This proposal aims to elucidate the catalytic mechanism of carbon monoxide dehydrogenase from Clostridium thermoaceticum. The structure of the enzyme's nickel and iron-sulfur clusters, their redox and spectroscopic properties, their roles in catalysis, and their relationship to the structure of the protein are of particular interest. Only four nickel containing enzymes are known, and the biochemical properties of Ni are not well understood. Ni complexes are widely used as industrial catalysts, and they serve to cleave DNA site specifically. More detailed knowledge of the catalytic properties of Ni may facilitate development of improved artificial enzymes with biomedical and biotechnological applications. The enzyme catalyses two types of reactions, the reversible oxidation of CO to CO2 and the synthesis of acetyl-coenzyme A. The enzyme has an a2b2 quaternary structure with 2Ni, 11-13 Fe, and approximately 14 S2- ions per ab. The metal ions are organized into three types of clusters, called A,B, and C. The C-and A-clusters are novel Ni-Fe-S clusters that serve as the active sites, while the B-cluster is an Fe4S4 cluster that serves to transfer electrons to external electron-accepting agents. The predominant approach to be taken involves and has involved separating the subunits of the enzyme in a manner that does not destroy the metal clusters. By studying individual subunits and other dissociation products, the desired properties of individual clusters will be elucidated. A variety of physical methods will be used, including Electron Paramagnetic Resonance, Mossbauer, Electron Nuclear Double Resonance, X-ray Absorption, Electronic Absorption, Circular Dichroism, Fluorescence, Mass Spectrometry, and possibly Nuclear magnetic Resonance. Other methods, including kinetics, potentiometric titrations, activity assays, radioactivity and isotope labeling, analytical ultracentrifugation, equilibrium dialysis, and peptide sequencing will also be employed.
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