The crystal structure of the tetrameric alpha2beta2 acetyl-coenzyme A synthase/carbon monoxide dehydrogenase from Moorella thermoacetica has been solved at 1.9 A resolution. Surprisingly, the two alpha subunits display different (open and closed) conformations. Furthermore, X-ray data collected from crystals near the absorption edges of several metal ions indicate that the closed form contains one Zn and one Ni at its active site metal cluster (A-cluster) in the alpha subunit, whereas the open form has two Ni ions at the corresponding positions. Alternative metal contents at the active site have been observed in a recent structure of the same protein in which A-clusters contained one Cu and one Ni, and in reconstitution studies of a recombinant apo form of a related acetyl-CoA synthase. On the basis of our observations along with previously reported data, we postulate that only the A-clusters containing two Ni ions are catalytically active.

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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 = 1/2 Ared-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 = 1/2 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 proposed.

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Acetyl-CoA synthase (also known as carbon monoxide dehydrogenase) is a bifunctional Ni-Fe-S-containing enzyme that catalyzes the reversible reduction of CO(2) to CO and the synthesis of acetyl-coenzyme A from CO, CoA, and a methyl group donated by a corrinoid iron-sulfur protein. The active site for the latter reaction, called the A-cluster, consists of an Fe(4)S(4) cubane bridged to the proximal Ni site (Ni(p)), which is bridged in turn to the so-called distal Ni site. In this review, evidence is presented that Ni(p) achieves a zero-valent state at low potentials and during catalysis. Ni(p) appears to be the metal to which CO and methyl groups bind and then react to form an acetyl-Ni(p) intermediate. Methyl group binding requires reductive activation, where two electrons reduce some site on the A-cluster. The coordination environment of the distal Ni suggests that it could not be stabilized in redox states lower than 2+. The rate at which the [Fe(4)S(4)](2+) cubane is reduced is far slower than that at which reductive activation occurs, suggesting that the cubane is not the site of reduction. An intriguing possibility is that Ni(p)(2+) might be reduced to the zero-valent state. Reinforcing this idea are Ni-organometallic complexes in which the Ni exhibits analogous reactivity properties when reduced to the zero-valent state. A zero-valent Ni stabilized exclusively with biological ligands would be remarkable and unprecedented in biology.