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

AutorCarro, Juan; Ferreira, Patricia ; Martínez, Ángel T. ; Gadda, Giovanni
Fecha de publicación20-mar-2018
EditorAmerican Chemical Society
CitaciónBiochemistry 57(11):1790-1797 (2018)
ResumenThe 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.
Descripción21 p.-5 fig.-1 tab.-1 schem.
Versión del editorhttp://dx.doi.org/10.1021/acs.biochem.8b00106
URIhttp://hdl.handle.net/10261/163253
DOI10.1021/acs.biochem.8b00106
ISSN0006-2960
E-ISSN1520-4995
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