Spectrally resolved infrared stimulated vibrational echo measurements are used to measure the vibrational dephasing of the CO stretching mode of carbonmonoxy-hemoglobin (HbCO), a myoglobin mutant (H64V), and a bacterial cytochrome c(552) mutant (Ht-M61A) in aqueous solution and trehalose glasses. The vibrational dephasing of the heme-bound CO is significantly slower for all three proteins embedded in trehalose glasses compared to that of aqueous protein solutions. All three proteins exhibit persistent but notably slower spectral diffusion when the protein surface is fixed by the glassy solvent. Frequency-frequency correlation functions (FFCFs) of the CO are extracted from the vibrational echo data to reveal that the structural dynamics, as sensed by the CO, of the three proteins in trehalose and aqueous solution are dominated by fast (tens of femtoseconds), motionally narrowed fluctuations. MD simulations of H64V in dynamic and "static" water are presented as models of the aqueous and glassy environments. FFCFs are calculated from the H64V simulations and qualitatively reproduce the important features of the experimentally extracted FFCFs. The suppression of long time scale (picoseconds to tens of picoseconds) frequency fluctuations (spectral diffusion) in the glassy solvent is the result of a damping of atomic displacements throughout the protein structure and is not limited to structural dynamics that occur only at the protein surface. The analysis provides evidence that some dynamics are coupled to the hydration shell of water, supporting the idea that the bioprotection offered by trehalose is due to its ability to immobilize the protein surface through a thin, constrained layer of water.

Enzyme structural dynamics play a pivotal role in substrate binding and biological function, but the influence of substrate binding on enzyme dynamics has not been examined on fast time scales. In this work, picosecond dynamics of horseradish peroxidase (HRP) isoenzyme C in the free form and when ligated to a variety of small organic molecule substrates is studied by using 2D-IR vibrational echo spectroscopy. Carbon monoxide bound at the heme active site of HRP serves as a spectroscopic marker that is sensitive to the structural dynamics of the protein. In the free form, HRP assumes two distinct spectroscopic conformations that undergo fluctuations on a tens-of-picoseconds time scale. After substrate binding, HRP is locked into a single conformation that exhibits reduced amplitudes and slower time-scale structural dynamics. The decrease in carbon monoxide frequency fluctuations is attributed to reduced dynamic freedom of the distal histidine and the distal arginine, which are key residues in modulating substrate binding affinity. It is suggested that dynamic quenching caused by substrate binding can cause the protein to be locked into a conformation suitable for downstream steps in the enzymatic cycle of HRP.horseradish peroxidase ͉ ultrafast

Ultrafast protein dynamics of the CO adduct of a myoglobin mutant with the polar distal histidine replaced by a nonpolar valine (H64V) have been investigated by spectrally resolved infrared stimulated vibrational echo experiments and molecular dynamics (MD) simulations. In aqueous solution at room temperature, the vibrational dephasing rate of CO in the mutant is reduced by ∼50% relative to the native protein. This finding confirms that the dephasing of the CO vibration in the native protein is sensitive to the interaction between the ligand and the distal histidine. The stimulated vibrational echo observable is calculated from MD simulations of H64V within a model in which vibrational dephasing is driven by electrostatic forces. In agreement with experiment, calculated vibrational echoes show slower dephasing for the mutant than for the native protein. However, vibrational echoes calculated for H64V do not show the quantitative agreement with measurements demonstrated previously for the native protein.