Protein thiyl radical mediates S-glutathionylation of complex I

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• 作者：Patrick T. Kang ; Liwen Zhang ; Chwen-Lih Chen ; Jingfeng Chen ; Kari B. Green ; Yeong-Renn Chen
• 关键词：O2&bull ; &minus ; superoxide anion radical ; ETC ; electron transport chain ; SMP ; submitochondrial particle ; Q1 ; ubiquinone-1 ; DMPO ; 5 ; 5-dimethylpyrroline N-oxide ; FMN ; flavin mononucleotide ; GSH ; glutathione ; SDS&ndash ; PAGE ; SDS&ndash ; polyacrylamide gel ele
• 刊名：Free Radical Biology and Medicine
• 出版年：2012
• 出版时间：15 August, 2012
• 年：2012
• 卷：53
• 期：4
• 页码：962-973
• 全文大小：2048 K

文摘

Complex I is a critical site of O₂^?? production and the major host of reactive protein thiols in mitochondria. In response to oxidative stress, complex I protein thiols at the 51- and 75-kDa subunits are reversibly S-glutathionylated. The mechanism of complex I S-glutathionylation is mainly obtained from insight into GSSG-mediated thiol-disulfide exchange, which would require a dramatic decline in the GSH/GSSG ratio. Intrinsic complex I S-glutathionylation can be detected in the rat heart at a relatively high GSH/GSSG ratio (J. Chen et al., J. Biol. Chem. 285:3168-3180, 2010). Thus, we hypothesized that reactive thiyl radical is more likely to mediate protein S-glutathionylation of complex I. Here we employed immuno-spin trapping and tandem mass spectrometry (LC/MS/MS) to test the hypothesis in the 75-kDa subunit from S-glutathionylated complex I. Under the conditions of O₂^?? production in the presence of GSH, we detected complex I S-glutathionylation at Cys-226, Cys-367, and Cys-727 of the 75-kDa subunit. Addition of a radical trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), significantly decreased complex I S-glutathionylation and subsequently increased the protein radical adduct of complex I-DMPO as detected by immunoblotting using an anti-DMPO antibody. LC/MS/MS analysis indicated that Cys-226, Cys-554, and Cys-727 were involved in DMPO binding, confirming that formation of the complex I thiyl radical mediates S-glutathionylation. LC/MS/MS analysis also showed that Cys-554 and Cys-727 were S-sulfonated under conditions of O₂^?? generation in the absence of DMPO. In myocytes (HL-1 cell line) treated with menadione to trigger mitochondrial O₂^?? generation, complex I protein radical and S-glutathionylation were increased. Thus mediation of complex I S-glutathionylation by the protein thiyl radical provides a unique pathway for the redox regulation of mitochondrial function.