A signature of the T ---> R transition in human hemoglobin.

Allosteric effects in hemoglobin arise from the equilibrium between at least two energetic states of the molecule: a tense state, T, and a relaxed state, R. The two states differ from each other in the number and energy of the interactions between hemoglobin subunits. In the T state, constraints between subunits oppose the structural changes resulting from ligand binding. In the R state, these constraints are released, thus enhancing ligand-binding affinity. In the present work, we report the presence of four sites in hemoglobin that are structurally stabilized in the R relative to the T state. These sites are His alpha 103(G10) and His alpha 122(H5) in each alpha subunit of hemoglobin. They are located at the alpha(1)beta(1) and alpha(2)beta(2) interfaces of the hemoglobin tetramer, where the histidine side chains form hydrogen bonds with specific residues from the beta chains. We have measured the solvent exchange rates of side chain protons of His alpha 103(G10) and His alpha 122(H5) in both deoxygenated and ligated hemoglobin by NMR spectroscopy. The exchange rates were found to be higher in the deoxygenated-T than in ligated-R state. Analysis of exchange rates in terms of the local unfolding model revealed that the structural stabilization free energy at each of these two histidines is larger by approximately 1.5 kcal/(mol tetramer) in the R relative to the T state. The location of these histidines at the intradimeric alpha(1)beta(1) and alpha(2)beta(2) interfaces also suggests a role for these interfaces in the allosteric equilibrium of hemoglobin.