Non-planar heme deformations and excited state displacements in horseradish peroxidase detected by Raman spectroscopy at Soret excitation

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 gamma(5),gamma(6), gamma(7)(A(2u)) modes, gamma(21) and gamma(22)(E-g), gamma(15)(B-2u) and gamma(11)(B-1u). 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 (c) 2005 John Wiley & Sons, Ltd.