Structural and Biochemical Investigations on Fe-Only Hydrogenases

Montana State University, Bozeman MT

Investigators

Abstract

The main objective of this projectl is to investigate the structural and catalytic properties of Fe-Only hydrogenases. Hydrogenases catalyze reversible hydrogen oxidation and function physiologically to either recycle the reduced electron carriers that accumulate during a fermentative metabolism or couple the oxidation of hydrogen to the generation of reduced electron carriers that can be utilized in energy yielding processes. The metal-containing hydrogenases are classified according to their metal content and include those that contain both Ni and Fe (NiFe-hydrogenases) and those that contain just Fe (Fe-Only hydrogenases). These enzymes are of paramount importance in the metabolism of numerous microorganisms. In addition, since hydrogen is being explored as a future alternative fuel source, there is considerable interest in the details of these hydrogen producing biological catalysts. The studies will focus primarily on testing hypotheses that were 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-specific amino acid substitution experiments on CpI to examine the role of the polypeptide environment of the H Cluster in catalysis and stability; (3) structural characterization of the second Fe-Only hydrogenase (CpII) from C. pasteurianum to ascertain its primary sequence and structural relationship to other Fe-Only hydrogenases and to probe the contributions of the environment of the H Cluster and the accessory FeS clusters in defining hydrogen oxidation versus proton reduction activities in the Fe-Only hydrogenases. The above objectives will be pursued by a multidisciplinary approach combining biochemical methods, spectroscopic methods and structure determination by X-ray diffraction methods to answer some of the key remaining questions with respect to the structure and function of Fe-Only hydrogenases.