Carbon monoxide dehydrogenase from Methanosarcina barkeri. Disaggregation, purification, and physicochemical properties of the enzyme.
Carbon monoxide dehydrogenase from acetate-grown cells of Methanosarcina barkeri exists in a high molecular weight form (approximately 3 X 10(6)) under conditions of high ionic strength but is converted to a much smaller form by dialysis. The enzyme was purified by a procedure which exploits isolation of the aggregated form by gel filtration and subsequent dissociation. Following this, the enzyme was purified to within 92% of homogeneity by chromatography on phenyl-Sepharose and finally on hydroxylapatite. Due to the extreme oxygen lability of the enzyme, the entire procedure was carried out within the anaerobic laboratory at the National Institutes of Health. The enzyme has an alpha 2 beta 2 oligomeric structure composed of subunits with molecular weights of 19,700 and 84,500. The amino acid compositions of the individual subunits were determined. Analysis of the metal content by plasma emission spectroscopy indicated 1.3 +/- 0.3 (n = 4) nickel and 15.6 +/- 5.6 (n = 5) iron per mol of alpha 2 beta 2. The enzyme did not contain significant amounts of cobalt or molybdenum. Ferredoxin, FAD, FMN, 2,3,5-triphenyltetrazolium chloride, methyl viologen, and phenazine methosulfate served as electron acceptors; however, the enzyme failed to reduce NAD+, NADP+, or the 8-hydroxy-5-deazaflavin factor F420. The optimum pH was between 7 and 9. The apparent Km for methyl viologen was 7.1 mM, whereas the value for 2,3,5-triphenyltetrazolium chloride was below 0.5 mM. Strong inhibition was observed by oxygen and cyanide. Inactivation by glyoxaldehyde required enzymatic turnover which suggested that a reactive group was formed, or exposed, on an enzyme intermediate in catalysis. A high degree of thermostability was noted. Carbon monoxide, however, rendered the enzyme more susceptible to temperature inactivation.
D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D019605:Euryarchaeota / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009097:Multienzyme Complexes; D011489:Protein Denaturation; D013816:Thermodynamics
D000596:Amino Acids; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase