Role of Substrate Positioning in the Catalytic Reaction of 4‑Hydroxyphenylpyruvate DioxygenaseA QM/MM Study

Ring
hydroxylation and coupled rearrangement reactions catalyzed
by 4-hydroxy­phenyl­pyruvate dioxygenase were studied with
the QM/MM method ONIOM­(B3LYP:AMBER). For electrophilic attack of the
ferryl species on the aromatic ring, five channels were considered:
attacks on the three ring atoms closest to the oxo ligand (C1, C2,
C6) and insertion of oxygen across two bonds formed by them (C1–C2,
C1–C6). For the subsequent migration of the carboxymethyl substituent,
two possible directions were tested (C1→C2, C1→C6),
and two different mechanisms were sought (stepwise radical, single-step
heterolytic). In addition, formation of an epoxide (side)­product and
benzylic hydroxylation, as catalyzed by the closely related hydroxymandelate
synthase, were investigated. From the computed reaction free energy
profiles it follows that the most likely mechanism of 4-hydroxyphenylpyruvate
dioxygenase involves electrophilic attack on the C1 carbon of the
ring and subsequent single-step heterolytic migration of the substituent.
Computed values of the kinetic isotope effect for this step are inverse,
consistent with available experimental data. Electronic structure
arguments for the preferred mechanism of attack on the ring are also
presented.

本研究采用量子力学/分子力学（QM/MM）方法ONIOM(B3LYP:AMBER)，对4-羟苯丙酮酸双加氧酶（4-hydroxyphenylpyruvate dioxygenase）催化的芳香环羟基化及偶联重排反应展开了系统性研究。针对铁氧物种（ferryl species）对芳环的亲电进攻过程，共考量五类反应通道：分别为进攻距氧配体最近的三个环碳原子（C1、C2、C6），以及通过该三原子形成的两个共价键（C1–C2、C1–C6）插入氧原子的路径。针对后续羧甲基取代基的迁移过程，本研究测试了两种可能的迁移方向（C1→C2、C1→C6），并探索了两类不同的反应机理：分步自由基机理（stepwise radical）与单步异裂机理（single-step heterolytic）。此外，本研究还针对结构密切相关的羟扁桃酸合酶（hydroxymandelate synthase）催化的环氧化物（epoxide）副产物生成反应及苄位羟基化反应进行了探究。基于计算得到的反应自由能剖面（reaction free energy profiles）可知，4-羟苯丙酮酸双加氧酶最可能的催化机理为：先对芳环的C1碳原子发生亲电进攻，随后伴随取代基的单步异裂迁移。该步骤的计算动力学同位素效应（kinetic isotope effect）数值为反向动力学同位素效应，与已公开的实验数据相符。本研究同时提出了针对优势芳环进攻机理的电子结构分析依据。