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Article

Structural Disorder of Native Horseradish Peroxidase C Probed by Resonance Raman and Low-Temperature Optical Absorption Spectroscopy

Qing Huang,^† Krisztian Szigeti,^‡ Judit Fidy,^‡ and Reinhard Schweitzer-Stenner*^†

Department of Chemistry, University of Puerto Rico, Rio Píedras Campus, San Juan, PR 00931, and Institute of Biophysics and Radiation Biology, Semmelweis University, Puskin u. 9, Hungary H-1088

J. Phys. Chem. B, 2003, 107 (12), pp 2822–2830

DOI: 10.1021/jp026935e

Publication Date (Web): March 1, 2003

†

University of Puerto Rico.

‡

Semmelweis University.

Corresponding author: Reinhard Schweitzer-Stenner, Department of Chemistry, University of Puerto Rico, Río Piedras Campus, P.O. Box 23346, Facundo Bueso Bldg. FB110, San Juan, PR 00931. Phone: 1-787-764-0000, Ext 2417. Fax: 1-787-756-8242. E-mail: rstenner_upr_chemistry@ gmx.net.

Abstract

We have measured the low-temperature absorption and the depolarization ratio dispersion of various intense resonance Raman lines of horseradish peroxidase C. The absorption spectra reveal significant splitting of the Q and charge-transfer bands whereas the Soret band solely exhibits a narrowing of the band profile. The depolarization ratios of all Raman lines investigated are different from expectation values in D₄_h symmetry and show significant dispersion in the preresonance and resonance region of the Q_v band. All these data indicate symmetry lowering distortions of the heme macrocycle. An analysis of the depolarization ratios shows that in-plane B₁_g- and B₂_g-type perturbations distort the heme along the N−Fe−N and C_m−Fe−C_m line of the heme. The B₁_g perturbation gives rise to the band splitting in the optical spectrum and to a rhombicity of the iron's ligand field detected by EPR experiments. On the basis of group theoretical arguments we propose that particularly the strong B₂_g distortion gives rise to the quantum mixed spin state characteristic of class III peroxidases.