The reaction mechanism of xanthine oxidase: Evidence for two-electron chemistry rather than sequential one-electron steps

Current research on xanthine oxidase has favored a mechanism involving base-catalyzed proton abstraction from a Mo-OH group, allowing nucleophilic attack on the substrate and hydride transfer from the substrate to Mo=S group in the active site. During the course of this reaction mechanism, the molybdenum redox cycles from Mo^VI to Mo^IV, with reoxidation of the Mo^IV speices to form the EPR active Mo^V intermediate. However, it has also been suggested that the reaction occurs in two subsequent one-electron steps. We have determined kinetic parameters k_red and k_red/K_d for a variety of plausible substrates as well as the one-electron reduction potentials for these substrates. Our data indicate no correlation between these kinetic parameters and their one-electron reduction potentials, as would be expected if the enzyme were using two subsequent one-electron reduction steps. Our results provide additional support to current evidence for the favored two-electron reduction mechanism.