The equilibration of reducing equivalents within milk xanthine oxidase.
The rate at which reducing equivalents equilibrate among the several oxidation-reduction active sites in xanthine oxidase has been investigated using a pH-jump technique in which partially reduced enzyme in dilute buffer is mixed with concentrated anaerobic buffer at a different pH in a conventional stopped flow apparatus. It is found that the rate constant associated with the observed spectral change varies with pH, doubling from 155 s-1 at pH 6 to 330 s-1 at pH 8.5, but is always found to be approximately 10-fold greater than kcat at the same pH. The observation of fast rates for the equilibration of reducing equivalents within xanthine oxidase is consistent with a great deal of indirect evidence from conventional kinetic studies of both the oxidative and reductive half-reactions of xanthine oxidase and lends support to the rapid equilibrium model that has been proposed for the oxidation-reduction interactions of the several centers in xanthine oxidase (Olson, J. S., Ballou, D. P., Palmer, G., and Massey, V. (1974) J. Biol. Chem. 249, 4363-4382). The present conclusions are in conflict, however, with the interpretation of recent flash photolysis experiments with xanthine oxidase (Battacharyya, A., Tollin, G., Davis, M. D., and Edmondson, D. E. (1983) Biochemistry 22, 5270-5279). Possible sources for the apparent inconsistencies between the flash photolysis results and those of the present experiments are discussed.
D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005260:Female; D005415:Flavins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008892:Milk / Q000201:enzymology*; D008982:Molybdenum / Q000378:metabolism; D010084:Oxidation-Reduction; D010117:Oxypurinol / Q000378:metabolism; D010782:Photolysis; D011621:Pteridines / Q000378:metabolism; D013696:Temperature; D014969:Xanthine Oxidase / Q000378:metabolism*
D005415:Flavins; D011621:Pteridines; C040826:violapterin; D008982:Molybdenum; D014969:Xanthine Oxidase; D010117:Oxypurinol