STRUCTURAL DETERMINANTS OF THE STRETCHING FREQUENCY OF CO BOUND TO MYOGLOBIN

In order to assess the relative importance of polar versus steric interactions, infrared spectra and overall CO binding properties were measured at room temperature for 41 different recombinant myoglobins containing mutations at His(64)(E7), Val(68)(E11), Phe(43)(CD1), Arg(45)(CD3), Phe(46)(CD4), and Leu(29)(B10). The results were compared to the crystal structures of wild-type, Phe(29), Val(46), Ala(68), Phe(68), Gln(64), Leu(64), and Gly(64) sperm whale CO-myoglobin and that of Thr(68) pig CO-myoglobin. As observed in several previous studies, replacement of the distal histidine (His(64)) With aliphatic amino acids results in the appearance of a single IR band in the 1960-1970-cm(-1) region and in large increases in CO affinity (K-CO). More complex behavior is observed for Gly, Ala, Gin, Met, and Trp substitutions at position 64, but in each case there is a net increase in the intensity of this high-frequency component. Replacement of Val(68) with Ala, Leu, Ile, and Phe produces little effect on the IR spectrum, whereas these mutations cause 20-fold changes in K-CO, presumably due to steric effects. Replacement of Val(68) with Thr decreases K-CO 4-5-fold, whereas the position of the major IR band increases from 1945 to 1961 cm(-1). Replacement of Val(68) with Asn also causes a large decrease in K-CO, but in this case, the peak position of the major IR band decreases from 1945 to 1916 cm(-1). Nine replacements were made in the CD corner at positions 43, 45, and 46. All of the resultant mutants show increased stretching frequencies that can be correlated with movement of the imidazole side chain of His(64) away from the bound ligand. All five substitutions at position 29 cause changes in the IR spectra. The Leu(29)-->Phe mutation had the largest effect, producing a single band centered at 1932 cm(-1). Together these data demonstrate that there is little direct correlation between affinity, nu(CO) and Fe-C-O geometry. The major factor governing nu(CO) appears to be the electrostatic potential surrounding the bound ligand and not steric hindrance. The presence of positive charges from proton donors, such as N-epsilon of His(64) and N-delta of Asn(68), causes a decrease in the bond order and stretching frequency of bound CO, In contrast, the negative portion of the Thr(68) dipole points directly toward the bound ligand and increases the C-O bond order and stretching frequency. Movement of His(64) away from the bound ligand or replacement of this residue with aliphatic amino acids prevents hydrogen-bonding interactions, causing nu(CO) to increase. Placement of the positive portion of the aromatic multipole of Phe(29) next to bound CO has the opposite effect, causing a decrease in the order of the C-O bond. Substitutions that increase the space available in the distal pocket cause more subtle alterations in the IR spectrum; which can be attributed to increased conformational flexibility and enhanced polar interactions of the bound ligand with solvent water molecules. Thus, the vibrational spectrum of bound CO is a sensitive gauge of electrostatic potentials near the ligand binding site in myoglobin.