This proposal focuses on structure-function studies on the key enzymes in the Wood-Ljungdahl pathway. Understanding the mechanisms of these enzymes is significant to the areas of enzymology, bioinorganic chemistry and microbiology. This pathway is important to human biochemistry since acetogens and methanogens are components of the intestinal flora. Studies of this pathway are uncovering the principles by which metal centers in enzymes perform nucleophilic reactions and couple redox reactions to chemical catalysis. The Clostridium thermoaceticum methyl transferase is the ideal paradigm for the class of B12-dependent methyl transferases. An important objective is to increase the resolution of the atomic model of methyl transferase and determine the structure with both B12 and CH3-H4folate bound. X-ray diffraction, site-directed mutagenesis, and kinetic studies will be used to identify the proton transfer pathway that leads to activation of CH3-H4folate for nucleophilic attack by the corrinoid iron-sulfur protein. Coordination chemistry of the cobalt center has been proposed to play a controlling role in redox chemistry and catalysis of B12-dependent methyl transferases. It is planned to measure the extent to which the lower axial ligand controls catalysis and redox chemistry and to determine the atomic structure of the corrinoid iron-sulfur protein. Mechanistic studies on CO dehydrogenase/acetyl-CoA synthase will include: determining its crystal structure; elucidating how the CO dehydrogenase and acetyl-CoA synthase active sites communicate to coordinate electron and carbon flow; determining whether the precursors of the carbonyl, methyl, and acetyl groups of acetyl-CoA bind to the Ni or Fe components of Cluster A in acetyl-CoA synthase; determining the binding order of methyl, carbonyl, and CoA moieties to Cluster A; and developing a way to express recombinant CO dehydrogenase/acetyl-CoA synthase in an active form. The electronic structure of a substrate-derived hydroxyethyl-TPP radical that has been shown to be a catalytic intermediate in the mechanism of the C. thermoaceticum pyruvate:ferredoxin oxidoreductase will be elucidated. The hypothesis that CO2 is channeled from pyruvate:ferredoxin oxidoreductase to CO dehydrogenase will be tested. Studies will be focused on determining how CoA binding affects electron transfer and catalysis and if a CoA radical may be a catalytic intermediate.
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