McCarty Kevin D, Tateishi Yasuhiro, Guengerich F Peter
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
J Biol Chem. 2025 Jul;301(7):110378. doi: 10.1016/j.jbc.2025.110378. Epub 2025 Jun 14.
The catalytic cycle of cytochrome P450 (P450) enzymes involves ferric peroxide anion (FeO, Compound 0) and perferryl oxygen (FeO, Compound I) intermediates. Compound I is generally viewed as responsible for most P450-catalyzed oxidations, but Compound 0 has been implicated in the oxidation of some carbonyl compounds, particularly deformylation reactions. We considered the hypothesis that Compound 0 could also attack other electrophilic carbon atoms and accordingly positioned keto groups at preferred hydroxylation sites of substrates for two P450s with well-defined catalytic reactions, bacterial P450 (102A1), and human P450 11A1. The predicted products of Compound I and Compound 0 reactions were analyzed. With the normally preferred ω-1 site blocked, P450 oxidized 12-oxotridecanoic acid (12-oxo C13:0) only at the ω-2 position (yielding 11-hydroxy,12-oxotridecanoic acid), indicative of a Compound I oxidation. P450 11A1 is highly selective for catalyzing the 22R-hydroxylation of cholesterol (and some other sterols) in the first step of its overall side-chain cleavage reaction. With 22-oxocholesterol as the substrate, P450 11A1 (slowly) generated only 23-hydroxy,22-oxocholesterol, indicative of Compound I oxidation. Neither P450 generated the products expected from nucleophilic Compound 0 reactions. We conclude that the strategic placement of electrophilic oxo substituents at sites of substrate hydroxylation failed to divert the oxidation mechanism to a Compound 0 pathway with either enzyme. Instead, the Compound I mechanism-blocked at the preferred reaction site-was redirected to neighboring carbons, suggesting that the basis for Compound 0-mediated reactions lies in chemical properties of the enzyme rather than those of the substrate.
细胞色素P450（P450）酶的催化循环涉及过氧化铁阴离子（FeO，化合物0）和过氧亚铁氧基（FeO，化合物I）中间体。化合物I通常被认为是大多数P450催化氧化反应的原因，但化合物0与某些羰基化合物的氧化反应有关，特别是脱甲酰基反应。我们考虑了这样一个假设，即化合物0也可以攻击其他亲电碳原子，并因此在具有明确催化反应的两种P450（细菌P450（102A1）和人P450 11A1）的底物的优选羟基化位点处定位酮基。分析了化合物I和化合物0反应的预测产物。由于正常优选的ω-1位点被阻断，P450仅在ω-2位置氧化12-氧代十三烷酸（12-氧代C13:0）（生成11-羟基-12-氧代十三烷酸），这表明是化合物I氧化反应。P450 11A1在其整个侧链裂解反应的第一步中对催化胆固醇（和其他一些甾醇）的22R-羟基化具有高度选择性。以22-氧代胆固醇为底物，P450 11A1（缓慢地）仅生成23-羟基-22-氧代胆固醇，这表明是化合物I氧化反应。两种P450均未生成亲核化合物0反应预期的产物。我们得出结论，在底物羟基化位点处亲电氧代取代基的策略性定位未能将氧化机制转向任何一种酶的化合物0途径。相反，在优选反应位点被阻断的化合物I机制被重定向到相邻的碳原子，这表明化合物0介导反应的基础在于酶的化学性质而非底物的化学性质。
