The reaction of reduced xanthine oxidase with oxygen. Kinetics of peroxide and superoxide formation.
Product formation during the oxidation of xanthine oxidase has been examined directly by using cytochrome c peroxidase as a trapping agent for hydrogen peroxide and the reduction of cytochrome c as a measure of superoxide formation. When fully reduced enzyme is mixed with high concentrations of oxygen, 2 molecules of H2O2/flavin are produced rapidly, while 1 molecule of O2-/flavin is produced rapidly and another produced much more slowly. Time courses for superoxide formation and those for the absorbance changes due to enzyme oxidation were fitted successfully to the mechanism proposed earlier (Olson, J. S., Ballou, D. P., Palmer, G., and Massey, V. (1974) J. Biol. Chem. 249, 4363-4382). In this scheme, each oxidative step is initiated by the very rapid and reversible formation of an oxygen.FADH2 complex (the apparent KD = 2.2 X 10(-4) M at 20 degrees C, pH 8.3). In the cases of 6- and 4-electron-reduced enzyme, 2 electrons are transferred rapidly (ke = 60 s-1) to generate hydrogen peroxide and partially oxidized xanthine oxidase. In the case of the 2-electron-reduced enzyme, only 1 electron is transferred rapidly and superoxide is produced. The remaining electron remains in the iron-sulfur centers and is removed slowly by a second order process (ks = 1 X 10(4) M-1 s-1). When the pH is decreased from 9.9 to 6.2, both the apparent KD for oxygen binding and the rapid rate of electron transfer are decreased about 20-fold. This result is suggestive of uncompetitive inhibition and implies that proton binding to the enzyme-flavin active site affects primarily the rate of electron transfer, not the formation of the initial oxygen complex.
D000818:Animals; D002417:Cattle; D003578:Cytochrome-c Peroxidase / Q000378:metabolism; D005260:Female; D007700:Kinetics; D008892:Milk / Q000201:enzymology; D010084:Oxidation-Reduction; D010100:Oxygen; D010545:Peroxides; D013481:Superoxides; D014969:Xanthine Oxidase / Q000378:metabolism*
D010545:Peroxides; D013481:Superoxides; D003578:Cytochrome-c Peroxidase; D014969:Xanthine Oxidase; D010100:Oxygen