A proteomic strategy combining 2DE, Western blot, and mass spectrometry was implemented to survey the status of tyrosine nitration in mouse heart mitochondria. Compared to normal mice, nitrated proteins in the heart mitochondria of the db/db mouse model were significantly augmented due to diabetic development. A total of 18 proteins were identified as the nitration targets. Of the nitrated proteins, succinyl-CoA:3-oxoacid CoA-transferase (SCOT) is a key enzyme involved in ketolysis and has yet to be explored how its catalysis is affected by nitration. We therefore initiated a systematic investigation toward the nitrated site(s) and the corresponding changes of SCOT catalysis. To monitor modification kinetics and nitrated residue(s), recombinant SCOT was incubated with peroxynitrite followed by examination of nitration development as well as catalytic activity changes. The nitration of recombinant SCOT steadily increased in response to increasing concentrations of peroxynitrite, while its catalysis was gradually attenuated. The nitrated sites of modified SCOT were further identified by LC-ESI-MS/MS. The MS/MS spectra indicated a +45 mass unit ion shift from [M + H](+) m/z at Tyr(4) and Tyr(76). Through site-directed mutagenesis, we found that mutation of tyrosine residues at Tyr(4) or Tyr(76) did not only significantly protect SCOT from peroxynitrite modification, but it also dramatically prevented loss of enzymatic activity. Taken together, these results indicate that the two tyrosine residues of SCOT are the priority sites attacked by NO, and their nitration status is a causal factor leading to inhibition of SCOT catalysis.