Orientational distribution of CO before and after photolysis of MbCO and HbCO: a determination using time-resolved polarized Mid-IR spectroscopy.

Abstract

The technique of time-resolved polarized mid-IR spectroscopy was used to probe the orientational distribution of carbon monoxide (CO) bound to and docked within horse myoglobin, sperm whale myoglobin, and human hemoglobin A in neutral pH solution at 283 K. An accurate determination of the orientation required that the experimentally measured polarization anisotropy be corrected for the effects of fractional photolysis in an optically thick sample. The experimental method measures the direction of the transition dipole, which is parallel to the CO bond axis when docked and nearly parallel when bound to the heme. The polarization anisotropy of bound CO is virtually the same for all protein systems investigated and is unchanging across its inhomogeneously broadened mid-IR absorption spectrum. From these results, it was concluded that the transition dipole moment of bound CO is oriented </=7 degrees from the heme plane normal. The polarized absorbance spectra of docked CO are similar for all protein systems investigated, but in stark contrast to bound CO, the polarization anisotropy is strongly correlated with vibrational frequency. The frequency-dependent anisotropy imposes severe constraints on the orientational probability distribution function of the transition dipole, which is well described as a dipole bathed in a Stark field whose out-of-plane motion is constrained by a simple double-well potential. The orientational and spatial constraints imposed on docked CO by the surrounding highly conserved amino acids serve to mediate ligand transport to and from the binding site and thereby control the rates and pathways for geminate ligand rebinding and ligand escape.