Characterization of adducts of ethanol metabolites with cytochrome c
Characterization of adducts of ethanol metabolites with cytochrome c
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DOI：
10.1111/j.1530-0277.1999.tb04020.x
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发表时间：
1999-01-01
影响因子：
3.2
通讯作者：
Israel, Y
中科院分区：
文献类型：
--
作者：
Anni, H;Israel, Y
细胞色素c（cyt c）存在于所有哺乳动物细胞的线粒体中，其中过氧化氢作为电子传递链的副产物产生。在过氧化物存在下，细胞色素c在Tyr残基处产生铁酰血红素和自由基（巴尔等人，1996年）。这些自由基可以转移到蛋白质内的Trp残基或转移到含Tyr和Trp的肽（Deterding等人，1998年）。我们报告说，乙醇添加到这个系统的细胞色素c加上过氧化物的结果在替换的Tyr/色氨酸自由基的1-羟乙基自由基（HER），和共价结合的每摩尔的细胞色素c高达10摩尔的乙醇。在不存在外源性添加的过氧化物，乙醇掺入细胞色素c也达到与乙醇诱导的细胞色素P-4502 E1同工酶的重建系统。与肌红蛋白和脱辅基肌红蛋白的比较研究表明，血红素是必要的乙醇加合的蛋白质发生。通过质谱法对加合的cyt c的胰蛋白酶消化级分进行的结构分析与在Lys残基上具有1至3个乙醛部分的几种肽和三种不同的含Tyr/Trp的肽一致：P[28-53]，P[56-73]，P[73-91]携带一至两个HER。cyt c的X射线晶体结构显示Tyr/加合肽中的色氨酸残基与血红素非常接近。总之，我们的数据表明，乙醇代谢产物烷基化细胞色素c氧化应激和指向HER-酪氨酸/色氨酸加合物作为合理的标志物酒精中毒。
Cytochrome c (cyt c) is found in the mitochondria of all mammalian cells where hydrogen peroxide is produced as a byproduct of the electron transport chain. In the presence of peroxide cyt c generates a ferryl heme and radicals at Tyr residues (Barr et al., 1996). These radicals can be transferred to Trp residues within the protein or to Tyr- and Trp-containing peptides (Deterding et al., 1998). We report that addition of ethanol to this system of cyt c plus peroxide results in replacement of the Tyr/Trp radicals by 1-hydroxyethyl radicals (HER), and covalent binding of up to 10 mol of ethanol per mol of cyt c. In the absence of exogenously added peroxide, ethanol incorporation to cyt c is attained also with a reconstituted system of the ethanol-inducible cytochrome P-4502E1 isozyme. Comparative studies with myoglobin and apomyoglobin suggest that the heme is necessary for ethanol adduction of the protein to occur. Structural analysis by mass spectrometry of the tryptic digestion fractions of adducted cyt c is consistent with several peptides bearing one-to-three acetaldehyde moieties on Lys residues, and three distinct Tyr/ Trp-containing peptides: P[28-53], P[56-73], P[73-91] carrying one-to-two HER. The x-ray crystallographic structure of cyt c shows that the Tyr/Trp residues in the adducted peptides are in close proximity to the heme. In conclusion, our data show that ethanol metabolites alkylate cyt c under oxidative stress and point to HER-Tyr/Trp adducts as plausible markers of alcoholism.