The effects of heme pocket hydrophobicity on the ligand binding dynamics in myoglobin as studied with leucine 29 mutants.

To examine the effects of heme pocket hydrophobicity on the ligand binding in myoglobin, some artificial mutants of human myoglobin have been prepared, in which less hydrophobic amino acid residue (Ala, Gly, Ser) is located at the Leu29 (10th residue of the B helix) position. CO rebinding rates for the mutants were markedly decelerated, while the 1H, and 15N NMR spectra of the mutants show that the structural changes around the heme iron for these mutants are rather small. The kinetic and structural properties of the mutants indicate that the ligand binding rate depends on the hydrophobicity inside the heme cavity for these mutants in addition to the volume of the side chain at the 29-position. On the basis of the IR stretching frequency of liganded CO, invasion of water molecules into the heme pocket in the mutants is suggested, which would be induced by the decrease in the hydrophobicity due to the amino acid substitution. A slight red shift of the position of the Soret peak for the serine mutant L29S also supports the reduced hydrophobicity inside the heme cavity. We can concluded that, together with the kinetic properties of the mutants, the hydrophobicity of the heme pocket is one of the key factors in regulating the ligand binding to the heme iron.