Genetic Engineering of Myoglobin as a Simple Prototype for Hemoglobin-Based Blood Substitutes

Abstract

Site-directed mutagenesis has been used to examine the structural and functional roles of distal pocket residues in regulating O₂ affinity, CO binding, rates of association and dissociation, autooxidation, and hemin loss in mammalian myoglobins and human hemoglobin. In myoglobin, His-E7 inhibits CO binding by requiring displacement of distal pocket water. In the case of O₂ binding, this displacement is compensated by a strong hydrogen bond between the bound ligand and the imidazole side chain. The isopropyl side chain Val-E11 also sterically restricts CO binding. The rates of ligand binding are regulated by distal pocket water displacement, steric restrictions near the iron atom, and an outer more global protein barrier. Autooxidation occurs by two mechanisms, direct dissociation of HO₂ and bimolecular reaction of external O₂ with unliganded heme. Both processes are inhibited markedly by hydrogen bonding interactions with His-E7. Double mutants have been constructed to decrease oxygen affinity, but still prevent oxidation. The apoprotein of His-E7 → Tyr myoglobin has been used to extract hemin from other myoglobins and hemoglobin, causing a brown to green color change. This assay has been used to show that polar interactions between residues CDS, E7, E10, F7, and the porphyrin propionates inhibit hemin dissociation markedly.