The reaction of xanthine oxidase with molecular oxygen.

The reaction of reduced xanthine oxidase with oxygen occurs in two distinct kinetic phases with the rate of the fast phase being about 10 times greater than that of the slow phase. Comparison of transient state and steady state kinetics demonstrates that only the fast phase of oxidation is catalytically significant. The time course of oxidation is quantitatively independent of the following factors: (a) the activity to flavin ratio; (b) presence of competitive inhibitors; (c) pretreatment of enzyme with either cyanide or allopurinol; and (d) addition of superoxide dismutase. Removal of flavin eliminates both oxidase activity and the reactivity of the reduced enzyme with molecular oxygen (KOMAI, H., MASSEY, V., AND PALMER, G. (1969) /. Biot. Chem. 244, 1692-1700). Both optical and EPR kinetic data support the interpretation that 5 electrons are consumed in the fast phase and 1 in the slow phase. The spectral properties of the slowly reacting species suggest that the residual electron is present principally as reduced iron-sulfur center II at pH 8.5. The simplest interpretation of the kinetic data requires that rapid reaction of the enzyme with oxygen occurs via fully reduced flavin and that the slow phase observed for 1 -electron reduced enzyme is a consequence of both the poor reactivity and small equilibrium concentration of flavin semiquinone. The absence of any effect of superoxide dismutase on both the optical and EPR data and the small amount of superoxide generated indicates that hydrogen peroxide is the major product formed during oxidation of fully reduced xanthine oxidase.