Abstract

Myoglobin (Mb) is an oxygen-binding protein found in muscle. The active binding site is an iron-II-containing porphyrin (heme) that is located within the hydrophobic interior of the protein. The X-ray structure of Mb reveals a distal cavity large enough to accommodate a ligand the size of O₂, but no channel large enough for the ligand to diffuse between the heme pocket and the surrounding solvent. Clearly, structural fluctuations of the protein are required to open channels for ligand transport to and from the binding site. What is the functional role of the heme pocket and the residues that circumscribe it? What pathways exist for ligand transport between the heme pocket and the solvent? On what time scale does the ligand escape into the surrounding solvent? We have sought to answer these questions by probing the time-resolved vibrational spectrum of CO, a ligand similar in size to O₂, following photodissociation of MbCO at physiological temperatures. We have developed a femtosecond time-resolved mid-IR spectrometer with the sensitivity required to measure the “free” CO absorbance. Ultrafast diffusion to a “docking” site has been observed, the orientation of the docked CO relative to the plane of the heme has been measured, and the rate of escape from the heme pocket has been determined. Additional studies with mutants of Mb are being conducted to probe the location of the docking site within the heme pocket and the pathways for ligand escape from the heme pocket.

© 1994 Optical Society of America