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

Abstract

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 distribution 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 decreases 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.

Identification of the TyrOH•+ Radical Cation in the Flavoenzyme TrmFO

“…Under our experimental conditions using a sample near the optically thin limit and <10% excitation energy, the deviation is estimated <∼3% (cf. ref ); hence substantially smaller than the variation in anisotropy used to discuss the results (see below).…”

Dynamics of Geminate Recombination of NO with Myoglobin in Aqueous Solution Probed by Femtosecond Mid-IR Spectroscopy

“…Due to the excellent short-term stability of the IR light source (<0.5% rms), the noise is less than 1 × 10 -4 rms in absorbance units after 0.5 s of signal averaging without single shot referencing with an independent detector. The pump spot (180 μm diameter) was made larger than the probe spot (120 μm diameter) to ensure spatially uniform photoexcitation across the spatial dimensions of the probe pulse . Moderate pump energy with an enlarged photolysis beam size also minimizes spatial thermal effects.…”

“…Recent advances in time-resolved IR spectroscopy − make it possible to probe subtle changes in absorbance on femtosecond time scales, enabling us to directly probe GR dynamics of MbNO in aqueous solution at room temperature. Here, we report the use of femtosecond IR spectroscopy in probing GR dynamics of NO after photolysis of MbNO and MpNO in aqueous solution at room temperature.…”

Orientational Distribution of CO before and after Photolysis of MbCO and HbCO: A Determination Using Time-Resolved Polarized Mid-IR Spectroscopy

“…We have shown that the appropriate correction factor for an optically thick sample can be calculated as a sum over optically thin ones, with parameters in each optically thin slice determined according to the fraction of molecules photolyzed within that slice, i.e where i is the index for each slice and f i is its fractional contribution to the total number of photolyzed molecules …”

“…It should be noted that if the photolysis pulse is sufficiently short in duration, A * corresponds to the excited-state absorbance of the sample, not the ground-state absorbance of the photoproduct. The correction factor becomes a strong function of A and A * at higher levels of photolysis, thereby causing the optically thick correction factor to differ significantly from the optically thin limit ( A → 0) …”