title: Role of active site histidines in the two half-reactions of the aryl-alcohol oxidase catalytic cycle.
abstract: The crystal structure of aryl-alcohol oxidase (AAO), a flavoenzyme involved in lignin degradation, reveals two active-site histidines, whose role in the two enzyme half-reactions was investigated. The redox state of flavin during turnover of the variants obtained show a stronger histidine involvement in the reductive than in the oxidative half-reaction. This was confirmed by the k(cat)/K(m(Al)) and reduction constants that are 2-3 orders of magnitude decreased for the His546 variants and up to 5 orders for the His502 variants, while the corresponding O(2) constants only decreased up to 1 order of magnitude. These results confirm His502 as the catalytic base in the AAO reductive half-reaction. The solvent kinetic isotope effect (KIE) revealed that hydroxyl proton abstraction is partially limiting the reaction, while the α-deuterated alcohol KIE showed a stereoselective hydride transfer. Concerning the oxidative half-reaction, directed mutagenesis and computational simulations indicate that only His502 is involved. Quantum mechanical/molecular mechanical (QM/MM) reveals an initial partial electron transfer from the reduced FADH(-) to O(2), without formation of a flavin-hydroperoxide intermediate. Reaction follows with a nearly barrierless His502H(+) proton transfer that decreases the triplet/singlet gap. Spin inversion and second electron transfer, concomitant with a slower proton transfer from flavin N5, yields H(2)O(2). No solvent KIE was found for O(2) reduction confirming that the His502 proton transfer does not limit the oxidative half-reaction. However, the small KIE on k(cat)/K(m(Ox)), during steady-state oxidation of α-deuterated alcohol, suggests that the second proton transfer from N5H is partially limiting, as predicted by the QM/MM simulations.
authors: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
authors: Aitor
authors: A
authors: Hernández-Ortega
authors: Fátima
authors: F
authors: Lucas
authors: Patricia
authors: P
authors: Ferreira
authors: Milagros
authors: M
authors: Medina
authors: Victor
authors: V
authors: Guallar
authors: Angel T
authors: AT
authors: Martínez
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D001592:Benzyl Alcohols / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D005415:Flavins / Q000378:metabolism; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D010084:Oxidation-Reduction; D020076:Pleurotus / Q000201:enzymology; D011789:Quantum Theory
chemical_list: D001592:Benzyl Alcohols; D005415:Flavins; D006639:Histidine; D000429:Alcohol Oxidoreductases; C077759:aryl-alcohol oxidase