Non-planar heme deformations and excited state displacements in horseradish peroxidase detected by Raman spectroscopy at Soret excitation
Article first published online: 15 APR 2005
Copyright © 2005 John Wiley & Sons, Ltd.
Journal of Raman Spectroscopy
Special Issue: Structure and dynamics of biomolecules
Volume 36, Issue 4, pages 363–375, April 2005
How to Cite
Huang, Q. and Schweitzer-Stenner, R. (2005), Non-planar heme deformations and excited state displacements in horseradish peroxidase detected by Raman spectroscopy at Soret excitation. J. Raman Spectrosc., 36: 363–375. doi: 10.1002/jrs.1326
- Issue published online: 15 APR 2005
- Article first published online: 15 APR 2005
- Manuscript Accepted: 2 FEB 2005
- Manuscript Received: 3 DEC 2004
- National Science Foundation. Grant Number: MCB-0318749.
- ACS-Petroleum Research Fund. Grant Number: PRF 37406-AC.
- NIH. Grant Number: P20 RR16439-01.
- vibronic coupling;
- Franck–Condon interaction;
- heme protein;
- non-planar distortions;
- resonance Raman spectroscopy
We studied the out-of-plane modes of horseradish peroxidase (HRP) in different spin, oxidation and ligation states by measuring the respective resonance Raman spectra with (near) Soret excitation. The non-planar modes of the heme are Raman inactive for planar macrocycles, but become resonance Raman active in the presence of out-of-plane deformations. The thus induced Raman scattering results mostly from Franck–Condon type coupling. We observed bands from a variety of out-of-plane modes such as γ5, γ6, γ7(A2u) modes, γ21 and γ22(Eg), γ15(B2u) and γ11(B1u). The appearance of these bands is clearly indicative of non-planar deformations of the same symmetry. We determined the relative intensities of all sufficiently intense Raman modes of ferric pentacoordinated, quantum mixed and hexacoordinated low-spin as well as of ferrous pentacoordinated high-spin HRP C. The apparent vibronic coupling parameters were obtained from a self-consistent analysis of the Raman intensities and the respective optical absorption spectrum. They revealed significantly reduced displacements of the excited B-state for the ferrous, deoxy-like state compared with the resting ferric state. An analysis of the obtained coupling strengths of out-of-plane modes revealed that they are predominantly induced by static non-planar deformations along the normal coordinates of the lowest wavenumber modes of a given symmetry type. We used the deformations of the heme in resting HRP C to determine the vibronic coupling strength of the experimentally detectable out-of-plane modes and subsequently employed this information to obtain the non-planar deformations for the investigated ferric hexacoordinated low-spin state. This yielded a substantial reduction of the ruffling distortion, whereas the doming deformation remained mostly unaffected by the change of the iron's spin and ligation state. Copyright © 2005 John Wiley & Sons, Ltd.