Mitochondrial respiratory chain is the main intracellular site of superoxide radical (O₂.-) production. Nitric oxide (NO^.) a diffusible free radical, is produced by the nitric oxide synthases or can be even produced inside mitochondria. Superoxide and NO. react at diffusion controlled-rates, to yield peroxynitrite (ONOO^-), which can efficiently oxidize and nitrate proteins. Cytochrome c (Cyt c) is a heme-protein that participates in both mitochondrial electron transport and apoptosome activation. The Cyt c porphyrin is covalently bound to C14 and C17, with the 5^th and 6^th coordination positions of the heme iron interacting with H18 and M80, respectively. Cyt c contains four highly conserved tyrosines, Y48, 67, 74 and 97, two close to the heme and two exposed to the solvent. We have previously observed that Cyt c is nitrated by both ONOO- and nitrite plus hydrogen peroxide (NO₂^- + H₂O₂). Herein, we report that low ONOO^- fluxes yield mononitrated Cyt c species (Y74 and Y97) whereas at higher concentrations heme-adjacent Y67 was preferentially nitrated and well-represented in dinitrated species. Spectral analysis of nitro-Cyt c showed a loss in 695 nm band at pH 7.4 reflecting the disappearance of heme-M80 coordination and consistent with an early "alkaline transition" and a gain of peroxidatic activity. High yields of pure Cyt c were obtained to perform structural and functional studies of nitrated forms: horse heart Cyt c was expressed and produced in E. coli BL21 (DE3) using the plasmid construct pJRhrsN2 allowing co-expression of Cyt c and yeast heme lyase. Site-directed mutagenesis (Y to F) was performed in order to obtain pure Cyt c mutants of one to four Y. In addition, we successfully expressed the M80A mutant that could be considered a model penta-coordinate heme. The purified Y67F mutant revealed to be a stable protein whose spectra and reducibility by ascorbate were similar to wild type but was more heavily nitrated by ONOO^-. Studies aimed to define nitration mechanisms in wild-type and mutated Cyt c and changes in redox activity and pro-apoptotic functions of the nitro- and M80A species are underway. We postulate that nitration of Cyt c yields a heme penta-coordinated protein with different biochemical properties than the hexa-coordinated form including a gain of peroxidase activity; importantly, conditions other than nitration may lead to penta-coordination and impact in the cellular functions mediated by Cyt c.