METHYLATION OF CARBON-MONOXIDE DEHYDROGENASE FROM CLOSTRIDIUM-THERMOACETICUM AND MECHANISM OF ACETYL-COENZYME-A SYNTHESIS

Carbon monoxide dehydrogenase from Clostridium thermoaceticum was meth ylated such that all bound methyl groups could subsequently react with CO and coenzyme A (or OH-) to yield acetyl-coenzyme A (acetyl-CoA) (o r acetate). Methyl groups could not bind enzyme lacking the labile Ni of the A-cluster, but could bind such samples after incubation in aque ous Ni2+, a process known to reinsert the labile Ni and reactivate the enzyme. Bound methyl groups inhibited the ability of 1,10-phenanthrol ine to remove the labile Ni, and the amount bound approximately correl ated with the amount of labile Ni. This is strong evidence that the me thyl 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 methy lation can occur. Both methyl and acetyl intermediates appear to be EP R-silent. The acetyl intermediate reacted slowly with OH- to yield ace tate and rapidly with CoAS(-) to yield acetyl-CoA. When enzyme in a st ate with the A-cluster reduced and bound with CO (the S = 1/2 A(red)-C O state) was methylated, the resulting acetyl intermediate was also EP R-silent, indicating that the order of substrate addition had no effec t 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 oxidiz ed A-cluster upon methylation and to oxidize the A-cluster as the prod uct acetyl-CoA dissociates. D is EPR-silent in both of its oxidation s tates 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 b e oxidized to cystine at unusually low potentials (similar to -530 mV vs NHE). Catalytic mechanisms that do not include D or its functional equivalent, or that employ the reduced S = 1/2 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 pro posed.