The reactions of NO2 with both oxidized and reduced cytochrome c at pH 7.2 and 7.4, respectively, and with N-acetyltyrosine amide and N-acetyltryptophan amide at pH 7.3 were studied by pulse radiolysis at 23 °C. NO2 oxidizes N-acetyltyrosine amide and N-acetyltryptophan amide with rate constants of (3.1±0.3)×10(5) and (1.1±0.1)×10(6) M(-1) s(-1), respectively. With iron(III)cytochrome c, the reaction involves only its amino acids, because no changes in the visible spectrum of cytochrome c are observed. The second-order rate constant is (5.8±0.7)×10(6) M(-1) s(-1) at pH 7.2. NO2 oxidizes iron(II)cytochrome c with a second-order rate constant of (6.6±0.5)×10(7) M(-1) s(-1) at pH 7.4; formation of iron(III)cytochrome c is quantitative. Based on these rate constants, we propose that the reaction with iron(II)cytochrome c proceeds via a mechanism in which 90% of NO2 oxidizes the iron center directly-most probably via reaction at the solvent-accessible heme edge-whereas 10% oxidizes the amino acid residues to the corresponding radicals, which, in turn, oxidize iron(II). Iron(II)cytochrome c is also oxidized by peroxynitrite in the presence of CO2 to iron(III)cytochrome c, with a yield of ~60% relative to peroxynitrite. Our results indicate that, in vivo, NO2 will attack preferentially the reduced form of cytochrome c; protein damage is expected to be marginal, the consequence of formation of amino acid radicals on iron(III)cytochrome c.