Ligand binding to heme proteins: a comparison of cytochrome c variants with globins

We have studied the binding of carbon monoxide (CO) in mutants of Cyt c having its methionine at position 80 replaced by alanine, aspartate, and arginine, so that the sixth coordination is available for ligand binding. We have employed Fourier transform infrared (FTIR) photolysis difference spectroscopy to examine interactions of the heme-bound and photolyzed CO (and also nitric oxide, NO) in the small heme pocket created by the mutations. By using FTIR temperature derivative spectroscopy (TDS) and nanosecond flash photolysis, the enthalpy barrier distributions for CO rebinding were determined. In flash photolysis experiments, the majority of ligands rebind to the heme iron on picosecond time scales so that only the high-barrier tail of the distributions is visible on the nanosecond scale. By continuous wave excitation prior to TDS characterization of the barriers, however, each Cyt c molecule is photoexcited multiple times and complete photodissociation can be achieved, which likely arises from a rotation of the CO within the heme pocket so that the oxygen faces the heme iron. Apparently, reorientation prior to rebinding constitutes an additional and significant contribution to the rebinding barrier. Our experiments reveal that the compact, rigid structure of Cyt c offers no alternative binding sites for photodissociated ligands in the protein matrix. A comparison of ligand binding in these Cyt c mutants and hemoglobins underscores the importance of internal ligand docking sites and ligand migration routes for conveying a ligand binding function to heme proteins.