Wang Xiaoshi, Ullrich René, Hofrichter Martin, Groves John T
Department of Chemistry, Princeton University, Princeton, NJ 08544; and.
Department of Bio- and Environmental Sciences, International Institute Zittau, Dresden University of Technology, 02763 Zittau, Germany.
Proc Natl Acad Sci U S A. 2015 Mar 24;112(12):3686-91. doi: 10.1073/pnas.1503340112. Epub 2015 Mar 10.
A kinetic and spectroscopic characterization of the ferryl intermediate (APO-II) from APO, the heme-thiolate peroxygenase from Agrocybe aegerita, is described. APO-II was generated by reaction of the ferric enzyme with metachloroperoxybenzoic acid in the presence of nitroxyl radicals and detected with the use of rapid-mixing stopped-flow UV-visible (UV-vis) spectroscopy. The nitroxyl radicals served as selective reductants of APO-I, reacting only slowly with APO-II. APO-II displayed a split Soret UV-vis spectrum (370 nm and 428 nm) characteristic of thiolate ligation. Rapid-mixing, pH-jump spectrophotometry revealed a basic pKa of 10.0 for the Fe(IV)-O-H of APO-II, indicating that APO-II is protonated under typical turnover conditions. Kinetic characterization showed that APO-II is unusually reactive toward a panel of benzylic C-H and phenolic substrates, with second-order rate constants for C-H and O-H bond scission in the range of 10-10(7) M(-1)⋅s(-1). Our results demonstrate the important role of the axial cysteine ligand in increasing the proton affinity of the ferryl oxygen of APO intermediates, thus providing additional driving force for C-H and O-H bond scission.
本文描述了来自皱盖乌芝的血红素硫醇盐过氧合酶（APO）的高铁中间体（APO-II）的动力学和光谱表征。APO-II是通过在硝酰基自由基存在下，使高铁酶与间氯过氧苯甲酸反应生成的，并使用快速混合停流紫外可见（UV-vis）光谱进行检测。硝酰基自由基作为APO-I的选择性还原剂，与APO-II反应缓慢。APO-II呈现出硫醇盐配位特征的分裂Soret紫外可见光谱（370 nm和428 nm）。快速混合、pH跃变分光光度法显示APO-II的Fe(IV)-O-H的碱性pKa为10.0，表明APO-II在典型的周转条件下是质子化的。动力学表征表明，APO-II对一系列苄基C-H和酚类底物具有异常高的反应活性，C-H和O-H键断裂的二级速率常数在10-10(7) M(-1)⋅s(-1)范围内。我们的结果证明了轴向半胱氨酸配体在增加APO中间体高铁氧的质子亲和力方面的重要作用，从而为C-H和O-H键断裂提供了额外的驱动力。
