Cytochrome c nitration by peroxynitrite

Abstract

Peroxynitrite (ONOO-), the product of superoxide (O2·-) 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 c3+ was nitrated by ONOO-, as indicated by spectral changes, Western blot analysis, and mass spectrometry. A dose-dependent loss of cytochrome c3+ 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 c3+ was more extensively nitrated than cytochrome c2+ 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 occuring during ·No- and ONOO-- mediated cell injury.