doi:10.1039/C7CP05904CengCarro, JuanMartínez-Julve, MartaMedina, MilagrosMartínez, Angel T.Ferreira, PatriciaProtein dynamics promote hydride tunnelling in substrate oxidation by aryl-alcohol oxidaseART-2020-102550The temperature dependence of hydride transfer from the substrate to the N5 of the FAD cofactor during the reductive half-reaction of Pleurotus eryngii aryl-alcohol oxidase (AAO) is assessed here. Kinetic isotope effects on both the pre-steady state reduction of the enzyme and its steady-state kinetics, with differently deuterated substrates, suggest an environmentally-coupled quantum-mechanical tunnelling process. Moreover, those kinetic data, along with the crystallographic structure of the enzyme in complex with a substrate analogue, indicate that AAO shows a pre-organized active site that would only require the approaching of the hydride donor and acceptor for the tunnelled transfer to take place. Modification of the enzyme''s active-site architecture by replacement of Tyr92, a residue establishing hydrophobic interactions with the substrate analogue in the crystal structure, in the Y92F, Y92L and Y92W variants resulted in different temperature dependence patterns that indicated a role of this residue in modulating the transfer reaction.2020http://zaguan.unizar.es/record/7542410.1039/C7CP05904Chttp://zaguan.unizar.es/record/75424oai:zaguan.unizar.es:75424info:eu-repo/grantAgreement/EC/FP7/613549/EU/Optimized oxidoreductases for medium and large scale industrial biotransformations/INDOXinfo:eu-repo/grantAgreement/EC/H2020/720297/EU/New enzymatic oxidation/oxyfunctionalization technologies for added value bio-based products/EnzOx2This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 720297-EnzOx2info:eu-repo/grantAgreement/ES/MINECO/BIO2016-75183-Pinfo:eu-repo/grantAgreement/ES/MINECO/NOESIS-BIO2014-56388-RPhysical Chemistry Chemical Physics 19 (2020), 28666All rights reservedhttp://www.europeana.eu/rights/rr-f/info:eu-repo/semantics/openAccess