The Reductive Half-reaction of Xanthine Oxidase

Abstract

The pH dependence and solvent isotope sensitivity of three discrete steps in the reductive half-reaction of xanthine oxidase have been investigated. The pH dependence of both k[IMG]_16812_tex2html_wrap77.xbm">/K[IMG]_16812_tex2html_wrap79.xbm"> from steady-state experiments and k[IMG]_16812_tex2html_wrap103.xbm">/K[IMG]_16812_tex2html_wrap83.xbm">from rapid reaction experiments with xanthine as substrate indicate that enzyme reacts preferentially with the neutral form of substrate and that an ionizable group in the active site having a pK[IMG]_16812_tex2html_wrap109.xbm"> of [IMG]_16812_tex2html_wrap87.xbm">6.6 must be unprotonated for reaction to take place. The solvent kinetic isotope effect on k[IMG]_16812_tex2html_wrap103.xbm">/K[IMG]_16812_tex2html_wrap83.xbm"> is 2.4, once a uniform shift on going to D[(2)]O of [IMG]_16812_tex2html_wrap87.xbm">1 unit for both pK[IMG]_16812_tex2html_wrap109.xbm"> values is taken into account. The pH dependence of the formation and decay of E[IMG]_16812_tex2html_wrap103.xbm">-P formed in the course the reaction of xanthine oxidase with lumazine has also been examined. Formation of this complex exhibits bell-shaped pH dependence, with pK[IMG]_16812_tex2html_wrap109.xbm"> values of 6.5 and 7.8, consistent with the results obtained with xanthine. Decay of the E[IMG]_16812_tex2html_wrap103.xbm">-P complex is base-catalyzed with a pK[IMG]_16812_tex2html_wrap109.xbm"> > 11 and exhibits a small solvent kinetic isotope effect of 1.7 at pH/D 8.5. By contrast, the catalytic intermediate giving rise to the ``very rapid'' EPR signal that is transiently observed in the course of the reaction of enzyme with the substrate 2-hydroxy-6-methylpurine is found to undergo acid-catalyzed breakdown with an associated pK[IMG]_16812_tex2html_wrap109.xbm"> < 6. Formation and decay of this species exhibit solvent kinetic isotope effects of 2.0 and 3.5 at pH 10. The results are discussed in the context of a specific reaction mechanism for the reductive half-reaction of xanthine oxidase, in which discrete ionizations associated with the molybdenum center of the active site play critical roles in determining the magnitude of the rate constants by which the Mo(IV)-P and Mo(V)-P intermediates form and decay.