Protein oxidation of cytochrome c by reactive halogen species enhances its peroxidase activity
Protein oxidation of cytochrome c by reactive halogen species enhances its peroxidase activity
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DOI：
10.1074/jbc.m200709200
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发表时间：
2002-08-16
影响因子：
4.8
通讯作者：
Mason, RP
中科院分区：
文献类型：
--
作者：
Chen, YR;Deterding, LJ;Mason, RP
反应性卤素物种（RHS; X-2和HOX，其中X代表Cl、Br或I）是由中性粒细胞活化及其伴随的呼吸爆发介导的代谢物。我们用电喷雾质谱和电子自旋共振（ESR）研究了RHS与线粒体细胞色素c（cyt c）的相互作用。当纯化的细胞色素c与过量的次氯酸（HOCl）在pH 7.4反应时，细胞色素c的过氧化物酶活性分别增加了4.5-，6.9-和8.6倍，摩尔比（HOCl/细胞色素c）为2，4和8，分别。与天然cyt c相比，从HOCl处理的cyt c获得的质谱显示，氧共价结合到蛋白质中，如分子离子m/z = 12，360（cyt c）、12，376（cyt c + O）和12，392（cyt c + 20）所示。使用串联质谱法，一种肽（从HOCl处理的细胞色素c的胰蛋白酶解物中获得）对应于氨基酸序列MIFAGIK，其中含有与血红素结合的甲硫氨酸，被鉴定为参与氧掺入。氧掺入的位置被明确地确定为甲硫氨酸残基，这表明HOCl氧化血红素配体（Met-80）导致Cytc的过氧化物酶活性增强。在亚硝基自旋捕获剂2-甲基-2-亚硝基丙烷（MNP）存在下，HOCl氧化的Cytc与H2 O2反应时，ESR谱产生了比天然Cytc更多的固定化MNP/酪氨酰加合物。在H2 O2存在下，HOCl氧化的Cytc的过氧化物酶活性表现出增加的能力，氧化酪氨酸为酪氨酰自由基直接测量的快速流动ESR。用不同量的H2 O2滴定天然cyt c和HOCl氧化的cyt c表明，后者具有降低的H2 O2的表观K-m，暗示cyt c的蛋白质氧化增加其对H2 O2的可及性。HOCl-氧化的细胞色素c也显示出受损的能力，以支持氧消耗的纯化的线粒体细胞色素c氧化酶，这表明蛋白质氧化的细胞色素c可能会打破电子传递链，抑制线粒体中的能量转导。
Reactive halogen species (RHS; X-2 and HOX, where X represents Cl, Br, or I) are metabolites mediated by neutrophil activation and its accompanying respiratory burst. We have investigated the interaction between RHS and mitochondrial cytochrome c (cyt c) by using electrospray mass spectrometry and electron spin resonance (ESR). When the purified cyt c was reacted with an excess amount of hypochlorous acid (HOC1) at pH 7.4, the peroxidase activity of cyt c was increased by 4.5-, 6.9-, and 8.6-fold at molar ratios (HOCl/cyt c) of 2, 4, and 8, respectively. In comparison with native cyt c, the mass spectra obtained from the HOCl-treated cyt c revealed that oxygen is covalently incorporated into the protein as indicated by molecular ions of m/z = 12,360 (cyt c), 12,376 (cyt c + O), and 12,392 (cyt c + 20). Using tandem mass spectrometry, a peptide (obtained from the tryptic digests of HOCl-treated cyt c) corresponding to the amino acid sequence MIFAGIK, which contains the methionine that binds to the heme, was identified to be involved in the oxygen incorporation. The location of the oxygen incorporation was unequivocally determined to be the methionine residue, suggesting that the oxidation of heme ligand (Met-80) by HOCl results in the enhancement of peroxidase activity of cyt c. ESR spectroscopy of HOCl-oxidized cyt c, when reacted with H2O2 in the presence of the nitroso spin trap 2-methyl-2-nitrosopropane (MNP), yielded more immobilized MNP/tyrosyl adduct than native cyt c. In the presence of H2O2, the peroxidase activity of HOCl-oxidized cyt c exhibited an increasing ability to oxidize tyrosine to tyrosyl radical as measured directly by fast flow ESR. Titration of both native cyt c and HOCl-oxidized cyt c with various amounts of H2O2 indicated that the latter has a decreased apparent K-m for H2O2, implicating that protein oxidation of cyt c increases its accessibility to H2O2. HOCl-oxidized cyt c also displayed an impaired ability to support oxygen consumption by the purified mitochondrial cytochrome c oxidase, suggesting that protein oxidation of cyt c may break the electron transport chain and inhibit energy transduction in mitochondria.