Stepwise Hydrogen Atom and Proton Transfers in Dioxygen Reduction by Aryl-Alcohol Oxidase

Carro, J. ; Ferreira, P. (Universidad de Zaragoza) ; Martínez, A.T. ; Gadda, G.

Resumen: 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 [a-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.

Idioma: Inglés

DOI: 10.1021/acs.biochem.8b00106

Año: 2018

Publicado en: Biochemistry 57, 11 (2018), 1790-1797
ISSN: 0006-2960

Factor impacto JCR: 2.952 (2018)

Categ. JCR: BIOCHEMISTRY & MOLECULAR BIOLOGY rank: 139 / 294 = 0.473 (2018) - Q2 - T2

Factor impacto SCIMAGO: 1.556 - Biochemistry (Q1)

Financiación: info:eu-repo/grantAgreement/EC/FP7/613549/EU/Optimized oxidoreductases for medium and large scale industrial biotransformations/INDOX
Financiación: info:eu-repo/grantAgreement/EC/H2020/720297/EU/New enzymatic oxidation/oxyfunctionalization technologies for added value bio-based products/EnzOx2
Financiación: info:eu-repo/grantAgreement/ES/MINECO/NOESIS-BIO2014-56388-R

Tipo y forma: Article (PostPrint)

Área (Departamento): Área Bioquímica y Biolog.Mole. (Dpto. Bioq.Biolog.Mol. Celular)