The mechanism of action of xanthine oxidase.

The changes in optical and EPR spectra observed during the anaerobic titration of xanthine oxidase with dithionite can be accounted for by simple oxidation-reduction equilibria involving Mo(VI) u MO(V) H Mo(IV); FAD cf FADH. ++ FADH2, and the l-electron reduction of each of the two ironsulfur centers. The equilibrium constants so obtained permit the calculation of the relative probabilities of the 36 possible states that occur on addition of a given quantity of reductant. pH jump experiments with this enzyme (EDMONDSON, D., BALLOU, D., VAN HEWELEN, A., PALMER, G., AND MASSEY, V. (1973) J. Biol. Chem. 248, 6129-6135) show that internal electron transfer is much more rapid than turnover and we therefore assume that the distributions described by these equilibrium constants are maintained during catalysis. By adopting a model in which 3 moles of xanthine each donate a pair of electrons in a consecutive irreversible process, and using the equilibrium distributions described above, it is possible to calculate accurately the time course of reduction of both the flavin and iron-sulfur centers and to account for the increase in the rate of bleaching of deflavoenzyme observed experimentally. By invoking the existence of enzyme-xanthine complexes we can account for (a) the participation and time dependence of “very rapid” and “rapid” molybdenum and (b) the deuterium isotope effects observed during steady state and transient state kinetics (EDMONDSON et al.). The same equilibrium model accounts for the optical and EPR data obtained during the reaction of reduced enzyme with oxygen; the biphasic kinetics are readily explained by assuming that FADHz is the species reacting rapidly with 02 in the fast phase. From these results we conclude that the iron-sulfur centers act as an electron reservoir functioning to maintain molybdenum as Mo(VI) (for efficient reduction) and flavin as FADH2 (for efficient oxidation).