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Met80 of cytochrome c (cyt c) has been shown to dissociate from its heme iron when cyt c interacts with cardiolipin (CL), which triggers the release of cyt c into the cytosol initiating apoptosis. We found that the mass of human cyt c increases by 16 Da in the Met80-Lys86 region by reaction with molecular oxygen in the presence of CL-containing liposomes and dithiothreitol (DTT). To investigate the effect of Met80 dissociation on the reaction of cyt c with molecular oxygen without affecting its secondary structures, a human cyt c mutant (Δ8384 cyt c) was constructed by removing two amino acids (Val83 and Gly84) from the loop containing Met80. According to MALDI-TOF-MS and tandem mass measurements, Met80 of Δ8384 cyt c was modified site-specifically to methionine sulfoxide when purified in the presence of molecular oxygen, whereas Met80 was not modified in the absence of molecular oxygen. A red-shift of the Soret band from 406 to 412 nm and absorption increase at ∼536 and ∼568 nm were observed for Δ8384 cyt c when it reacted with DTT and molecular oxygen, followed by a further red-shift of the Soret band to 416 nm and absorption increase at ∼620 and ∼650 nm. These results indicate that Met80 of cyt c is oxidized site-specifically by formation of the oxy and subsequent compound I-like species when Met80 dissociates from the heme iron, where the Met80 modification may affect its peroxidase activity related to apoptosis.