Characterization of the microsomal cytochrome P4502B4O2 activation intermediates by cryoreduction and electron paramagnetic resonance
Characterization of the microsomal cytochrome P4502B4O2 activation intermediates by cryoreduction and electron paramagnetic resonance
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DOI：
10.1021/bi800926x
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发表时间：
2008-09-09
期刊：
影响因子：
2.9
通讯作者：
Hoffrnan, Brian M.
中科院分区：
文献类型：
--
作者：
Davydov, Roman;Razeghifard, Reza;Hoffrnan, Brian M.
在-40℃下制备了细胞色素P450 - 2B4 （2B4）的氧-亚铁配合物，并在77 K下进行了单电子冷还原。电子顺磁共振（EPR）显示，在这两种情况下，观察到的冷冻还原产物都是氢过氧铁血红素，这表明微粒体P450包含一个有效的远端口袋质子传递网络。在没有底物的情况下，观察到两种不同的氢过氧铁血红素信号，反映了在含氧铁前驱体中存在两种主要构象底态。当BHT结合时，只观察到一种物种，表明活性位点更有序。BHT结合还改变了氢过氧铁2B4中间体的g张量分量，表明底物调节了该中间体的性质。在>= 175 K下对低温还原叔铁2B4配合物进行步进退火，导致氢过氧铁2B4的损失和高自旋铁2B4的平行出现；液相色谱-串联质谱（LC-MS/MS）分析表明，在此过程中BHT被定量转化为两种产物：羟甲基BHT(1)和3-羟基叔丁基BHT(2)。这表明低温还原制备的氢过氧铁具有催化活性，退火后观察到的高自旋态含有BHT单氧合的酶结合产物。低温还原退火的产物比（6.2/1）与常温下的产物比（2.5/1）有显著差异。这些发现表明，相对于氧化物质，底物在低温下保持更牢固，并且/或者FeOOH的解离在低温下被抑制。与环境条件下的实验一样，失活的f429h2b4突变体没有观察到产物的形成。
The oxy-ferrous complex of cytochrome P450 2B4 (2B4) has been prepared at -40 degrees C with and without bound substrate [butylated hydroxytoluene (BHT)] and radiolytically one-electron cryoreduced at 77 K. Electron paramagnetic resonance (EPR) shows that in both cases the observed product of cryoreduction is the hydroperoxo-ferriheme species, indicating that the microsomal P450 contains an efficient distal-pocket proton-delivery network. In the absence of substrate, two distinct hydroperoxo-ferriheme signals are observed, reflecting the presence of two major conformational substates in the oxy-ferrous precursor. Only one species is observed when BHT is bound, indicating a more ordered active site. BHT binding also changes the g-tensor components of the hydroperoxo-ferric 2B4 intermediate, indicating that the substrate modulates the properties of this intermediate. Step annealing the cryoreduced tertiary 2B4 complex at >= 175 K causes the loss of hydroperoxo-ferric 2B4 and the parallel appearance of high-spin ferric 2B4; liquid chromatography-tandem mass spectroscopy (LC-MS/MS) analysis shows that in this process BHT is quantitatively converted to two products, hydroxymethyl BHT (1) and 3-hydroxy-tert-butyl BHT (2). This implies that the hydroperoxo-ferric 2B4 prepared by cryoreduction is catalytically active and that the high-spin state observed after annealing contains an enzyme-bound product of BHT monooxygenation. The ratio of products generated during cryoreduction and annealing (6.2/1) is significantly different from the ratio (2.5/1) at ambient temperature. These findings Suggest that substrate is held more rigidly relative to the oxidizing species at low temperatures and/or that dissociation of FeOOH is inhibited at low temperature. As in experiments under ambient conditions, product formation is not observed with the inactive F429H 2B4 mutant.