Time-resolved resonance Raman study on the binding of carbon monoxide to recombinant human myoglobin and its distal histidine mutants

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), Gln (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 microseconds 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 approximately 510 cm-1 shoulder appeared much later. The equilibrium relative intensity of the two bands was attained at 500 microseconds, suggesting that the interconversion between the two forms is slower than 100 microseconds.(ABSTRACT TRUNCATED AT 250 WORDS)