Bioinorganic Chemistry of Carbon Monoxide Dehydrogenase

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DESCRIPTION (provided by applicant): The objective of this project is to elucidate the catalytic mechanism of CO dehydrogenase (a.k.a. acetyl-CoA synthase - ACS), emphasizing the structure and function of the unique Ni-Fe-S clusters contained therein. The enzyme is an alpha-2-beta-2 tetramer that catalyzes two reactions; the reversible reduction of CO2 to CO and the synthesis of acetyl-CoA. The former occurs at one Ni-Fe-S cluster in the beta subunit, while the latter reaction occurs at another Ni-Fe-S site in alpha. A tunnel through which CO migrates connects the two sites. The reaction mechanism is "organometallic" in nature; how does Ni promote this within an aqueous protein matrix? Mechanistic steps occurring at the two sites are synchronized, making it an attractive system to investigate details of metabolic channeling and active-site coupling. ACS is useful biotechnologically; organisms containing it reduce atmospheric levels of CO and degrade TNT. ACS is found in Clostridium difficile, a pathogenic organism responsible for the deaths of about 2000 people annually. Given the complexity of this enzyme, the general strategy used in this project will be to study each activity separately, and then compare observed properties with those of the bifunctional enzyme; such an approach may reveal some of the complexities of channeling and catalytic coupling. This approach is possible because isolated recombinant alpha subunits able to catalyze the synthesis of acetyl-CoA can be prepared, as can a homolog of the beta subunit that is able to catalyze CO/CO2 redox. Many of these developments (recombinant biosynthesis of active alpha, discovery of the tunnel and of active site coupling) arose from efforts of the previous granting period which resulted in 11 publications. The methodologies used for the project include enzyme kinetics (stopped-flow, rapid freeze-quench, and steady-state), spectroscopy (EPR, Mossbauer, and XAS), and X-ray diffraction. A rapid-freeze-quench instrument that allows samples to be prepared under reliably anaerobic conditions has been constructed, and should be an improvement over existing technology. Kinetic data will be simulated, ultimately leading to a comprehensive mechanistic model describing the general kinetic behavior of the enzyme. Crystals of alpha have been obtained, suggesting that an X-ray diffraction structure may be achievable. These studies require repetitive purification of at least 5 different proteins; funding for a preparative FPLC/HPLC is requested.