Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
J Am Chem Soc. 2009 Mar 4;131(8):2971-6. doi: 10.1021/ja808982g.
Cytochromes P450 are ubiquitous heme-containing enzymes that catalyze a wide range of reactions in nature including many oxidation reactions. The active oxidant species in P450 enzymes are widely thought to be iron(IV)-oxo porphyrin radical cations, termed Compound I species, but these intermediates have not been observed under turnover conditions. We prepared Compounds I of the mammalian hepatic P450 enzyme CYP2B4 and three mutants (E301Q, T302A, and F429H) by laser flash photolysis of the Compound II species that, in turn, were prepared by reaction of the resting enzymes with peroxynitrite. The PN treatment resulted in a small amount of nitration of the P450 as determined by mass spectrometry but no change in reactivity of the P450 in a test reaction. CYP2B4 Compound I oxidized benzphetamine to norbenzphetamine in high yield in bulk studies. In direct kinetic studies of benzphetamine oxidations, Compounds I displayed saturation kinetics with similar binding equilibrium constants (K(bind)) for each. The first-order oxidation rate constants (k(ox)) were comparable for Compounds I of CYP2B4, the E301Q mutant, and the T302A mutant, whereas the k(ox) for Compound I of the F429H mutant was reduced by a factor of 2. CYP119 Compound I, studied for comparison purposes, reacted with benzphetamine with a binding constant that was nearly an order of magnitude smaller than that of CYP2B4 but a rate constant that was similar. Substrate binding constants for P450 Compound I are important for controlling overall rates of oxidation reactions, and the intrinsic reactivities of Compounds I from various P450 enzymes are comparable.
细胞色素 P450 是广泛存在的含血红素酶，可催化自然界中多种反应，包括许多氧化反应。P450 酶中的活性氧化剂物种被广泛认为是铁（IV）-氧卟啉自由基阳离子，称为化合物 I 物种，但这些中间体在周转条件下尚未观察到。我们通过激光闪光光解制备了哺乳动物肝 P450 酶 CYP2B4 及其三个突变体（E301Q、T302A 和 F429H）的化合物 I，而化合物 II 则通过酶与过氧亚硝酸盐的反应来制备。PN 处理导致 P450 的少量硝化，这可以通过质谱法来确定，但在测试反应中 P450 的反应性没有变化。CYP2B4 化合物 I 在批量研究中高收率地氧化苯丙胺为去甲苯丙胺。在苯丙胺氧化的直接动力学研究中，化合物 I 显示出相似的结合平衡常数（K(bind)）的饱和动力学。对于 CYP2B4、E301Q 突变体和 T302A 突变体的化合物 I，一级氧化速率常数（k(ox)）是可比的，而 F429H 突变体的化合物 I 的 k(ox)则降低了 2 倍。为了进行比较研究，我们研究了 CYP119 化合物 I，其与苯丙胺的结合常数比 CYP2B4 小一个数量级，但反应速率常数相似。P450 化合物 I 的底物结合常数对于控制氧化反应的总速率很重要，并且各种 P450 酶的化合物 I 的固有反应性是可比的。
