A study has been conducted on carbon monoxide dehydrogenase (CODH) from acetate-grown cells of Methanosarcina barkeri. Under conditions of high ionic strength the enzyme exists in an aggregated state along with a discrete set of other proteins (total mol. wt. approx. 3,000,000). Dissociation of the aggregate occurred when the ionic strength was decreased by dialysis. The disaggregated form of CODH retained full activity and exhibited a molecular weight of approximately 161,000. An efficient purification procedure was developed which produced high yields of pure CODH. The method consisted of the following steps which were carried out under strictly anaerobic conditions in the NIH Anaerobic Laboratory: 1. Isolation of the CODH aggregate by gel-filtration and subsequent dissociation; 2. Chromatography on Phenyl Sepharose; and 3. Hydroxylapatite chromatography. CODH appeared to be less hydrophobic than any of the other protein components of the aggregate. It bound tightly to hydroxylapatite and, thus, may have a relatively large number of exposed carboxylic acid residues. Pure CODH is composed of two subunits of molecular weights 93,800 and 20,400. Among various compounds tested, oxygen and cyanide are potent inactivators. Glyoxaldehyde inactivation occurred only during enzymatic turnover, which suggests that a reactive group is formed, or exposed, on an enzyme intermediate in catalysis.
