SERINE(92) (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 myoglobi n crystal structures, the hydroxyl group of serine92 is within hydroge n-bonding distance of the N(delta)-H of histidine 93, whose N(epsilon) coordinates the iron atom of the heme prosthetic group. The associati on 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 wit h the mutant ferric proteins at neutral pH are decreased by factors of 2-5 consistent with an increased affinity for the iron-bound water mo lecule which must be displaced. The dissociation rates for azide appea r to be decreased 4-10-fold, suggesting that the affinity of the mutan t proteins for this ligand is also higher. Thus, the overall affinitie s are increased regardless of the chemical nature of the liganded spec ies, indicating that the reactivity of the heme iron itself has been r aised. 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 trac es with spectra following the reaction of isocyanide ligands with chel ated proton heme in soap micelles indicates that the slow phase is ass ociated with the breaking of the iron-proximal histidine bond and the binding of a second isocyanide species in the proximal heme pocket. Th e rates of hemin dissociation from the Ala92 and Leu92 mutants are 10- 20-fold faster than that for the wild-type protein at pH 5. The X-ray structure of the aquomet form of the Leu92 mutant has been solved to a nominal d(min) = 2.7 angstrom. Hydrogen-bonding and electrostatic int eractions involving residue92, the proximal histidine, the heme-7-prop ionate, and histidine97 have been disrupted, leading to a more exposed proximal heme pocket which allows access of the second isocyanide lig and and lowers the stability of the heme with respect to dissociation.