Out‐of‐plane deformations of the heme group in different ferrocytochrome c proteins probed by resonance Raman spectroscopy

Abstract

AbstractWe measured the low‐wavenumber polarized resonance Raman spectra of horse heart (hhc), chicken (chc) and yeastC102T (yc) ferrocytochromes c with Soret excitation. We examined the out‐of‐plane (oop) deformations of the heme groups by virtue of relative intensities and depolarization ratios of a variety of oop and in‐plane (ip) Raman active bands. Analysis of relative Raman intensities shows differences in deviation from planarity of the heme groups of yeast, horse heart and chicken cytochromes c. The heme groups in cytochrome c proteins have been shown by normal coordinate static deformation (NSD) analysis from crystal structures to exhibit a dominant ruffling (B1u) deformation. As a consequence the B1u modes, γ10 − γ12, become resonance Raman active. We used normalized Raman intensity ratios and depolarization ratios of oop Raman active modes, whose intensities are attributable to specific nonplanar deformations, to estimate and compare their Franck‐Condon‐type and Jahn‐Teller‐type coupling magnitudes for horse heart, chicken and yeast ferrocytochrome c at neutral pH. These coupling magnitudes allow for a quantitative comparison of oop deformations between individual heme groups. Chicken ferrocytochrome was found to have the largest ruffling deformation of the three investigated proteins, followed by horse heart and yeast cytochrome c. The heme group of the former is slightly more ruffled than the corresponding active site of the latter, while saddling in both proteins is substantially larger than in chicken ferrocytochrome c. The Raman data are sensitive enough to allow a comparison of lesser deformations. Doming, which is a kinetic coordinate in many heme proteins, is largest in chicken and smallest in yeast cytochrome c. Waving is largest in yeast, followed by horse heart and chicken cytochrome c. Propellering deformations could be compared for chicken and horse heart cytochrome c and were found to be substantially larger in the latter. A comparison with heme deformations obtained from X‐ray structures (for horse heart and yeast cytochrome c) and from molecular dynamics simulations (MDS) (performed for all three proteins) yields some agreement with the main ruffling and saddling deformations derived from the crystal structures, whereas the heme conformations produced by MDS seem to account better for smaller deformations like doming and propellering. The present study demonstrates the usefulness of resonance Raman spectroscopy for the analysis of nonplanar deformations in heme proteins. Copyright © 2008 John Wiley & Sons, Ltd.