Carbon monoxide:methylene blue oxidoreductase from Pseudomonas carboxydovorans.
The enzyme carbon monoxide:methylene blue oxidoreductase from CO autotrophically grown cells of Pseudomonas carboxydovorans strain OM5, was purified to homogeneity. The enzyme was obtained in 26% yield and was purified 36-fold. The enzyme was stable for at least 6 days, had a molecular weight of 230,000, gave a single protein and activity band on polyacrylamide gel electrophoresis, and was homogeneous by the criterion of sedimentation equilibrium. Sodium dodecyl sulfate gel electrophoresis revealed a single band of molecular weight 107,000. Carbon monoxide:methylene blue oxidoreductase did not catalyze reduction of pyridine or flavin nucleotides but catalyzed the oxidation of CO to CO2 in the presence of methylene blue, thionine, toluylene blue, dichlorophenolindophenol, or pyocyanine under strictly anaerobic conditions. The visible spectrum revealed maxima at 405 and 470 nm. The millimolar extinction coefficients were 43.9 (405 nm) and 395.5 (275 nm), respectively. Absorption at 470 nm decreased in the presence of dithionite, and the spectrum was not affected by the substrate CO. Maximum reaction rates were found at pH 7.0 and 63 degrees C; temperature dependence followed the Arrhenius equation, with an activation energy (delta H degree) of 36.8 kJ/mol (8.8 kcal/mol). The apparent Km was 53 microM for CO. The purified enzyme was incapable of oxidizing methane, methanol, or formaldehyde in the presence of methylene blue as electron acceptor.
D002248:Carbon Monoxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008751:Methylene Blue / Q000378:metabolism; D008970:Molecular Weight; D010088:Oxidoreductases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D002248:Carbon Monoxide; D010088:Oxidoreductases; D008751:Methylene Blue