Time-resolved resonance Raman study of intermediates generated after photodissociation of wild-type and mutant co-myoglobins Original Research Article

Time-resolved resonance Raman (TR3) spectroscopy was applied to elucidate transient structures of myoglobin (Mb) involved in its ligand binding. Pump/probe Raman measurements of the Fe–CO stretching bands (νFe–CO) were carried out for various delay times (Δt=−20 ns–1 ms with a time resolution of 7 ns) after laser photolysis of native and mutant COMb complexes. His64(E7) and Leu29(B10) were replaced with an aliphatic and aromatic residues. The static νFe–CO frequencies of the mutants depended strongly on the environments around the bound CO and correlated more with the hydropathy indices of the replaced residues than with their sizes. The kinetics of bimolecular CO recombination correlate with the static νFe–CO frequencies; a lower frequency generally results in faster rebinding. Despite these differences, all the proteins exhibited the shift of a porphyrin band from 370 to 379 cm−1 upon binding of CO and also a transient Raman band at ∼497 cm−1, which occurred before recovery of the original νFe–CO band. The latter frequency was unaffected by isotopically labeling the ligand with 13C18O. The 497 cm−1 band was absent in the spectrum at Δt=0 ns for all of the myoglobins examined except for the His64→Leu (H64L) mutant which shows the band immediately after photolysis. The 370 and 497 cm−1 bands are associated with the Cβ–Cc–Cd in-plane bending of the propionic side chains and the out-of-plane γ12 containing pyrrole swiveling and propionic bending motions, respectively. The 497 cm−1 transient band appears to reflect a deoxyheme intermediate in which the hydrogen bonding lattice between Arg45(CD3), His64(E7), the heme-6-propionate, and an external distal pocket water molecule is temporarily disrupted. This disruption allows larger movements of the propionate side chain, explaining intensity enhancement of the 497 cm−1 band. Recovery of the hydrogen bonding lattice dampens the movements of the propionate Cβ–Cc–Cd bond system and finally fixes it in the heme plane in the CO-bound form, causing the frequency shift of the bending mode from 370 cm−1 back to 379 cm−1. In the Leu64 mutant, the external water molecule is already absent, facilitating rapid movement of the heme-6-propionate after photolysis. Larger-scale movements of all three side chains could create an open conformation with a channel from the heme iron to the solvent, allowing ligand escape and/or rebinding.