CARBON-MONOXIDE DEHYDROGENASE FROM CLOSTRIDIUM-THERMOACETICUM - QUATERNARY STRUCTURE, STOICHIOMETRY OF ITS SDS-INDUCED DISSOCIATION, AND CHARACTERIZATION OF THE FASTER-MIGRATING FORM

The molecular mass (M(r)) of the nickel- and iron-sulfur-containing en zyme CO dehydrogenase from Clostridium thermoaceticum was determined b y sedimentation equilibrium ultracentrifugation to be 300000 +/- 30000 Da. Since the enzyme is known to contain equal numbers of two types o f subunits (M(r) = 82000 Da for alpha and 73000 Da for beta), this ind icates an alpha(2) beta(2) quaternary structure. The enzyme was previo usly thought to have an alpha(3) beta 3 structure because it migrates through calibrated size-exclusion chromatographic columns with an appa rent M(r) of about 420000 Da. The disproportionately fast migration ra te suggests that the enzyme is nonspherical. SDS induces the dissociat ion of an a subunit, yielding a stable species called FM-CODH. FM-CODH had a molecular mass of 210000 +/- 30000 Da, indicating an alpha(1) b eta(2) structure. It contained 2.1 +/- 0.3 Ni and 16 +/- 3 Fe per alph a(1) beta(2), exhibited S --> Fe charge-transfer transitions typical o f Fe-S proteins, and afforded the g(av) = 1.82, 1.86, and 1.94 EPR sig nals. Quantitation of the 1.82 and {1.94 + 1.86} signals afforded 0.35 and 1.9 spin/alpha(1) beta(2), respectively. FM-CODH samples exhibite d CO oxidation activity, but little CO/acetyl-CoA exchange activity. S ome FM-CODH samples exhibited CO oxidation activities as high as nativ e 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 beta subunit. The alpha subunit that dissociated during formation of FM-CODH is not requ ired for CO oxidation activity. FM-CODH is either devoid of A-clusters , or if such clusters are present, they have lost their ability to exh ibit substantial NiFeC signals and CO/acetyl-CoA exchange activity. In cubating FM-CODH and alpha yielded a species that migrated through pol yacrylamide gels at the same rate as native enzyme, and had a molecula r mass indicating an alpha(2) beta(2) structure. Thus, the SDS-induced dissociation of the enzyme appears to be reversible.