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

[Image: see text] 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.