Structural Assignments and Dynamics of the A Substates of MbCO: Spectrally Resolved Vibrational Echo Experiments and Molecular Dynamics Simulations

Spectrally resolved three-pulse stimulated vibrational echo experiments are used as the basis for structural assignments of the A1 and A3 spectroscopic substates in the IR spectrum of the carbon monoxide (CO) stretch of carbonmonoxymyoglobin (MbCO). The measured dephasing dynamics of these substates is compared to the dephasing dynamics of MbCO predicted from molecular dynamics (MD) simulations. We assign the A1 and A3 substates to different protein conformations on the basis of the agreement between the measured and computed vibrational echoes. In the A1 substate, the N-H proton and N‰ of His64 are equidistant from the ligand, whereas in the A3 substate, the N-H of His64 is oriented toward the CO.