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The characteristics of the oxidation reaction and of the thermodynamics of electron transfer in xanthine oxidase were analyzed by stopped flow spectrophotometry and room temperature potentiometric titrations. Product formation during the oxidation of xanthine oxidase was examined directly by using cytochrome c peroxidase and cytochrome c as trapping agents for H(,2)O(,2) and O(')(,2) respectively. When fully reduced enzyme is mixed with high concentrations of oxygen, 2 molecules of H(,2)O(,2)/flavin are produced rapidly, while 1 molecule of O(')(,2)/flavin is produced rapidly and another much more slowly. Time courses for superoxide formation and those for the absorbance changes due to the 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, fully reduced enzyme is reoxidized by a series of steps in which sets of 2, 2, 1 and 1 electrons are removed in succession to form 2 equivalents of H(,2)O(,2) and 2 equivalents of O(')(,2). The observed pH effects on the reaction rates can be attributed to proton binding to the FAD-H(,2)O(,2) adduct. A series of potentiometric titration of xanthine oxidase was carried out at room temperature in the pH range 6.2-9.9. Reduction of the two Fe/S centers was monitored by CD, and that of the FAD and Mo center by EPR. The Fe/S centers behave as centers having a protonable group whose pK(,a) changes with reduction state (E = -344 mV, pK(,o) = 6.4, pK(,r) = 8.1 for Fe/S I; and E = -249 mV, pK(,o) = 6.4, pK(,r) = 8.0 for Fe/S II). The flavin and the two types of molybdenum centers show varying behavior but in all the cases electron addition is accompanied by protonation. The sequence for FAD is: reduction, protonation, reduction, protonation with E(,1) = -398 mV, E(,2) = -240 mV, pK(,1) = 9.5, pK(,2) = 7.4. For "rapid" molybdenum the sequence is: protonation, reduction, protonation, reduction with E(,1) = -369 mV, E(,2) = -301 mV, pK(,1) = 7.9, pK(,2) = 8.4; and for "slow" molybdenum: protonation, reduction, reduction, protonation with E(,1) = -320 mV, E(,2) = -477 mV, pK(,1) = 7.5, pK(,2) = 9.5. Comparison to data obtained previously at cryogenic temperatures (Cammack, R., Barber, M. J., and Bray, R. C., (1976), Biochem. J. 157, 469-478 and Barber, M. J., and Siegel, L. M., (1981) in "Flavins and Flavoproteins", (Massey, V., and Williams, C. H., eds.) in press) showed the centers to have significant temperature dependence.... (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of school.) UMI |
