Purification and characterization of carbon monoxide dehydrogenase, a nickel, zinc, iron-sulfur protein, from Rhodospirillum rubrum.
Carbon monoxide dehydrogenase (CO dehydrogenase) from Rhodospirillum rubrum was shown to be an oxygen-sensitive, nickel, iron-sulfur, and zinc-containing protein that was induced by carbon monoxide (CO). The enzyme was purified 212-fold by heat treatment, ion-exchange, and hydroxylapatite chromatography and preparative gel electrophoresis. The purified protein, active as a monomer of Mr = 61,800, existed in two forms that were comprised of identical polypeptides and differed in metal content. Form 1 comprised 90% of the final activity, had a specific activity of 1,079 mumol CO oxidized per min-1 mg-1, and contained 7 iron, 6 sulfur, 0.6 nickel, and 0.4 zinc/monomer. Form 2 had a lower specific activity (694 mumol CO min-1 mg-1) and contained 9 iron, 8 sulfur, 1.4 nickel, and 0.8 zinc/monomer. Reduction of either form by CO or dithionite resulted in identical, rhombic ESR spectra with g-values of 2.042, 1.939, and 1.888. Form 2 exhibited a 2-fold higher integrated spin concentration, supporting the conclusion that it contained an additional reducible metal center(s). Cells grown in the presence of 63NiCl2 incorporated 63Ni into CO dehydrogenase. Although nickel was clearly present in the protein, it was not ESR-active under any conditions tested. R. rubrum CO dehydrogenase was antigenically distinct from the CO dehydrogenases from Methanosarcina barkeri and Clostridium thermoaceticum.
D000445:Aldehyde Oxidoreductases / Q000032:analysis* / Q000378:metabolism; D002248:Carbon Monoxide / Q000494:pharmacology; D002845:Chromatography; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D007501:Iron / Q000032:analysis*; D008970:Molecular Weight; D009097:Multienzyme Complexes; D009532:Nickel / Q000032:analysis*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000494:pharmacology; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D013053:Spectrophotometry; D013455:Sulfur / Q000032:analysis*; D015032:Zinc / Q000032:analysis*
D009097:Multienzyme Complexes; D013455:Sulfur; D009532:Nickel; D002248:Carbon Monoxide; D007501:Iron; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; D015032:Zinc; D010100:Oxygen