Understanding the mechanisms of key enzymes in the reductive acetyl-CoA pathway is significant to the areas of biochemistry, inorganic chemistry, and microbiology. Studies of this pathway are uncovering new biochemical mechanisms of one-carbon transfer and carbon-carbon and carbon-sulfur bond formation. New roles for metals in biology are being elucidated since key intermediates are enzyme bound, organometallic species. These organometallic reactions bear close analogy to processes that have been well studied in inorganic solution chemistry and play a notable role in industrial chemistry. This pathway plays a crucial role in the global carbon cycle. It is the major mechanism of CO2 fixation under anaerobic conditions, occurring in acetogenic, methanogenic, and sulfate reducing bacteria. Understanding this pathway is important to human biochemistry since acetogens and methanogens are components of our intestinal flora. The goal of the proposed research is to define the reaction mechanisms of the three key enzymes that constitute the unique character of the acetyl-CoA pathway: a corrinoid/iron-sulfur protein (C-Fe-SP), a methyl- H4folate: C/Fe-SP methyltransferase (MeTr), and carbon monoxide dehydrogenase (CODH). In studying these enzymes from Clostridium thermoaceticum, the specific aims over the next five years are to: (1) define how the methyl group from CH3-H4folate is transferred to the C/Fe-SP by focusing on (i) how the methyl group of CH3-H4folate is activated and (ii) determining the crystal structure of MeTr. (2) elucidate the role(s) of the [4Fe-4S] cluster in the C/Fe-SP by evaluating the effects of mutating the cluster ligands on rates of electron transfer and methyl transfer reactions. (3) determine the mechanism and the structure of the active site of CO oxidation and CO2 reduction (Center C) and define how CO, cyanide, cyanate, thiocyanate, and azide bind to this site. (4) characterize, by kinetics and spectroscopy, the elementary steps in assembly of acetyl-CoA at Center A (the [Ni-X-Fe3-4S4] cluster) of CODH. Objectives include: (i) identifying the bridging ligand, """"""""X"""""""" between nickel and iron and determining specifically to which metal (i.e., nickel or iron) the methyl and acetyl groups bind in their adducts with Center A; (ii) defining the mode of binding of CO and its competitive inhibitor, CS2; (iii) elucidating how CODH is methylated by the methylated C/FE-SP; (iv) determining the mechanism of formation of the acetyl-CODH intermediate; and (v) elucidating the mechanism of the disassembly of acetyl-CoA. (5) Define the nickel and zinc coordination environment by X-ray absorption spectroscopic methods.
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