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

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• 作者：Aitor Hern谩ndez-Ortega ; F谩tima Lucas ; Patricia Ferreira ; Milagros Medina ; Victor Guallar ; Angel T. Mart铆nez
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：August 21, 2012
• 年：2012
• 卷：51
• 期：33
• 页码：6595-6608
• 全文大小：610K
• 年卷期：v.51,no.33(August 21, 2012)
• ISSN：1520-4995

文摘

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鈥? 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 伪-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^{鈥?/sup> 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 k_cat/K_m(Ox), during steady-state oxidation of 伪-deuterated alcohol, suggests that the second proton transfer from N5H is partially limiting, as predicted by the QM/MM simulations.}