Investigations of photolysis and rebinding kinetics in myoglobin using proximal ligand replacements.

We use laser flash photolysis and time-resolved Raman spectroscopy of CO-bound H93G myoglobin (Mb) mutants to study the influence of the proximal ligand on the CO rebinding kinetics. In H93G mutants, where the proximal linkage with the protein is eliminated and the heme can bind exogenous ligands (e.g., imidazole, 4-bromoimidazole, pyridine, or dibromopyridine), we observe significant effects on the CO rebinding kinetics in the 10 ns to 10 ms time window. Resonance Raman spectra of the various H93G Mb complexes are also presented to aid in the interpretation of the kinetic results. For CO-bound H93G(dibromopyridine), we observe a rapid large-amplitude geminate phase with a fundamental CO rebinding rate that is approximately 45 times faster than for wild-type MbCO at 293 K. The absence of an iron proximal ligand vibrational mode in the 10 ns photoproduct Raman spectrum of CO-bound H93G(dibromopyridine) supports the hypothesis that proximal ligation has a significant influence on the kinetics of diatomic ligand binding to the heme.