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 His64(E7), Val68(E11), Phe43(CD1), Arg45(CD3), Phe46(CD4), and Leu29(B10). The results were compared to the crystal structures of wild-type, Phe29, Val46, Ala68, Phe68, Gln64, Leu64, and Gly64 sperm whale CO-myoglobin and that of Thr68 pig CO-myoglobin. As observed in several previous studies, replacement of the distal histidine (His64) 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 (KCO). More complex behavior is observed for Gly, Ala, Gln, 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 Val68 with Ala, Leu, Ile, and Phe produces little effect on the IR spectrum, whereas these mutations cause 20-fold changes in KCO, presumably due to steric effects. Replacement of Val68 with Thr decreases KCO 4-5-fold, whereas the position of the major IR band increases from 1945 to 1961 cm-1. Replacement of Val68 with Asn also causes a large decrease in KCO, 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 His64 away from the bound ligand. All five substitutions at position 29 cause changes in the IR spectra. The Leu29-->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, vCO, and Fe-C-O geometry. The major factor governing vCO 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 His64 and N delta of Asn68, cause a decrease in the bond order and stretching frequency of bound CO. In contrast, the negative portion of the Thr68 dipole points directly toward the bound ligand and increases the C-O bond order and stretching frequency. Movement of His64 away from the bound ligand or replacement of this residue with aliphatic amino acids prevents hydrogen-bonding interactions, causing vCO to increase.(ABSTRACT TRUNCATED AT 250 WORDS)

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Formation of a water-expelling nonpolar core is the paradigm of protein folding and stability. Although experiment largely confirms this picture, water buried in “hydrophobic” cavities is required for the function of some proteins. Hydration of the protein core has also been suggested as the mechanism of pressure-induced unfolding. We therefore are led to ask whether even the most nonpolar protein core is truly hydrophobic (i.e., water-repelling). To answer this question we probed the hydration of an ≈160-Å 3 , highly hydrophobic cavity created by mutation in T4 lysozyme by using high-pressure crystallography and molecular dynamics simulation. We show that application of modest pressure causes approximately four water molecules to enter the cavity while the protein itself remains essentially unchanged. The highly cooperative filling is primarily due to a small change in bulk water activity, which implies that changing solvent conditions or, equivalently, cavity polarity can dramatically affect interior hydration of proteins and thereby influence both protein activity and folding.