Cytochrome c nitration by peroxynitrite.
Peroxynitrite (ONOO(-)), the product of superoxide (O(2)) and nitric oxide (.NO) 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 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-(-NO(2)) 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 (ONOOCO(2)(-)) 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- and ONOO(-)-mediated cell injury.
Cassina
A M
AM
Departamento de Bioquimica, Facultad de Medicina and Laboratorio de Enzimologia, Instituto de Quimica Biológica, Facultad de Ciencias, Universidad de la República, 11800 Montevideo, Uruguay.
Hodara
R
R
Souza
J M
JM
Thomson
L
L
Castro
L
L
Ischiropoulos
H
H
Freeman
B A
BA
Radi
R
R
D000818:Animals; D001665:Binding Sites; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism*; D006736:Horses; D007700:Kinetics; D013058:Mass Spectrometry; D008929:Mitochondria, Heart / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D009566:Nitrates / Q000737:chemistry* / Q000494:pharmacology; D016877:Oxidants / Q000737:chemistry / Q000494:pharmacology; D010449:Peptide Mapping; D011487:Protein Conformation; D051381:Rats; D013053:Spectrophotometry; D014357:Trypsin
D003574:Cytochrome c Group; D009566:Nitrates; D016877:Oxidants; C039642:peroxynitric acid; D014357:Trypsin