BIOINORGANIC CHEMISTRY OF CARBON MONOXIDE DEHYDROGENASE

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The long-term objectives for this proposal are to enumerate and characterize the novel nickel and iron-containing complexes and clusters in carbon monoxide dehydrogenase from Clostridium thermoaceticum, and to elucidate the functional roles they play in the catalytic mechanism of the enzyme. The enzyme catalyzes two major reactions, the oxidation of CO to CO2 and the synthesis of acetyl-coenzyme A from CO, a methyl group, and coenzyme A. The metal ions are organized into various complexes and clusters. the nickel in the so-called NiFe complex can be removed by the bidentate ligand 1,10-phenanthroline. Removal results in loss of the enzyme's synthase activity but not in its CO oxidation activity. The nickel can be reinserted and activity recovered by adding Ni2+ to the phenanthroline-treated enzyme. The substrate CO appears to bind at the labile Ni, suggesting that it is the active site for the synthase activity. This is an extraordinary discovery that will allow the mechanism of the enzyme to be probed in unexpected detail. Removal and reinsertion of Ni will be optimized, to determine how much can be removed and reinserted, and how tightly it is bound. The attributes which makes the phenanthroline ligand so effective in removing the nickel will be evaluated by screening a variety of ligands with different structural properties. Foreign metals such as Fe2+ can be inserted into the labile site. The conditions for their insertion will be optimized. Utilizing the properties of particular foreign metals, the number and nature of the coordinating ligands, and the geometry of the labile site will be evaluated using various spectroscopic methods, including EPR, Mossbauer, EXAFS, and NMR. The mechanism of the enzyme will be probed by determining whether the methyl substrate binds to this labile Ni. The structures and functions of the enzyme's other metal complex and clusters will be evaluated as well. Using an inhibitor to the CO oxidation activity, the site at which CO is oxidized will be determined and characterized. The redox and spectroscopic properties of the complexes and clusters will be determined. These properties will be incorporated into establishing a better understanding of the mechanism of the enzyme. The proposed research is of general significance to the study of enzymes, since nickel is an unusual constituent of enzymes, and its biochemical roles are poorly understood.