Purification and some properties of carbon monoxide dehydrogenase from Acinetobacter sp. strain JC1 DSM 3803.
A brown carbon monoxide dehydrogenase from CO-autotrophically grown cells of Acinetobacter sp. strain JC1, which is unstable outside the cells, was purified 80-fold in seven steps to better than 95% homogeneity, with a yield of 44% in the presence of the stabilizing agents iodoacetamide (1 mM) and ammonium sulfate (100 mM). The final specific activity was 474 mumol of acceptor reduced per min per mg of protein as determined by an assay based on the CO-dependent reduction of thionin. Methyl viologen, NAD(P), flavin mononucleotide, flavin adenine dinucleotide, and ferricyanide were not reduced by the enzyme, but methylene blue, thionin, and dichlorophenolindophenol were reduced. The molecular weight of the native enzyme was determined to be 380,000. Sodium dodecyl sulfate-gel electrophoresis revealed at least three nonidentical subunits of molecular weights 16,000 (alpha), 34,000 (beta), and 85,000 (gamma). The purified enzyme contained particulate hydrogenase-like activity. Selenium did not stimulate carbon monoxide dehydrogenase activity. The isoelectic point of the native enzyme was found to be 5.8; the Km of CO was 150 microM. The enzyme was rapidly inactivated by methanol. One mole of native enzyme was found to contain 2 mol of each of flavin adenine dinucleotide and molybdenum and 8 mol each of nonheme iron and labile sulfide, which indicated that the enzyme was a molybdenum-containing iron-sulfur flavoprotein. The ratio of densities of each subunit after electrophoresis (alpha:beta:gamma = 1:2:6) and the number of each cofactor in the native enzyme suggest a alpha 2 beta 2 gamma 2 structure of the enzyme. The carbon monoxide dehydrogenase of Acinetobacter sp. strain JC1 was found to have no immunological relationship with enzymes of Pseudomonas carboxydohydrogena and Pseudomonas carboxydovorans.
Kim
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KS
Department of Biology, College of Science, Yonsei University, Seoul, Korea.
Ro
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Kim
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D000150:Acinetobacter / Q000201:enzymology*; D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004795:Enzyme Stability; D006882:Hydroxyapatites; D005779:Immunodiffusion; D007202:Indicators and Reagents; D007501:Iron / Q000032:analysis; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D008982:Molybdenum / Q000032:analysis; D009097:Multienzyme Complexes; D013045:Species Specificity
D006882:Hydroxyapatites; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D008982:Molybdenum; D017886:Durapatite; D007501:Iron; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase