Carbon monoxide dehydrogenase from Clostridium thermoaceticum: Quaternary structure, stoichiometry of its SDS-induced dissociation, and characterization of the faster-migrating form

被引:36
作者
Xia, JQ
Sinclair, JF
Baldwin, TO
Lindahl, PA
机构
[1] TEXAS A&M UNIV,DEPT CHEM,COLLEGE STN,TX 77843
[2] TEXAS A&M UNIV,DEPT BIOCHEM & BIOPHYS,COLLEGE STN,TX 77843
[3] TEXAS A&M UNIV,CTR MACROMOLEC DESIGN,COLLEGE STN,TX 77843
关键词
D O I
10.1021/bi9511853
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The molecular mass (M(r)) of the nickel- and iron-sulfur-containing enzyme CO dehydrogenase from Clostridium thermoaceticum was determined by sedimentation equilibrium ultracentrifugation to be 300000 +/- 30000 Da. Since the enzyme is known to contain equal numbers of two types of subunits (M(r) = 82000 Da for alpha and 73000 Da for beta), this indicates an alpha(2) beta(2) quaternary structure. The enzyme was previously thought to have an alpha(3) beta 3 structure because it migrates through calibrated size-exclusion chromatographic columns with an apparent M(r) of about 420000 Da. The disproportionately fast migration rate suggests that the enzyme is nonspherical. SDS induces the dissociation 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) beta(2) structure. It contained 2.1 +/- 0.3 Ni and 16 +/- 3 Fe per alpha(1) beta(2), exhibited S --> Fe charge-transfer transitions typical of Fe-S proteins, and afforded the g(av) = 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/alpha(1) beta(2), 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 beta subunit. The alpha 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 alpha yielded a species that migrated through polyacrylamide gels at the same rate as native enzyme, and had a molecular mass indicating an alpha(2) beta(2) structure. Thus, the SDS-induced dissociation of the enzyme appears to be reversible.
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页码:1965 / 1971
页数:7
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