Carbon Monoxide Dehydrogenase from Clostridium thermoaceticum: Quaternary Structure, Stoichiometry of Its SDS-Induced Dissociation, and Characterization of the Faster-Migrating Form

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Abstract
The molecular mass (Mr) of the nickel- and iron-sulfur-containing enzyme CO dehydrogenase from Clostridium thermoaceticum was determined by sedimentation equilibrium ultracentrifugation to be 300 000 ± 30 000 Da. Since the enzyme is known to contain equal numbers of two types of subunits (Mr = 82 000 Da for α and 73 000 Da for β), this indicates an α2β2 quaternary structure. The enzyme was previously thought to have an α3β3 structure because it migrates through calibrated size-exclusion chromatographic columns with an apparent Mr of about 420 000 Da. The disproportionately fast migration rate suggests that the enzyme is nonspherical. SDS induces the dissociation of an α subunit, yielding a stable species called FM-CODH. FM-CODH had a molecular mass of 210 000 ± 30 000 Da, indicating an α1β2 structure. It contained 2.1 ± 0.3 Ni and 16 ± 3 Fe per α1β2, exhibited S → Fe charge-transfer transitions typical of Fe-S proteins, and afforded the gav = 1.82, 1.86, and 1.94 EPR signals. Quantitation of the 1.82 and {1.94 + 1.86} signals afforded 0.35 and 1.9 spin/α1β2, respectively. FM-CODH samples exhibited CO oxidation activity, but little CO/acetyl-CoA exchange activity. Some FM-CODH samples exhibited CO oxidation activities as high as native enzyme. These results, along with the quantified spin intensities of the EPR signals, indicate that FM-CODH contains the B- and C-clusters and suggest that these clusters are located in the β subunit. The α subunit that dissociated during formation of FM-CODH is not required for CO oxidation activity. FM-CODH is either devoid of A-clusters, or if such clusters are present, they have lost their ability to exhibit substantial NiFeC signals and CO/acetyl-CoA exchange activity. Incubating FM-CODH and α yielded a species that migrated through polyacrylamide gels at the same rate as native enzyme, and had a molecular mass indicating an α2β2 structure. Thus, the SDS-induced dissociation of the enzyme appears to be reversible.