Deformations of the Heme Group of Different Ferrocytochrome c Proteins Probed by Resonance Raman Spectroscopy

We measured the low-frequency polarized resonance Raman spectra of horse heart, chicken, and yeast(C102T) ferrocytochromes c with Soret excitation. We examined the out-of-plane deformations of the heme groups by determining the relative intensities and depolarization ratios of a variety of out-of-plane and in-plane Raman active bands. Analysis of relative Raman intensities shows differences in non-planarity of the heme groups of yeast(C102T), horse heart and chicken cytochrome c. Cytochrome c has been shown to have a dominant ruffling (B1u) deformation by means of normal coordinate structural decomposition (NSD) analysis of the heme group in crystal structures. The presence and intensity of B1u modes, γ10-γ12, support the indication of ruffling being the major contribution to the non-planar deformations in cytochrome c. Other types of non-planar deformations like doming (A2U) and waving (Eg) can be deduced from the Raman activity of γ5 (A2u), γ21 and γ22 (Eg). The depolarization ratios of γ5, γ10, γ11 and γ12 are larger than 0.125, indicating the presence of other deformations such as saddling (B2u) and propellering (A1u), which is again in agreement with the crystal structures of horse heart and yeast ferrocytochrome c. An analysis of the intensities and depolarization ratios of out-of-plane modes revealed that ruffling is comparable in yeast and horse heart cytochrome c, saddling is larger and doming as well as propellering are lower in yeast cytochrome c. With respect to doming and ruffling our results contradict values obtained from the NSD analysis of the corresponding crystal structures. With respect to saddling, our data are in agreement with the crystal structure. The NSD analysis of heme structures resulting from MD simulations did not correlate very well with the spectroscopically obtained results concerning the ruffling and doming coordinate, whereas a qualitative agreement was again obtained for saddling.