Role of Active Site Histidines in the Two Half-Reactions of the Aryl-Alcohol Oxidase Catalytic Cycle

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 <i>k</i>_cat/<i>K</i>_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 O₂ 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 O₂, 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 H₂O₂. No solvent KIE was found for O₂ reduction confirming that the His502 proton transfer does not limit the oxidative half-reaction. However, the small KIE on <i>k</i>_cat/<i>K</i>_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