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.

The influence of solvent viscosity on the surface and internal structural dynamics of the protein myoglobin is studied using ultrafast infrared vibrational echo measurements of the pure dephasing of the A 1 CO stretching mode of myoglobin-CO (Mb-CO). The dephasing reflects protein structural fluctuations as sensed by the CO ligand bound at the protein's active site. Measurements made as a function of solvent viscosity at 295 K show that the pure dephasing has a marked dependence on viscosity. In addition, the pure dephasing of Mb-CO in the solvents trehalose and 50:50 ethylene glycol:water are compared as a function of temperature T (10-295 K). The pure dephasing data in the two solvents have identical T 1.3 temperature dependences at low temperatures, where both solvents are glassy solids. At higher temperatures, the Mb-CO pure dephasing has a much steeper temperature dependence in ethylene glycol:water, which is a liquid, than in trehalose, which is a glass at all temperatures studied. The steep temperature dependence in liquid ethylene glycol: water is described as a combination of a viscosity-dependent component and a temperature-dependent component. The viscosity-dependent data are analyzed using a theory that connects the fluctuations of the protein surface to the solvent's viscoelastic response. When the solvent's viscosity is lowered, the increased rate of fluctuation of the protein's surface allows more rapid internal protein dynamics, which result in more rapid dephasing. Good agreement is obtained for physically reasonable parameters. The experimental echo decay times are proportional to the cube root of the solvent viscosity η 1/3 . This proportionality is characteristic of protein structural fluctuations that give rise to CO frequency fluctuations that are in the spectral diffusion regime (relatively slow evolution).

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