Reaction of cytochrome P450 with cumene hydroperoxide: ESR spin trapping evidence for the homolytic scission of the peroxide O-O bond by ferric cytochrome P450 1A2
Reaction of cytochrome P450 with cumene hydroperoxide: ESR spin trapping evidence for the homolytic scission of the peroxide O-O bond by ferric cytochrome P450 1A2
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DOI：
10.1021/tx9501501
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发表时间：
1996-01-01
影响因子：
4.1
通讯作者：
Mason, RP
中科院分区：
文献类型：
--
作者：
Barr, DP;Martin, MV;Mason, RP
用ESR自旋捕捉法研究了兔细胞色素P450(P450)1A2与过氧化氢异丙苯的反应。异丙苯过氧化氢衍生的过氧基、烷氧基和碳心自由基在反应过程中生成并被捕获。各自由基加合物对复合ESR谱的相对贡献受自旋陷阱浓度的影响。用计算机模拟了不同5，5-二甲基-1-吡咯啉N-氧化物(DMPO)浓度下的实验数据，定量计算了每个自由基加合物对复合ESR谱的贡献。烷氧基是反应过程中产生的初始自由基。用2-甲基-2-亚硝基丙烷进行的实验确定了碳中心加合物为甲基自由基、羟甲基自由基和二次碳中心自由基的加合物。该反应不需要NADPH-细胞色素P450还原酶或NADPH。结果表明，该反应涉及过氧化氢O-O键的初始均解断裂，生成了异丙氧基。甲基自由基是由异丙氧基的β-裂解生成的。在没有分子氧的情况下，没有观察到过氧基加合物。我们的结论是，在有氧条件下检测到的DMPO过氧基加合物是由于甲基自由基与氧反应生成的甲基过氧基。在较高的P450浓度下，还检测到蛋白质衍生的自由基加合物。
ESR spin trapping was used to investigate the reaction of rabbit cytochrome P450 (P450) 1A2 with cumene hydroperoxide. Cumene hydroperoxide-derived peroxyl, alkoxyl, and carbon-centered radicals were formed and trapped during the reaction. The relative contributions of each radical adduct to the composite ESR spectrum were influenced by the concentration of the spin trap. Computer simulation of the experimental data obtained at various 5,5-dimethyl-1-pyrroline N-oxide (DMPO) concentrations was used to quantitate the contributions of each radical adduct to the composite ESR spectrum. The alkoxyl radical was the initial radical produced during the reaction. Experiments with 2-methyl-2-nitrosopropane identified the carbon-centered adducts as those of the methyl radical, hydroxymethyl radical, and a secondary carbon-centered radical. The reaction did not require NADPH-cytochrome P450 reductase or NADPH. It is concluded that the reaction involves the initial homolytic scission of the peroxide O-O bond to produce the cumoxyl radical. Methyl radicals were produced from the beta-scission of the cumoxyl radical. The peroxyl adduct was not observed in the absence of molecular oxygen. We conclude that the DMPO peroxyl radical adduct detected in the presence of oxygen was due to the methylperoxyl radical formed by the reaction of the methyl radical with oxygen. At a higher P450 concentration, a protein-derived radical adduct was also detected.