Stepwise hydrogen atom and proton transfers in dioxygen reduction by aryl-alcohol oxidase

Abstract

21 p.-5 fig.-1 tab.-1 schem.The mechanism of dioxygen reduction by the flavoenzyme aryl-alcohol oxidase was investigated with kinetic isotope, viscosity, and pL (pH/pD) effects in rapid kinetics experiments by stopped-flow spectrophotometry of the oxidative half-reaction of the enzyme. Double mixing of the enzyme in a stopped-flow spectrophotometer with [α-2H2]- p-methoxybenzyl alcohol and oxygen at varying aging times established a slow rate constant of 0.0023 s-1 for the wash-out of the D atom from the N5 atom of the reduced flavin. Thus, the deuterated substrate could be used to probe the cleavage of the N-H bond of the reduced flavin in the oxidative half-reaction. A significant and pH-independent substrate kinetic isotope effect (KIE) of 1.5 between pH 5.0 and 8.0 demonstrated that H transfer is partially limiting the oxidative half-reaction of the enzyme; a negligible solvent KIE of 1.0 between pD 5.0 and 8.0 proved a fast H+ transfer reaction that does not contribute to determining the flavin oxidation rates. Thus, a mechanism for dioxygen reduction in which the H atom originating from the reduced flavin and a H+ from a solvent exchangeable site are transferred in separate kinetic steps is proposed. The spectroscopic and kinetic data presented also showed a lack of stabilization of transient flavin intermediates. The substantial differences in the mechanistic details of O2 reduction by aryl-alcohol oxidase with respect to other alcohol oxidases like choline oxidase, pyranose 2-oxidase, and glucose oxidase further demonstrate the high level of versatility of the flavin cofactor in flavoenzymes.This work was supported by the European INDOX (KBBE-2013-7-613549, www.indoxproject.eu) and EnzOx2 (H2020-BBI-PPP-2015-720297,www.enzox2.eu) projects, the NOESIS project (BIO2014-56388-R) of the Spanish Ministry of Economy and Competitiveness (MINECO), and National Science Foundation Grant CHE-1506518. J.C. acknowledges an FPU fellowship (FPU2012-0241) as well as an Estancias Breves FPU grant (EST14/00358) from the Spanish Ministry of Education, Culture and Sports.Peer reviewe