Time-resolved resonance Raman (RR) spectra of the recombined species of photodissociated CO with recombinant human myoglobin (Mb) and several E7 mutants, in which distal His was replaced by Gly (H64G), Gin (H64Q), Ala (H64A), Ile (H64I), Val (H64V), and Leu (H64L) through site-directed mutagenesis, were observed in the time range −20 ns to 1 ms following photolysis. The Fe-CO stretching (vFe-co) RR band was observed successfully with pulse excitation when the laser power was greatly reduced. H64H, H64G, and H64Q gave the VFe-co band at 505–510 cm−1 in their stationary states. In their recovery processes 1–100/µs after photodissociation, a broad transient band was observed at slightly lower frequencies than those of their equilibrium structures for H64G and H64Q, but a transient VFe-co band corresponding to the so-called “open” form was not identified around 490 cm−1 for any of the three species. A second group, H64A, H64I, H64V, and H64L, gave the main vFe-co band at 490–495 cm−1 with a shoulder around 510 cm−1 (except for H64L) in the stationary state and exhibited a much faster recovery than the first group. These latter four species gave a broad transient band around 492–500 cm−1 in the time range of 100–1000 ns, while the ∼ 510 cm−1 shoulder appeared much later. The equilibrium relative intensity of the two bands was attained at 500 µs, suggesting that the interconversion between the two forms is slower than 100 µs. For all MbCO examined here, the recovery, determined from the area intensity of the VFe-co band, exhibited two phases irrespective of the presence of one or two vFe-co bands. The vFe-co frequencies could be correlated with the hydropathy index of the E7 residue but not with its physical size. It is inferred that the more hydrophobic environment around CO reduces the polarization of CO and lowers the vFe-co frequency to ∼490 cm−1 on the one hand and stabilizes the intermediate state called the protein-separated pair on the other. The latter increases the contribution of recombination from this intermediate, resulting in faster recombination of CO.
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