Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 275, Issue 28, Pages 21409-21415Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M909978199
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Funding
- FIC NIH HHS [R03-TW0099] Funding Source: Medline
- NHLBI NIH HHS [HL54926] Funding Source: Medline
- NIA NIH HHS [AG13966] Funding Source: Medline
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Peroxynitrite (ONOO-), the product of superoxide (O-2(.-)) and nitric oxide ((NO)-N-.) reaction, inhibits mitochondrial respiration and can stimulate apoptosis. Cytochrome c, a mediator of these two aspects of mitochondrial function, thus represents an important potential target of ONOO- during conditions involving accelerated rates of oxygen radical and (NO)-N-. generation. Horse heart cytochrome c(3+) was nitrated by ONOO-, as indicated by spectral changes, Western blot analysis, and mass spectrometry. A dose-dependent loss of cytochrome c(3+) 695 nm absorption occurred, inferring that nitration of a critical heme-vicinal tyrosine (Tyr-67) promoted a conformational change, displacing the Met-80 heme ligand. Nitration was confirmed by cross-reactivity with a specific antibody against 3-nitrotyrosine and by increased molecular mass compatible with the addition of a nitro-(-NO2) group. Mass analysis of tryptic digests indicated the preferential nitration of Tyr-67 among the four conserved tyrosine residues in cytochrome c. Cytochrome c(3+) was more extensively nitrated than cytochrome c(2+) because of the preferential oxidation of the reduced heme by ONOO-. Similar protein nitration patterns were obtained by ONOO- reaction in the presence of carbon dioxide, whereupon secondary nitrating species arise from the decomposition of the nitroso-peroxocarboxylate (ONOOCO2-) intermediate. Peroxynitrite-nitrated cytochrome c displayed significant changes in redox properties, including (a) increased peroxidatic activity, (b) resistance to reduction by ascorbate, and (c) impaired support of state 4-dependent respiration in intact rat heart mitochondria. These results indicate that cytochrome c nitration may represent both oxidative and signaling events occurring during (NO)-N-.- and ONOO--mediated cell injury.
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