The first picosecond infrared vibrational echo experiments on a protein, myoglobin-CO, are described. These vibrational dephasing experiments examine the influence of protein dynamics on the CO ligand bound to the active site of the protein at physiologically relevant temperatures. The experiments were performed with a mid-IR free electron laser tuned to the CO stretch mode at 1945 cm -1. The vibrational echo results are combined with infrared pump-probe measurements of the CO vibrational lifetime to yield the homogenous pure dephasing, the Fourier transform of the homogeneous line width with the lifetime contribution removed. The measurements were made from 60 to 300 K. The results show that the CO vibrational spectrum is inhomogeneously broadened, even at room temperature. Above the glycerol/water solvent's glass transition temperature, ∼185 K, the temperature dependence can be fit as an activated process with ∆E ≈ 1000 cm -1 . Below 185 K, the pure dephasing displays a power law temperature dependence, T 1.3 . This temperature dependence is reminiscent of that associated with the properties of low-temperature glasses (<5 K) but is observed at much higher temperatures. A two-level system model of protein dynamics is considered. The nature of the temperature dependence and the mechanism of the coupling of the protein fluctuations to the CO vibrational transition energy are discussed.

Picosecond mid-IR pump-probe measurements of vibrational relaxation (VR) of CO bound to the active sites of wild-type and mutant myoglobins (Mb) reveal that an approximately linear relationship exists between the protein matrix-induced CO frequency shift and the VR rate. This relation parallels a similar linear relationship seen in a series of heme model compounds where Fe was replaced by Ru and Os. The VR rate of CO in the Mb is sensitive only to the magnitude of protein-induced carbonyl frequency shifts and apparently is not sensitive to the specific details of how the shift is induced, e.g., hydrogen bonding to CO or electrostatic interactions in the heme pocket. CO VR is insensitive to substantial changes in protein structure that do not affect the CO vibrational frequency. These observations suggest that the mechanism of carbon monoxide VR in heme proteins such as Mb occurs by through-π-bond anharmonic coupling, which, as shown in prior work, is also the dominant coupling in the model compounds. The experiments indicate that differing protein structures influence VR of CO bound at the active site not by opening and closing channels for vibrational energy flow from CO to the protein but by affecting the rate of energy flow from CO to heme. The rates are determined by the extent of back-bonding, which determines the magnitude of through-π-bond anharmonic coupling between CO and heme. The back-bonding and, therefore, the extent of anharmonic coupling are influenced by the electric fields in the heme pocket, which likely differ in the proteins studied here.

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Ultrafast infrared vibrational echo measurements of the temperature-dependent pure dephasing of the A 1 CO stretching mode of myoglobin-CO (Mb-CO) were performed in the solvents trehalose and 50:50 ethylene glycol:water. The results are compared to previously reported data in 95:5 glycerol:water. The temperature dependence (11-300 K) of the pure dephasing in trehalose (a glass at all temperatures studied) is a power law, T 1.3 , below T = 200 K, while at higher temperature it becomes dramatically steeper. The change in functional form occurs although the solvent does not go through its glass transition. In the other two solvents, the breaks in the temperature dependences occur at lower temperatures, and the temperature dependences are even steeper above the power law region. The results are discussed in terms of a combination of a temperature and viscosity dependence of protein dynamics.