The reaction of xanthine oxidase with lumazine. Characterization of the reductive half-reaction.

The optical and fluorescence characteristics of lumazine (2, 4-dihydroxypteridine) and violapterin (2,4,6-trihydroxypteridine) are described. The reduction of xanthine oxidase by excess lumazine is markedly biphasic. The initial rapid phase is about 100-fold more rapid than Vmax and reflects the reaction of the enzyme with the first equivalent of lumazine; it is accompanied by the appearance of long wavelength absorbance of the Mo(IV) violapterin charge transfer species together with partial reduction in absorbance at 450 nm, reflecting a partial transfer of electrons from the Mo to the flavin and iron-sulfur center. A loss of substrate fluorescence and a burst of fluorescence due to enzyme bound product also occur during this phase. The second phase is rate-controlled by the dissociation of violapterin from the reduced enzyme. It is accompanied by a variable loss of the charge-transfer band, further reduction of the flavin and iron-sulfur chromophores and of substrate fluorescence, and the appearance of fluorescence of unbound product. This phase correlates well with the steady state parameters. The variation in kinetic behavior with pH is qualitatively explained by a marked decrease in the affinity of the enzyme as the pH is raised; Vmax is almost unaffected by pH. The temporal sequence of kinetic events reveals at least three steps in the reaction of xanthine oxidase with the first equivalent of lumazine.