Serine92 (F7) contributes to the control of heme reactivity and stability in myoglobin

The effects of mutation of the conserved serine92 residue to alanine, valine, and leucine in pig myoglobin have been determined. In myoglobin crystal structures, the hydroxyl group of serine92 is within hydrogen-bonding distance of the N delta-H of histidine93, whose N epsilon coordinates the iron atom of the heme prosthetic group. The association equilibrium constants of the ferrous forms of the mutant myoglobins for O2, CO, and methyl and ethyl isocyanide are increased 1.3-13-fold relative to the wild-type protein. The rates of azide association with the mutant ferric proteins at neutral pH are decreased by factors of 2-5 consistent with an increased affinity for the iron-bound water molecule which must be displaced. The dissociation rates for azide appear to be decreased 4-10-fold, suggesting that the affinity of the mutant proteins for this ligand is also higher. Thus, the overall affinities are increased regardless of the chemical nature of the liganded species, indicating that the reactivity of the heme iron itself has been raised. Time courses for association of methyl and ethyl isocyanide at high concentrations show fast and slow phases in which the absorbance at 445 nm drops and then rises, respectively. Comparison of these traces with spectra following the reaction of isocyanide ligands with chelated proton heme in soap micelles indicates that the slow phase is associated with the breaking of the iron-proximal histidine bond and the binding of a second isocyanide species in the proximal heme pocket.