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

Abstract

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 g 5 , g 6 , g 7 .A 2u / modes, g 21 and g 22 .E g /, g 15 .B 2u / and g 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.

Resonance Raman Spectroscopy Reveals That Substrate Structure Selectively Impacts the Heme-Bound Diatomic Ligands of CYP17

“…7-9,41-43 The propionate bending modes typically occur in the region of ~360-380 cm −1 with features observed in the higher range being ascribed to stronger H-bonding of the terminal carboxyl groups with active site residues. 28,29,44 Similarly, the frequencies of the so-called “vinyl bending” modes, which occur in the 400-440 cm −1 region, like their ν(C=C) counterparts mentioned above, are normally considered to be sensitive the orientation of the vinyl groups with respect to the planes of the pyrrole rings to which they are attached.…”

Low-Frequency Mode Activity of Heme: Femtosecond Coherence Spectroscopy of Iron Porphine Halides and Nitrophorin

“…This “symmetry-lowering” mechanism has been studied in detail recently using heme model compounds. For example, the depolarization ratio of higher frequency (>200 cm -1 ) OOP bands were measured and analyzed using group theoretical arguments 86,88. In another study,87 the OOP Raman intensities were simulated using the Kramers-Kronig transform approach with computationally estimated values for Δ, the Condon coupling.…”

Enzymatic activity and thermal stability of metallo proteins in hydrated ionic liquids

“…14 HRP is a heme containing enzyme which catalyses the oxidation of various organic compounds with hydrogen peroxide. 25,26 Figure 2a shows the As shown in Figure 2b, the RR modes near 400 cm 21 ). In the far UV region (250-250 nm), the CD spectrum is an effective probe for protein secondary structure such as a-helix and b-sheet.…”