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 f
unction, thus represents an important potential target of ONOO- during cond
itions involving accelerated rates of oxygen radical and (NO)-N-. generatio
n. Horse heart cytochrome c(3+) was nitrated by ONOO-, as indicated by spec
tral changes, Western blot analysis, and mass spectrometry. A dose-dependen
t loss of cytochrome c(3+) 695 nm absorption occurred, inferring that nitra
tion of a critical heme-vicinal tyrosine (Tyr-67) promoted a conformational
change, displacing the Met-80 heme ligand. Nitration was confirmed by cros
s-reactivity with a specific antibody against 3-nitrotyrosine and by increa
sed molecular mass compatible with the addition of a nitro-(-NO2) group. Ma
ss 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 pre
ferential 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 h
eart mitochondria. These results indicate that cytochrome c nitration may r
epresent both oxidative and signaling events occurring during (NO)-N-.- and
ONOO--mediated cell injury.