Peroxynitrite-mediated oxidative modifications of complex II: relevance in myocardial infarction.
Increased O(2)(*-) and NO production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. In complex II, oxidative impairment and enhanced tyrosine nitration of the 70 kDa FAD-binding protein occur in the post-ischemic myocardium and are thought to be mediated by peroxynitrite (OONO(-)) in vivo [Chen, Y.-R., et al. (2008) J. Biol. Chem. 283, 27991-28003]. To gain deeper insights into the redox protein thiols involved in OONO(-)-mediated oxidative post-translational modifications relevant in myocardial infarction, we subjected isolated myocardial complex II to in vitro protein nitration with OONO(-). This resulted in site-specific nitration at the 70 kDa polypeptide and impairment of complex II-derived electron transfer activity. Under reducing conditions, the gel band of the 70 kDa polypeptide was subjected to in-gel trypsin/chymotrypsin digestion and then LC-MS/MS analysis. Nitration of Y(56) and Y(142) was previously reported. Further analysis revealed that C(267), C(476), and C(537) are involved in OONO(-)-mediated S-sulfonation. To identify the disulfide formation mediated by OONO(-), nitrated complex II was alkylated with iodoacetamide. In-gel proteolytic digestion and LC-MS/MS analysis were conducted under nonreducing conditions. The MS/MS data were examined with MassMatrix, indicating that three cysteine pairs, C(306)-C(312), C(439)-C(444), and C(288)-C(575), were involved in OONO(-)-mediated disulfide formation. Immuno-spin trapping with an anti-DMPO antibody and subsequent MS was used to define oxidative modification with protein radical formation. An OONO(-)-dependent DMPO adduct was detected, and further LC-MS/MS analysis indicated C(288) and C(655) were involved in DMPO binding. These results offered a complete profile of OONO(-)-mediated oxidative modifications that may be relevant in the disease model of myocardial infarction.
Zhang
Liwen
L
Campus Chemical Instrument Center, Proteomics and Mass Spectrometry Facility, The Ohio State University, Columbus, Ohio 43210, USA.
Chen
Chwen-Lih
CL
Kang
Patrick T
PT
Garg
Vivek
V
Hu
Keli
K
Green-Church
Kari B
KB
Chen
Yeong-Renn
YR
D000595:Amino Acid Sequence; D000818:Animals; D015687:Cell Hypoxia; D003497:Cyclic N-Oxides / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D042963:Electron Transport Complex II / Q000737:chemistry / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D032342:Muscle Cells / Q000378:metabolism / Q000473:pathology; D009203:Myocardial Infarction / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000096:biosynthesis / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014443:Tyrosine / Q000378:metabolism
D003497:Cyclic N-Oxides; D004220:Disulfides; D021122:Protein Subunits; D005182:Flavin-Adenine Dinucleotide; D030421:Peroxynitrous Acid; D014443:Tyrosine; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D042963:Electron Transport Complex II; D003545:Cysteine