Resonance Raman studies of oriented chromophores: Metmyoglobin single crystals

Abstract

We report Soret enhanced resonance Raman studies of single crystals of metmyoglobin. Differing scattering intensities of the 345 cm−1 mode in solution and crystalline forms of the protein indicate a structural perturbation of the heme upon crystallization, possibly involving a propionate substituent of the heme. The immobilization of the heme in the crystal also allows observation of low-quantum-yield photoreduction of the heme that appears to be driven by Soret band absorption. We present experimental investigations of the orientational dependence of the intensity and polarization of several strong heme Raman modes along with calculations that explore the effects of vibrational mode symmetry and of differential coupling to x-, y-, and z-polarized electronic transitions. Orientation studies provide evidence that the vinyl C=C stretching band at 1622 cm−1 is a superposition of two independent modes that couple selectively to the x- and y-polarized Soret transitions. We also demonstrate how modes of nominal B1g and B2g symmetry can be distinguished by crystal studies. Modes that couple preferentially to charge transfer transitions polarized perpendicular to the heme plane are shown to be easily distinguishable from the majority of heme modes, which couple to the in-plane polarized Soret transition; we observe no modes of the former type in metmyoglobin. Finally, we present calculations showing that single-crystal polarization studies of resonance Raman scattering as a function of excitation frequency should be sensitive to the splitting of the nearly xy-degenerate Soret band as well as to the orientation of the x- and y-polarized transitions.