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.

Mid-infrared transient absorption ("pump-probe") measurements on the amide I band of myoglobin in D 2 O and in a glass-forming D 2 O/glycer(ol-d 3 ) solvent mixture reveal very rapid vibrational energy relaxation. At 300 K, the exponential decay time is 1.3 ( 0.2 ps in D 2 O. The temperature dependence of the vibrational relaxation in the solvent mixture is slight, changing from 1.9 ( 0.2 ps below 100 K to 1.2 ( 0.2 ps at 310 K. The lack of a strong temperature dependence is indicative of a low-order relaxation process where energy transfers into high-energy modes of the system rather than directly to low-energy solvent or protein "bath" modes. The pump-probe signal is also strongly wavelength-dependent. As the laser is tuned to the lowenergy side of the absorption band, transient absorption contributions to the signal increase, indicating an anharmonicity of 15 ( 2 cm -1 for the amide I mode. The time-resolved polarization anisotropies at 300 and 100 K show a decay of about 10 ps, independent of temperature, which is attributed to energy transfer within the amide I band.

Picosecond infrared vibrational echo measurements from 60 to 300 K on CO bound to the active site of a mutant myoglobin, H93G(N-MeIm), are presented and compared to measurements on native myoglobin and on the mutant H64V. Although in H93G(N-MeIm) (the proximal histidine replaced by glycine, with exogenous N-methylimidazole in the proximal histidine pocket, covalently bound to Fe), the only covalent linkage between heme−CO and the protein is broken, there is no change in the temperature-dependent vibrational pure dephasing time, T 2*. The results demonstrate that severing the only covalent bond between the heme and the globin has little or no effect on the protein dynamics detected by vibration of the CO at the active site of myoglobin.