Resonance Raman Studies on Heme Ligand Stretching Modes in Methionine80-Depleted Cytochrome <i>c</i>: Fe–His, Fe–O₂, and O–O Stretching Modes

Abstract

High Resolution Image Download MS PowerPoint Slide 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.

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