Ultrafast mid-IR spectroscopy of photodissociated carbonmonoxymyoglobin

Myoglobin (Mb) is an oxygen-storage protein that consists of a ligand-binding heme group buried in the hydrophobic interior of a globular protein. Essential to the function of Mb is the diffusion of oxygen between the heme binding site and the surrounding solution. While it is not possible to probe directly the dynamics of O₂ motion with ultrafast time resolution, it has recently become possible to probe that of CO by measuring its vibrational spectrum with femtosecond time resolution. Because CO has a small permanent dipole moment and is similar in size to O₂, the dynamics of CO motion are expected to be similar to that of O₂. We have developed a time-resolved mid-IR (approx.3.3 - 5.5 μm) spectrometer that achieves 3 cm^-1 spectral resolution, ≤300 fs time resolution, and high sensitivity (see Figure 1). The spectral evolution of 'free' CO measured with this apparatus from fs to ms times reveals many details regarding ligand motion: the dynamics of ligand diffusion within the heme pocket; conformational relaxation of the protein; ligand escape from the heme pocket; diffusion to another pocket within the protein; escape into the surrounding solvent; and finally, bimolecular rebinding. Measurements on Mb and mutants of Mb have been carried out to probe the functional role of the heme pocket and the pathway for ligand escape.