The peroxidase activity of cytochrome (cyt) c increases when Met80 dissociates from the heme iron, which is related to the initial cyt c membrane permeation step of apoptosis. Met80-dissociated cyt c can form an oxygenated species. Herein, resonance Raman spectra of Met80-depleted horse cyt c (M80A cyt c) were analyzed to elucidate the heme ligand properties of Met80-dissociated cyt c. The Fe-His stretching (νFe-His) mode of ferrous M80A cyt c was observed at 236 cm-1, and this frequency decreased by 1.5 cm-1 for the 15N-labeled protein. The higher νFe-His frequency of M80A cyt c than of other His-ligated heme proteins indicates strong heme coordination and the imidazolate character of His18. Peaks attributed to the Fe-O2 stretching (νFe-O) and O-O stretching (νO-O) modes of the oxygenated species of M80A cyt c were observed at 576 and 1148 cm-1, respectively, under an 16O2 atmosphere, whereas the frequencies decreased to 544 and 1077 cm-1, respectively, under an 18O2 atmosphere. The νFe-O mode of Hydrogenobacter thermophilus (HT) M59A cyt c552 was observed at 580 cm-1 under an 16O2 atmosphere, whereas the frequency decreased to 553 cm-1 under an 18O2 atmosphere, indicating that relatively high νFe-O frequencies are characteristic of c-type cyt proteins. By comparison of the simultaneously observed νFe-O and νO-O frequencies of oxygenated cyt c and other oxygenated His-ligated heme proteins, the frequencies tend to have a positive linear relationship; the νFe-O frequency increases when the νO-O frequency increases. The imidazolate character of the heme-coordinated His and strong Fe-O and O-O bonds are characteristic of cyt c and apparently related to the peroxidase activity when Met80 dissociates from the heme iron.