source, there is considerable interest in the details of thesehydrogen producing biological catalysts. The studies will focus primarily on testing hypotheses thatwere developed as a result of the detailed structural analysis of the Fe-Only hydrogenase(CpI) isolated from the anaerobic soil microorganism Clostridium pasteurianum. The specific objectives are (1) additional structural and biochemical studies on CpI probing a number of remaining key questions with respect to the H Cluster structure and the role of the accessory [FeS] clusters; (2) site-specificamino acid substitution experiments on CpI to examine the role of the polypeptideenvironment of the H Cluster in catalysis and stability; (3) structural characterization ofthe second Fe-Only hydrogenase (CpII) from C. pasteurianum to ascertain its primarysequence and structural relationship to other Fe-Only hydrogenases and to probe thecontributions of the environment of the H Cluster and the accessory FeS clusters indefining hydrogen oxidation versus proton reduction activities in the Fe-Onlyhydrogenases. The above objectives will be pursued by a multidisciplinary approach combiningbiochemical methods, spectroscopic methods and structure determination by X-raydiffraction methods to answer some of the key remaining questions with respect to thestructure and function of Fe-Only hydrogenases.