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Marked difference in cytochrome c oxidation mediated by HO and/or O_2~(·-) free radicals in vitro

机译:HO和/或O_2〜(·-)自由基介导的细胞色素c氧化的显着差异

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Cytochrome c (cyt c) is an electron carrier involved in the mitochondrial respiratory chain and a critical protein in apoptosis. The oxidation of cytochrome c can therefore be relevant biologically. We studied whether cytochrome c underwent the attack of reactive oxygen species (ROS) during ionizing irradiation-induced oxidative stress. ROS were generated via water radiolysis under ionizing radiation (IR) in vitro. Characterization of oxidation was performed by mass spectrometry, after tryptic digestion, and UV-visible spectrophotometry. When both hydroxyl and superoxide free radicals were generated during water radiolysis, only five tryptic peptides of cyt c were reproducibly identified as oxidized according to a relation that was dependent of the dose of ionizing radiation. The same behavior was observed when hydroxyl free radicals were specifically generated (N_2O-saturated solutions). Specific oxidation of cyt c by superoxide free radicals was performed and has shown that only one oxidized peptide (MIFAGIK + 16), corresponding to the oxidation of Met80 into methionine sulfoxide, exhibited a radiation dose-dependent formation. In addition, the enzymatic site of cytochrome c was sensitive to the attack of both superoxide and hydroxyl radicals as observed through the reduction of Fe~(3+), the degradation of the protoporphyrin IX and the oxidative disruption of the Met80-Fe~(3+) bond. Noteworthy, the latter has been involved in the conversion of cyt c to a peroxidase. Finally, Met80 appears as the most sensitive residue towards hydroxyl but also superoxide free radicals mediated oxidation.
机译:细胞色素c(cyt c)是一种参与线粒体呼吸链的电子载体,是细胞凋亡中的关键蛋白。因此,细胞色素c的氧化在生物学上可能是相关的。我们研究了细胞色素c在电离辐射诱导的氧化应激过程中是否经历了活性氧(ROS)的攻击。 ROS是在体外电离辐射(IR)下通过水的裂解作用产生的。胰蛋白酶消化后,通过质谱法和紫外可见分光光度法进行氧化的表征。当在水辐射分解过程中同时产生羟基自由基和超氧化物自由基时,根据依赖于电离辐射剂量的关系,可再现地将cyt c的五个胰蛋白酶肽鉴定为被氧化。当专门生成羟基自由基(N_2O饱和溶液)时,观察到相同的行为。 cyt c被超氧化物自由基特异性氧化,结果表明只有一个氧化的肽(MIFAGIK + 16),对应于Met80氧化成蛋氨酸亚砜,表现出辐射剂量依赖性。此外,通过还原Fe〜(3+),原卟啉IX的降解和Met80-Fe〜(的氧化破坏)观察到的细胞色素c的酶促位点对超氧化物和羟基自由基的攻击都敏感。 3+)键。值得注意的是,后者参与了将cyt c转化为过氧化物酶。最后,Met80似乎是对羟基最敏感的残基,也是超氧自由基介导的氧化反应。

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