AbstractOften, deregulation of protein activity and turnover by tyrosine nitration drives cells toward pathogenesis. Hence, understanding how the nitration of a protein affects both its function and stability is of outstanding interest. Nowadays, most of the in vitro analyses of nitrated proteins rely on chemical treatment of native proteins with an excess of a chemical reagent. One such reagent, peroxynitrite, stands out for its biological relevance. However, given the excess of the nitrating reagent, the resulting in vitro modification could differ from the physiological nitration. Here, we determine unequivocally the configuration of distinct nitrated‐tyrosine rings in single‐tyrosine mutants of cytochrome c. We aimed to confirm the nitration position by a non‐destructive method. Thus, we have resorted to 1H‐15N heteronuclear single quantum coherence(HSQC) spectra to identify the 3J(NH) correlation between a 15N‐tagged nitro group and the adjacent aromatic proton. Once the chemical shift of this proton was determined, we compared the 1H‐13C HSQC spectra of untreated and nitrated samples. All tyrosines were nitrated at ε positions, in agreement to previous analysis by indirect techniques. Notably, the various nitrotyrosine residues show a different dynamic behaviour that is consistent with molecular dynamics computations.