000078009 001__ 78009
000078009 005__ 20200117221654.0
000078009 0247_ $$2doi$$a10.1021/acs.biochem.8b00106
000078009 0248_ $$2sideral$$a105446
000078009 037__ $$aART-2018-105446
000078009 041__ $$aeng
000078009 100__ $$aCarro, J.
000078009 245__ $$aStepwise Hydrogen Atom and Proton Transfers in Dioxygen Reduction by Aryl-Alcohol Oxidase
000078009 260__ $$c2018
000078009 5060_ $$aAccess copy available to the general public$$fUnrestricted
000078009 5203_ $$aThe 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.
000078009 536__ $$9info:eu-repo/grantAgreement/ES/MINECO/NOESIS-BIO2014-56388-R$$9This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 720297-EnzOx2$$9info:eu-repo/grantAgreement/EC/H2020/720297/EU/New enzymatic oxidation/oxyfunctionalization technologies for added value bio-based products/EnzOx2$$9info:eu-repo/grantAgreement/EC/FP7/613549/EU/Optimized oxidoreductases for medium and large scale industrial biotransformations/INDOX
000078009 540__ $$9info:eu-repo/semantics/openAccess$$aAll rights reserved$$uhttp://www.europeana.eu/rights/rr-f/
000078009 590__ $$a2.952$$b2018
000078009 591__ $$aBIOCHEMISTRY & MOLECULAR BIOLOGY$$b139 / 294 = 0.473$$c2018$$dQ2$$eT2
000078009 592__ $$a1.556$$b2018
000078009 593__ $$aBiochemistry$$c2018$$dQ1
000078009 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/acceptedVersion
000078009 700__ $$0(orcid)0000-0003-4076-6118$$aFerreira, P.$$uUniversidad de Zaragoza
000078009 700__ $$aMartínez, A.T.
000078009 700__ $$aGadda, G.
000078009 7102_ $$11002$$2060$$aUniversidad de Zaragoza$$bDpto. Bioq.Biolog.Mol. Celular$$cÁrea Bioquímica y Biolog.Mole.
000078009 773__ $$g57, 11 (2018), 1790-1797$$pBiochemistry$$tBiochemistry$$x0006-2960
000078009 8564_ $$s1276077$$uhttps://zaguan.unizar.es/record/78009/files/texto_completo.pdf$$yPostprint
000078009 8564_ $$s41149$$uhttps://zaguan.unizar.es/record/78009/files/texto_completo.jpg?subformat=icon$$xicon$$yPostprint
000078009 909CO $$ooai:zaguan.unizar.es:78009$$particulos$$pdriver
000078009 951__ $$a2020-01-17-22:10:25
000078009 980__ $$aARTICLE