Rapid kinetics investigations of peracid oxidation of ferric cytochrome P450cam: nature and possible function of compound ES.
Rapid kinetics investigations of peracid oxidation of ferric cytochrome P450cam: nature and possible function of compound ES.
复制标题
三价铁细胞色素 P450cam 过酸氧化的快速动力学研究：化合物 ES 的性质和可能功能。
DOI：
10.1016/j.jinorgbio.2006.09.026
复制
发表时间：
2006
影响因子：
3.9
通讯作者：
Ballou,DavidP
中科院分区：
文献类型：
--
作者：
Spolitak,Tatyana;Dawson,JohnH;Ballou,DavidP
在此之前，我们报道了细胞色素P450cam化合物I（铁基铁+卟啉π-阳离子自由基（FeIV=O/Por+））和化合物ES （FeIV=O/Tyr）在无底物铁酶与间氯过苯甲酸反应中的光谱性质。Spolitak， J.H. Dawson， D.P. Ballou， J. Biol。化学，280(2005)20300-9]。化合物ES是通过分子内电子从附近的酪氨酸转移到化合物I的卟啉π-阳离子自由基上而产生的，并通过rapid-freeze-quench-Mössbauer/EPR光谱对其进行了表征；野生型的Tyr96和Tyr96Phe变体的Tyr75的酪氨酸基自由基被分配[V]。sch<e:1> nemann， F. Lendzian， C. Jung， J. Contzen， A.L. Barra， S.G. Sligar， A.X. Trautwein， J. Biol。化学，279(2004)10919-10930]。在这里，我们报告了对过酸与无底物铁Y75F、Y96F和Y96F/Y75F P450cam变异体反应的快速扫描停流研究，显示了这些活性位点的变化如何影响附近酪氨酸的电子转移和中间体的形成。奇怪的是，这两个单突变体的化合物I和ES的产生率与野生型没有太大差异。与早期的EPR结果相比，Y96F/Y75F变异也显示形成es样物种，但更慢。当底物不存在或结合不当时，化合物I迅速转化为化合物ES，化合物ES可以被还原成H2O和铁P450，从而避免了附近蛋白基团的修饰或活性氧的释放。
Previously, we reported spectroscopic properties of cytochrome P450cam compound I, (ferryl iron plus a porphyrin π-cation radical (FeIV=O/Por+)), as well as compound ES (FeIV=O/Tyr) in reactions of substrate-free ferric enzyme with m-chloroperbenzoic acid [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300-9]. Compound ES arises by intramolecular electron transfer from nearby tyrosines to the porphyrin π-cation radical of Compound I, and has been characterized by rapid-freeze-quench-Mössbauer/EPR spectroscopy; the tyrosyl radical was assigned to Tyr96 for wild type or to Tyr75 for the Tyr96Phe variant [V. Schünemann, F. Lendzian, C. Jung, J. Contzen, A.L. Barra, S.G. Sligar, A.X. Trautwein, J. Biol. Chem. 279 (2004) 10919–10930]. Here we report rapid-scanning stopped-flow studies of the reactions of peracids with substrate-free ferric Y75F, Y96F, and Y96F/Y75F P450cam variants, showing how these active site changes influence electron transfer from nearby tyrosines and affect formation of intermediates. Curiously, rates of generation of Compounds I and ES for both single mutants were not very different from wild type. Contrasting with the earlier EPR results, the Y96F/Y75F variant was also shown to form an ES-like species, but more slowly. When substrate is not present, or is improperly bound, compound I rapidly converts to compound ES, which can be reduced to form H2O and ferric P450, thus avoiding the modification of nearby protein groups or release of reactive oxygen species.