Spectroscopic characterization of the iron-oxo intermediate in cytochrome P450
Spectroscopic characterization of the iron-oxo intermediate in cytochrome P450
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DOI：
10.1515/bc.2005.120
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发表时间：
2005-10-01
影响因子：
3.7
通讯作者：
Barra, AL
中科院分区：
文献类型：
--
作者：
Jung, C;Schünemann, V;Barra, AL
与烟热霉中的氯过氧化物酶类比，认为细胞色素 P450 催化循环中中间铁-氧物种的电子结构对应于铁（IV）卟啉-π-阳离子自由基（化合物 1）。然而，我们最近对 P450cam 的研究表明，8 毫秒后，P450 铁与分流通路中的外部氧化剂反应形成酪氨酸自由基和铁 (IV)。目前对 P450BM3 (P450BMP) 血红素结构域的研究显示了类似的结果。除了酪氨酸自由基之外，在 P450BMR 的电子顺磁共振 (EPR) 谱中还发现了色氨酸自由基的贡献。在这里，我们介绍了使用过氧乙酸作为两种 P450 细胞色素的氧化剂产生的冷冻淬灭中间体的比较多频率 EPR（9.6、94 和 285 GHz）和穆斯堡尔光谱研究。 8 毫秒后，两个系统中都出现了氨基酸自由基，而不是所提出的铁（IV）卟啉-π-阳离子自由基，后者可能在更快的时间尺度上瞬时形成。这些发现是针对其他血红素硫醇蛋白进行讨论的。我们的研究表明，芳香族氨基酸的分子内电子转移是这些酶的共同特征。电子转移会猝灭可能瞬时形成的卟啉-π-阳离子自由基，这使得捕获化合物 I 变得极其困难。
From analogy to chloroperoxidase from Caldariomyces fumago, it is believed that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of cytochrome P450 corresponds to an iron(IV) porphyrin-pi-cation radical (compound 1). However, our recent studies on P450cam revealed that after 8 ms a tyrosine radical and iron(IV) were formed in the reaction of ferric P450 with external oxidants in the shunt pathway. The present study on the heme domain of P450BM3 (P450BMP) shows a similar result. In addition to a tyrosine radical, a contribution from a tryptophan radical was found in the electron paramagnetic resonance (EPR) spectra of P450BMR Here we present comparative multi-frequency EPR (9.6, 94 and 285 GHz) and Mossbauer spectroscopic studies on freeze-quenched intermediates produced using peroxy acetic acid as oxidant for both P450 cytochromes. After 8 ms in both systems, amino acid radicals occurred instead of the proposed iron(IV) porphyrin-pi-cation radical, which may be transiently formed on a much faster time scale. These findings are discussed with respect to other heme thiolate proteins. Our studies demonstrate that intramolecular electron transfer from aromatic amino acids is a common feature in these enzymes. The electron transfer quenches the presumably transiently formed porphyrin-pi-cation radical, which makes it extremely difficult to trap compound I.