Nuclear magnetic resonance study of the isotope exchange of the proximal histidyl ring labile protons in hemoglobin A. The exchange rates and mechanisms of individual subunits in deoxy and oxy-hemoglobin.

Proton nuclear magnetic resonance spectroscopy has been used to investigate the rates and mechanism of exchange with deuterium of the proximal histidyl imidazole labile ring proton in deoxy and oxy-hemoglobin A. The resolved signals for the two subunits indicate dynamic heterogeneity, with the exchange rate always faster in the alpha than the beta subunits, suggesting a lower dynamic stability for the alpha subunit. The activation energy for the exchange in both subunits (approximately 25 kcal; 1 cal = 4.184 J) indicates that exchange proceeds via an intermediate far from denaturation or global unfolding. The pH profiles for both hemoglobin states reflect the EX2 mechanism for both subunits. While the base catalysis expected for an iron-bound imidazole is observed in all cases, there are important differences in both rates and mechanisms between the subunits. In deoxy-hemoglobin, both base-catalyzed and water-assisted exchange contribute to the alpha subunit, but only the former to the beta subunit. For oxy-hemoglobin, the base-catalysis is retained for both subunits, but the slope is considerably less for the alpha relative to the beta subunit. Thus the two subunits in the two states of hemoglobin differ both in mechanisms and in the inherent dynamic stability reflected in any one mechanism. The relationships of the proximal histidyl ring NH exchange rates to previously characterized subsets of allosterically responsive protons in hemoglobin A is briefly discussed.