TIME-RESOLVED RESONANCE RAMAN-STUDY ON THE BINDING OF CARBON-MONOXIDETO RECOMBINANT HUMAN MYOGLOBIN AND ITS DISTAL HISTIDINE MUTANTS

Time-resolved resonance Raman (RR) spectra of the recombined species o f photodissociated CO with recombinant human myoglobin (Mb) and severa l E7 mutants, in which distal His was replaced by Gly (H64G), Gln (H64 Q), Ala (H64A), Ile (H641), Val (H64V), and Leu (H64L) through site-di rected mutagenesis, were observed in the time range -20 ns to 1 ms fol lowing photolysis. The Fe-CO stretching (nu(Fe-CO)) RR band was observ ed successfully with pulse excitation when the laser power was greatly reduced. H64H, H64G, and H64Q gave the nu(Fe-CO) band at 505-5 10 cm- 1 in their stationary states. In their recovery processes 1-100 mus af ter photodissociation, a broad transient band was observed at slightly lower frequencies than those of their equilibrium structures for H64G and H64Q, but a transient nu(Fe-CO) band corresponding to the so-call ed ''open'' form was not identified around 490 cm-1 for any of the thr ee species. A second group, H64A, H641, H64V, and H64L, gave the main nu(Fe-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 recover y than the first group. These latter four species gave a broad transie nt band around 492-500 cm-1 in the time range of 100-1000 ns, while th e approximately 510 cm-1 shoulder appeared much later. The equilibrium relative intensity of the two bands was attained at 500 mus, suggesti ng that the interconversion between the two forms is slower than 100 m us. For all MbCO examined here, the recovery, determined from the area intensity of the P(Fe-CO) band, exhibited two phases irrespective of the presence of one or two nu(Fe-CO) bands. The nu(Fe-CO) frequencies could be correlated with the hydropathy index of the E7 residue but no t with its physical size. It is inferred that the more hydrophobic env ironment around CO reduces the polarization of CO and lowers the nu(Fe -CO) frequency to approximately 490 cm-1 on the one hand and stabilize s the intermediate state called the protein-separated pair on the othe r. The latter increases the contribution of recombination from this in termediate, resulting in faster recombination of CO.