Dynamics of ultrafast rebinding of CO to carboxymethyl cytochrome c.

Rebinding dynamics of CO to carboxymethyl cytochrome c (Ccytc), a chemically modified cytochrome c to bind ligands in its ferrous form, in D(2)O solution at 283 K after photodeligation, was investigated using femtosecond vibrational spectroscopy. The stretching mode of CO bound to the protein shows four stretching bands near 1962 cm(-1). Time-resolved spectra of the bound CO revealed a slight band-position-dependent rebinding kinetics, suggesting that the geminate rebinding of CO depends on the conformation of the protein. The overall rebinding kinetics of CO to Ccytc was more than 1000 times faster than that to myoglobin (Mb), a ligand-binding protein, and is also faster than a model heme, microperoxidase-8 in viscous solvent. The efficient rebinding of CO to Ccytc was attributed to the longer retention of the dissociated CO near the active binding site by the organized protein matrix of Ccytc. The spectra of the dissociated CO reveal a fast-growing band in the picosecond time scale that is assigned to CO in D(2)O solvent. The ultrafast CO escape to bulk solution is consistent with its 3D structure showing a sizable opening in the active site. It appears that most of the dissociated CO rebinds within 1 ns, except for those that escape to the bulk solution through the opening. The CO rebinding in Ccytc indicates that the primary heme pocket in Mb, located near the active site and holding the dissociated ligand for longer than tens of nanoseconds, has a specific structure to suppress CO rebinding.