Resonance Raman Studies on Heme Ligand Stretching Modes in Methionine80-Depleted Cytochrome c : Fe-His, Fe-O 2 , and O-O Stretching Modes.

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 (ν _Fe-His ) mode of ferrous M80A cyt c was observed at 236 cm ^-1 , and this frequency decreased by 1.5 cm ^-1 for the ¹⁵ N-labeled protein. The higher ν _Fe-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-O ₂ stretching (ν _{Fe-O 2} ) and O-O stretching (ν _O-O ) modes of the oxygenated species of M80A cyt c were observed at 576 and 1148 cm ^-1 , respectively, under an ¹⁶ O ₂ atmosphere, whereas the frequencies decreased to 544 and 1077 cm ^-1 , respectively, under an ¹⁸ O ₂ atmosphere. The ν _{Fe-O 2} mode of Hydrogenobacter thermophilus (HT) M59A cyt c ₅₅₂ was observed at 580 cm ^-1 under an ¹⁶ O ₂ atmosphere, whereas the frequency decreased to 553 cm ^-1 under an ¹⁸ O ₂ atmosphere, indicating that relatively high ν _{Fe-O 2} frequencies are characteristic of c -type cyt proteins. By comparison of the simultaneously observed ν _{Fe-O 2} and ν _O-O frequencies of oxygenated cyt c and other oxygenated His-ligated heme proteins, the frequencies tend to have a positive linear relationship; the ν _{Fe-O 2} frequency increases when the ν _O-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.