Coherence Spectroscopy Investigations of the Low-Frequency Vibrations of Heme: Effects of Protein-Specific Perturbations

Abstract

Femtosecond coherence spectroscopy is used to probe the low-frequency (20–200 cm⁻¹) vibrational modes of heme proteins in solution. Horseradish peroxidase (HRP), myoglobin (Mb), and Campylobacter jejuni globin (Cgb) are compared and significant differences in the coherence spectra are revealed. It is concluded that hydrogen bonding and ligand charge do not strongly affect the low-frequency coherence spectra and that protein-specific deformations of the heme group lower its symmetry and control the relative spectral intensities. Such deformations potentially provide a means for proteins to tune heme reaction coordinates, so that they can perform a broad array of specific functions. Native HRP displays complex spectral behavior above ∼50 cm⁻¹ and very weak activity below ∼50 cm⁻¹. Binding of the substrate analog, benzhydroxamic acid, leads to distinct changes in the coherence and Raman spectra of HRP that are consistent with the stabilization of a heme water ligand. The CN derivatives of the three proteins are studied to make comparisons under conditions of uniform heme coordination and spin-state. MbCN is dominated by a doming mode near 40 cm⁻¹, while HRPCN displays a strong oscillation at higher frequency (96 cm⁻¹) that can be correlated with the saddling distortion observed in the X-ray structure. In contrast, CgbCN displays low-frequency coherence spectra that contain strong modes near 30 and 80 cm⁻¹, probably associated with a combination of heme doming and ruffling. HRPNO displays a strong doming mode near 40 cm⁻¹ that is activated by photolysis. The damping of the coherent motions is significantly reduced when the heme is shielded from solvent fluctuations by the protein material and reduced still further when T ≲ 50 K, as pure dephasing processes due to the protein–solvent phonon bath are frozen out.