Myoglobin-CO Substate Structures and Dynamics: Multidimensional Vibrational Echoes and Molecular Dynamics Simulations

Spectrally resolved infrared stimulated vibrational echo data were obtained for sperm whale carbonmonoxymyoglobin (MbCO) at 300 K. The measured dephasing dynamics of the CO ligand are in agreement with dephasing dynamics calculated with molecular dynamics (MD) simulations for MbCO with the residue histidine-64 (His64) having its imidazole ε nitrogen protonated (N _ε-H). The two conformational substate structures B _ε and R_ε observed in the MD simulations are assigned to the spectroscopic A₁ and A₃ conformational substates of MbCO, respectively, based on the agreement between the experimentally measured and calculated dephasing dynamics for these substates. In the A₁ substate, the N_ε-H proton and N _δ of His64 are approximately equidistant from the CO ligand, while in the A₃ substate, the N_ε-H of His64 is oriented toward the CO, and the N_δ is on the surface of the protein. The MD simulations show that dynamics of His64 represent the major source of vibrational dephasing of the CO ligand in the A₃ state on both femtosecond and picosecond time scales. Dephasing in the A₁ state is controlled by His64 on femtosecond time scales, and by the rest of the protein and the water solvent on longer time scales.