Barr D P, Mason R P
Laboratory of Molecular Biophysics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
J Biol Chem. 1995 May 26;270(21):12709-16. doi: 10.1074/jbc.270.21.12709.
The mechanism for the reaction of cytochrome c with t-butyl hydroperoxide and cumene hydroperoxide was investigated. ESR spin trapping studies using 5,5-dimethyl-1-pyrroline N-oxide were performed to demonstrate the presence of hydroperoxide-derived peroxyl, alkoxyl, and methyl radicals. Computer simulation of the experimental data obtained at various 5,5-dimethyl-1-pyrroline N-oxide concentrations was used to determine the relative contributions of each radical adduct to each composite ESR spectrum. From these analyses, it was concluded that the alkoxyl radical of the hydroperoxide was the initial radical produced, presumably by homolytic scission of the O-O bond by ferric cytochrome c. This was in contrast to a previous ESR study that proposed a heterolytic peroxidase-type mechanism for the reaction of cytochrome c with organic hydroperoxides. Methyl radicals were produced from the beta-scission of the alkoxyl radical. The peroxyl radicals are shown to be secondary products formed from the reaction of oxygen with the methyl radical to produce the methyl peroxyl radical. In separate experiments, visible absorption spectroscopy revealed that the heme center was destroyed during the reaction. Both the heme destruction and production of radical adducts were inhibited by cyanide, presumably due to the formation of a cyanoheme complex.
研究了细胞色素c与叔丁基过氧化氢和异丙苯过氧化氢反应的机制。使用5,5-二甲基-1-吡咯啉N-氧化物进行电子顺磁共振（ESR）自旋捕获研究，以证明存在过氧化氢衍生的过氧自由基、烷氧自由基和甲基自由基。通过对在不同5,5-二甲基-1-吡咯啉N-氧化物浓度下获得的实验数据进行计算机模拟，确定每种自由基加合物对每个复合ESR光谱的相对贡献。从这些分析得出的结论是，过氧化氢的烷氧自由基是最初产生的自由基，可能是由细胞色素c（III）通过O-O键的均裂产生的。这与之前的一项ESR研究形成对比，该研究提出细胞色素c与有机过氧化氢反应的异裂过氧化物酶型机制。甲基自由基是由烷氧自由基的β-断裂产生的。过氧自由基被证明是由氧与甲基自由基反应生成甲基过氧自由基形成的次级产物。在单独的实验中，可见吸收光谱显示反应过程中血红素中心被破坏。血红素的破坏和自由基加合物的产生均受到氰化物的抑制，这可能是由于形成了氰合血红素复合物。
