Xanthine Dehydrogenase Electrocatalysis: Autocatalysis and Novel Activity

Abstract

The enzyme xanthine dehydrogenase (XDH) from the purple photosynthetic bacterium Rhodobacter capsulatus catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid as part of purine metabolism. The native electron acceptor is NAD(+) but herein we show that uric acid in its 2-electron oxidized form is able to act as an artificial electron acceptor from XDH in an electrochemically driven catalytic system. Hypoxanthine oxidation is also observed with the novel production of uric acid in a series of two consecutive 2-electron oxidation reactions via xanthine. XDH exhibits native activity in terms of its pH optimum and inhibition by allopurinol.

Low Potential Catalytic Voltammetry of Human Sulfite Oxidase

“…So utilizing the mass transport limitations presented by the membrane, the linear response of the electrode to sulfite is increased by at least 2 orders of magnitude. We have observed similar observations in other Mo enzyme systems [25,26,42].…”

The Reductive Half-reaction of Xanthine Dehydrogenase from Rhodobacter capsulatus

“…The pK a values for k cat /K m with wild-type enzyme determined here are in fair agreement with the values of 6.3 and 7.7 reported previously by Aguey-Zinsou et al (27), who monitored catalysis electrochemically (the pH dependence of k cat was not reported in this work). However, here the autocatalytic reaction with uric acid was measured directly at the electrode, which might explain the difference in the pH profile, because the latter is rather related to the ionization of uric acid (28). The maximal theoretical value for k cat /K m , obtained from the fit to the pH profile as described under "Experimental Procedures," decreases from 2.0 ϫ 10 …”

The Mononuclear Molybdenum Enzymes

“…Given that the xanthine oxidase reaction involves obligatory two-electron chemistry, 132 and that the enzyme is known to catalyze the dehydroxylation of uric acid to xanthine under strongly reducing conditions, 260 the possibility cannot be excluded that the 4-hydroxybenzoyl-CoA dehydrogenase reaction in fact runs simply in the reverse of the hydroxylation pathway for xanthine oxidase, with hydride transfer from an equatorial Mo-SH to C-4 of molybdenum-coordinated substrate, followed directly by dehydroxylation and rearomatization. It has been proposed, however, that the enzyme operates via a radical-based Birch-like mechanism, 259 in which a first reducing equivalent is added to molybdenum-coordinated substrate, followed by protonation at C-4 of substrate and addition of a second reducing equivalent, which leads to dehydroxylation and rearomatization.…”