Infrared Study of Carbon Monoxide Migration among Internal Cavities of Myoglobin Mutant L29W.

Myoglobin, a small globular heme protein that binds gaseous ligands such asO(2), CO and NO reversibly at the heme iron, provides an excellent modelsystem for studying structural and dynamic aspects of protein reactions. Flashphotolysis experiments, performed over wide ranges in time and temperature, reveal a complex ligand binding reaction with multiple kinetic intermediates, resulting from protein relaxation and movements of the ligand within the protein. Our recent studies of carbonmonoxy-myoglobin (MbCO) mutant L29W, using time-resolved infrared spectroscopy in combination with x-ray crystallography, have correlated kinetic intermediates with photoproduct structures that are characterized by the CO residing in different internal protein cavities, so-called xenon holes. Here we have used Fourier transform infrared temperature derivative spectroscopy (FTIR-TDS) to further examine the role of internal cavities in the dynamics. Different cavities can be accessed by the CO ligands at different temperatures, and characteristic infrared absorption spectra have been obtained for the different locations of the CO ligand within the protein, enabling us to monitor ligand migration through the protein as well as conformational changes of the protein.