Detection and kinetic characterization of a highly reactive heme-thiolate peroxygenase compound I.
Detection and kinetic characterization of a highly reactive heme-thiolate peroxygenase compound I.
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DOI：
10.1021/ja3049223
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发表时间：
2012-08-08
影响因子：
15
通讯作者：
Groves JT
中科院分区：
文献类型：
--
作者：
Wang X;Peter S;Kinne M;Hofrichter M;Groves JT
来自绿草草的细胞外血红素硫酸盐过氧酶（AaeAPO）已被证明使用过氧化氢作为末端氧化剂羟基化烷烃和许多其他底物。我们描述了AaeAPO化合物I与mCPBA反应时的形成和分解动力学。AaeAPO-I （361,694 nm）的紫外可见光谱特征与氯过氧化物酶- i和最近报道的细胞色素P450-I相似。在pH 5.0, 4°C条件下，AaeAPO-I生成的二级速率常数为1.0（±0.4）×107 M−1s−1。相对较慢的分解速率为1.4（±0.03）s−1，可以测量其对底板的反应性。观察到的速率常数k2′跨越5个数量级，与强碳氢键底物的键解离焓呈线性相关，对数k2′与BDE斜率为~ 0.4。然而，在90 kcal/mol以下，羟基化速率对C-H BDE不敏感，类似于t-丁氧基自由基的行为。Brønsted-Evans-Polanyi图的形状和斜率表明，较强的碳氢键处于对称过渡态，较弱的碳氢键在早期过渡态的速率受到熵控制。AaeAPO-II fevo - h BDE估计为~ 103 kcal/mol。所有结果都支持高活性AaeAPO氧铁（IV）卟啉自由基阳离子中间体的形成，该中间体是这些羟基化反应中的活性氧。
The extracellular heme-thiolate peroxygenase from Agrocybe aegerita (AaeAPO) has been shown to hydroxylate alkanes and numerous other substrates using hydrogen peroxide as the terminal oxidant. We describe the kinetics of formation and decomposition of AaeAPO compound I upon its reaction with mCPBA. The UV–vis spectral features of AaeAPO–I (361, 694 nm) are similar to those of chloroperoxidase–I and the recently–described cytochrome P450–I. The second–order rate constant for AaeAPO–I formation was 1.0 (±0.4) ×107 M−1s−1 at pH 5.0, 4 °C. The relatively slow decomposition rate, 1.4 (±0.03) s−1, allowed the measurement of its reactivity toward a panel of substrates. The observed rate constants, k2’, spanned five orders of magnitude and correlated linearly with bond dissociation enthalpies of strong C–H bond substrates with a log k2’ vs. BDE slope of ~ 0.4. However, the hydroxylation rate was insensitive to C–H BDE below 90 kcal/mol, similar to the behavior of the t-butoxy radical. The shape and slope of the Brønsted-Evans-Polanyi plot indicate a symmetrical transition state for the stronger C–H bonds and suggest entropy control of the rate in an early transition state for weaker C–H bonds. The AaeAPO–II FeIVO–H BDE was estimated to be ~ 103 kcal/mol. All results support the formation of a highly reactive AaeAPO oxoiron(IV) porphyrin radical cation intermediate that is the active oxygen species in these hydroxylation reactions.