MECHANISM OF METHYL TRANSFERS IN ACETYL-COA SYNTHESIS

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Clostridium thermoaceticum and other acetogenic bacteria produce acetate as the sole product of growth on H2/CO2, CO, or organic substrates. The acetyl-CoA pathway, also called the Wood pathway, is a major mechanism of C02 fixation under anaerobic conditions. It is a noncyclic pathway of C-C bond formation in which the major intermediates occur as enzyme-bound (often organometallic) intermediates. There are three parts to the Wood pathway: (1) reduction of CO 2 to methyltetrahydrofolate (methyl-H4folate); (2) transfer of the methyl group of methyl-H4folate first to the C/Fe-SP and then to CODH, a reaction sequence involving four enzymes- a corrinoid/iron-sulfur protein (C-Fe-SP), a methyl-H4folate: C/Fe-SP methyl- transferase (MeTr), carbon monoxide dehydrogenase (CODH), and ferredoxin (Fd); and (3) the final steps of the synthesis in which methylated CODH binds CO and CoA and synthesizes acetyl-CoA from the bound methyl, CO, and CoA groups. This proposal focuses on the mechanistic enzymology of the transfer of the methyl group of methylH4folate to the C/Fe-SP, forming a methylcobalt species, and then to CODH, forming methyl-CODH. Formation of the methylcobalt species on the C/Fe-SP is catalyzed by a methyltransferase which is unique in that it apparently does not contain bound prosthetic groups such as cobalamin or S-adenosyl-L-methionine. The reaction involves reduction of the C/Fe-SP followed by a nucleophilic attack of Co 1+ on the N-methyl bond of the tertiary amine, 5-methyl-H4folate. Formation of methyl-CODH appears to be the rate limiting step in the acetyl-CoA pathway and involves reduction of a metal center on CODH at potentials < - 450 mV followed by nucleophilic attack of the reduced metal center on the methylcobamide center to form a methyl-metal intermediate on CODH. In the work described here, these methyl transfer steps and the proteins catalyzing them will be fully characterized by (1) steady-state and presteady-state kinetic analyses, (2) analyses of the primary structures of the C/Fe-SP and MeTr, (3) determination of the X-ray crystal structures of MeTr and the C/Fe-SP and analyses of the cobalt coordination environment by X-ray absorption studies, (4) elucidation of the role of the [4Fe-4S] cluster in the C/Fe-SP, (5) identification of the cobamide binding site and the location of the [4Fe-4S] cluster in the C/Fe-SP, and (6) examination of the steric and coordination state requirements for the cobamide by reconstitution of the C/Fe-SP with various cobamides.