Protein thiyl radical mediates S-glutathionylation of complex I.
Complex I is a critical site of O(2)(•-) 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 O(2)(•-) 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 O(2)(•-) generation in the absence of DMPO. In myocytes (HL-1 cell line) treated with menadione to trigger mitochondrial O(2)(•-) 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.
Department of Integrative Medical Sciences, College of Medicine, Northeast Ohio Medical University, Rootstown, OH 44272, USA.
Patrick T
PT
Kang
Liwen
L
Zhang
Chwen-Lih
CL
Chen
Jingfeng
J
Chen
Kari B
KB
Green
Yeong-Renn
YR
Chen
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D003497:Cyclic N-Oxides / Q000737:chemistry / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D042967:Electron Transport Complex I / Q000737:chemistry / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000737:chemistry / Q000494:pharmacology; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008929:Mitochondria, Heart / Q000201:enzymology / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D032342:Muscle Cells / Q000187:drug effects / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D018384:Oxidative Stress; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D051381:Rats; D012402:Rotenone / Q000494:pharmacology; D040681:Structural Homology, Protein; D013481:Superoxides / Q000378:metabolism
D003497:Cyclic N-Oxides; D016166:Free Radical Scavengers; D005609:Free Radicals; D009861:Onium Compounds; D010446:Peptide Fragments; D012402:Rotenone; D013481:Superoxides; C007517:diphenyleneiodonium; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D042967:Electron Transport Complex I; D005978:Glutathione; D003545:Cysteine