Discrimination between oxygen and carbon monoxide and inhibition of autooxidation by myoglobin. Site-directed mutagenesis of the distal histidine

Sperm whale myoglobin mutants were constructed using site-directed mutagenesis to replace the highly conserved distal histidine residue (His(E7)-64). His-64 was substituted with Gly, Val, Phe, Cys, Met, Lys, Arg, Asp, Thr, and Tyr, and all 10 mutant proteins expressed to approximately 10% of the total soluble cell protein in Escherichia coli as heme containing myoglobin. With the exception of His-64 → Tyr, which did not form a stable oxygen (O₂) complex, all mutant proteins could be reduced and bound O₂ and carbon monoxide (CO) reversibly. However, removal of the distal histidine increased the rate of autooxidation 40-350-fold. The His-64 → Gly, Val, Phe, Met, and Arg mutants all showed markedly increased O₂ dissociation rate constants which were ~50-1500-fold higher than those for wild-type myoglobin and increased O₂ association rate constants which were ~5-15-fold higher than those for the native protein. All mutants studied (except His-64 → Tyr) showed ~10-fold increased CO association rates and relatively unchanged CO dissociation rates. These altered O₂ and CO association and dissociation rate constants resulted in 3-14-fold increased CO affinities, 10-200-fold decreased O₂ affinities, and 50-300-fold greater K (K(CO)/K(O₂)) values for the mutants compared to the wild-type protein. Thus, the distal histidine of myoglobin discriminates between CO and O₂ binding by both sterically hindering bound CO and stabilizing bound O₂ through hydrogen bonding. The increased autooxidation rates observed for the mutants appear to be due to a decrease in oxygen affinity and an increase in solvent anion accessibility to the distal pocket.