Chemical and spectral properties of carbon monoxide: methylene blue oxidoreductase. The molybdenum-containing iron-sulfur flavoprotein from Pseudomonas carboxydovorans.
Carbon monoxide:methylene blue oxidoreductase, the key enzyme of CO-oxidation in energy metabolism of the carboxydobacterium Pseudomonas carboxydovorans, has been isolated in good yield and purity and found to contain FAD, molybdenum, iron, and labile sulfide in the ratio of 1:1:4:4. The enzyme is, therefore, a new molybdenum-containing iron-sulfur flavoprotein, exhibiting chemical and spectral properties quite similar to those of xanthine oxidase. Analytical data on the spectral characteristics of the enzyme in the oxidized and various reduced states are presented. Carbon monoxide:methylene blue oxidoreductase turned out to be photoreducible in the presence of EDTA and urea and was subject to reoxidation by air oxygen; no flavoprotein semiquinone was formed. Unphysiological electron acceptors, e.g. methylene blue, were used as oxidizing substrates whereas NAD or NADP turned out to be ineffective. Methylene blue reduction with CO was not affected by the presence of allopurinol, and carbon monoxide:methylene blue oxidoreductase was not able to catalyze the reduction of methylene blue with xanthine, adenine, or aldehydes. CO was the only reducing substrate used by the enzyme. Carbon monoxide:methylene blue oxidoreductase formed no sulfite adduct, and the reactivity with ferricyanide or cytochrome c was significant but slow. As known for other molybdenum hydroxylases, carbon monoxide:methylene blue oxidoreductase was rapidly inactivated by methanol, but the enzyme exhibited no ability to catalyze the oxidation of NADH with methylene blue, and NAD was not able to overcome methanol inhibition.
D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D000693:Anaerobiosis; D002248:Carbon Monoxide / Q000494:pharmacology; D003574:Cytochrome c Group; D005292:Ferricyanides; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D007700:Kinetics; D011549:Pseudomonas / Q000201:enzymology*; D013053:Spectrophotometry; D013379:Substrate Specificity
D003574:Cytochrome c Group; D005292:Ferricyanides; D007506:Iron-Sulfur Proteins; D002248:Carbon Monoxide; D000445:Aldehyde Oxidoreductases; C033598:carbon monoxide-methylene blue oxidoreductase