Structural determinants of functional behavior in distal pocket mutants of myoglobin

Abstract

The physiological role of myoglobin depends on the modulation of heme activity by the protein. The hypothesis that functional properties are governed by conserved residues in the distal pocket has been tested by site-directed mutagenesis of three residues: Leu$\sp{29}$, His$\sp{64}$, and Val$\sp{68}$. To facilitate interpretation of functional data, structures of several mutant myoglobins have been determined by X-ray crystallography. Leu$\sp{29}$ controls the volume of the distal pocket. Since Val$\sp{29}$ does not contact bound ligands, this substitution does not affect ligand affinities significantly. It does permit solvent approach to the heme, thereby increasing the rate of autooxidation. Although the Phe$\sp{29}$ mutant was constructed to reduce the volume of the binding site, dipole-multipole interactions stabilize bound oxygen and reduce the rate of autooxidation substantially. His$\sp{64}$ inhibits oxygen dissociation and autooxidation by hydrogen bonding to the ligand. In conjunction with a distal water molecule, it sterically hinders carbon monoxide association. Mutation of this residue eliminates hydrogen-bonding interactions in all cases except Gln$\sp{64}$, producing low oxygen affinities and high rates of autooxidation. In the Gly$\sp{64}$ mutant, the solvent-filled distal cavity partially restores binding site polarity. In contrast, the distal pockets of Val$\sp{64}$, Thr$\sp{64}$, and Leu$\sp{64}$ are completely apolar, leading to marked increases in rates of ligand binding. Val$\sp{68}$ governs the ligand accessibility of the iron. In the Ala$\sp{68}$ mutant, only slight rate enhancements occur because the distal water molecule is retained in the deoxygenated protein. The larger side chains of Ile$\sp{68}$ and Leu$\sp{68}$ displace this water molecule and occlude the binding site in unliganded structures. The lower affinities observed in Ile$\sp{68}$ compared to Leu$\sp{68}$ are due to the decreased ability of this residue to accommodate the bound ligand. In contrast, the Phe$\sp{68}$ side chain is directed away from the iron atom and does not inhibit binding directly. Nevertheless, the reduced volume in this mutant is filled with a water molecule, retarding ligand association. In all mutants, structural perturbations are limited to the site of the substitution and the flexible corner regions of myoglobin. Furthermore, the stereochemistry of the heme-ligand complex is little influenced by changes in the distal pocket.