The radical chemistry of milk xanthine oxidase as studied by radiation chemistry techniques.

The kinetics of electron transfer within the molybdoflavoenzyme xanthine oxidase has been investigated using the technique of pulse radiolysis. Subsequent to one-electron reduction of native enzyme at 20 degrees C in 20 mM pyrophosphate buffer, pH 8.5, using the CO-.2 species as reductant, a spectral change is observed having a rate constant of approximately 290 s-1. From its wavelength dependence, this spectral change is assigned to the transfer of an electron from flavin semiquinone (formed on reaction with the CO2-. species) to one of the iron-sulfur centers of the enzyme in an intramolecular equilibration process. The value for this rate constant agrees well with the 330 s-1 observed in previous stopped-flow pH-jump experiments carried out at 25 degrees C (Hille, R., and Massey, V. (1986) J. Biol. Chem. 261, 1241-1247). Experimental results with fully reduced enzyme reacting with the radiolytically generated N.3 species also support the conclusion that the equilibration of reducing equivalents among the oxidation-reduction centers of xanthine oxidase is a rapid process. Evidence is also found that xanthine oxidase possesses an unusually reactive disulfide bond that is reduced rapidly by radiolytically generated radicals. The ramifications of the present results with regard to the interpretation of experiments involving chemically reactive radical species, generated either by photolysis or radiolysis, are discussed.