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.
Campus Chemical Instrument Center, Proteomics and Mass Spectrometry Facility, The Ohio State University, Columbus, Ohio 43210, USA.
Liwen
L
Zhang
Chwen-Lih
CL
Chen
Patrick T
PT
Kang
Vivek
V
Garg
Keli
K
Hu
Kari B
KB
Green-Church
Yeong-Renn
YR
Chen
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