The Orientation of CO in Heme Proteins Determined by Time-Resolved Mid-IR Spectroscopy: Anisotropy Correction for Finite Photolysis of an Optically Thick Sample

A systematic way of determining the equilibrium orientation of carbon monoxide (CO) in heme proteins using time-resolved polarized mid-IR spectroscopy is presented. The polarization anisotropy at pump-probe delay time of zero in the limit of zero photolysis and the angular distrbution function of CO are required to obtain the equilibrium orientation of CO. An approach is developed for determining the polarization anisotropy in the zero-photolysis limit from the anisotropy measured under finite photolysis conditions in an optically thick sample where the fraction of molecules photolyzed decreased as the pump pulse passes through and is absorbed by the sample. This approach is verified by measuring the polarization anisotropy of CO of carbonmonoxy myoglobin at various levels of photolysis. This method can be readily applied to other photoselection experiments determining precise angle between transition dipoles.