Methylation of carbon monoxide dehydrogenase from Clostridium thermoaceticum and mechanism of acetyl coenzyme A synthesis
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Carbon monoxide dehydrogenase from Clostridium thermoaceticum was methylated such that all bound methyl groups could subsequently react with CO and coenzyme A (or OH-) to yield acetyl-coenzyme A (acetyl-CoA) (or acetate). Methyl groups could not bind enzyme lacking the labile Ni of the A- cluster, but could bind such samples after incubation in aqueous Ni2+, a process known to reinsert the labile Ni and reactivate the enzyme. Bound methyl groups inhibited the ability of 1,10-phenanthroline to remove the labile Ni, and the amount bound approximately correlated with the amount of labile Ni. This is strong evidence that the methyl group used in acetyl-CoA synthesis binds the labile Ni. Evidence is presented that a redox site (called the D site) other than the spin-coupled metals that define the A- cluster must be reduced before methylation can occur. Both methyl and acetyl intermediates appear to be EPR-silent. The acetyl intermediate reacted slowly with OH to yield acetate and rapidly with CoAS- to yield acetyl-CoA. When enzyme in a state with the A-cluster reduced and bound with CO (the S = 1/4 A(red)-CO state) was methylated, the resulting acetyl intermediate was also EPR-silent, indicating that the order of substrate addition had no effect on the EPR silence of the resulting acetyl intermediate. The D site appears to be an n = 2 redox agent that functions to reduce the oxidized A-cluster upon methylation and to oxidize the A-cluster as the product acetyl-CoA dissociates. D is EPR-silent in both of its oxidation states and is not any of the known metal clusters in the enzyme. D may be a special pair of cysteines coordinated to the labile Ni that can be oxidized to cystine at unusually low potentials (~-530 mV vs NHE). Catalytic mechanisms that do not include D or its functional equivalent, or that employ the reduced S = 1/4 CO- bound form of the A-cluster as an intermediate, are inconsistent with the present data. A new catalytic mechanism incorporating the results of this study is proposed.