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ONIOM(B3LYP:AMBER)量子力学/分子力学（QM/MM）联用方法，对4-羟苯基丙酮酸双加氧酶（4-hydroxyphenylpyruvate dioxygenase）催化的芳环羟基化与耦合重排反应展开了系统探究。针对铁氧物种（ferryl species）对芳香环的亲电进攻过程，本研究考量了五类反应路径：分别进攻距离氧配体最近的三个环碳原子（C1、C2、C6），以及在这三个原子形成的两条C1–C2、C1–C6键间插入氧原子。对于后续羧甲基取代基的迁移步骤，本研究测试了C1→C2、C1→C6两种可能的迁移方向，并探索了分步自由基、单步异裂两类不同的反应机理。此外，本研究还针对由亲缘关系相近的羟扁桃酸合酶（hydroxymandelate synthase）催化的环氧化物副产物生成反应与苄基羟基化反应进行了研究。通过计算得到的反应自由能剖面可知，4-羟苯基丙酮酸双加氧酶最可能的催化机理为：先对芳环的C1碳原子进行亲电进攻，随后发生单步异裂的取代基迁移过程。该步骤的计算动力学同位素效应（kinetic isotope effect）数值为逆同位素效应，与已公开的实验数据相符。本研究还提出了针对优势芳环进攻机理的电子结构相关论证。