Allosteric free energy changes at the α₁β₂ interface of human hemoglobin probed by proton exchange of Trpβ37

The energetic changes that occur on ligand binding in human hemoglobin have been investigated by measurements of the exchange rates of the indole proton of Trpβ37(C3). The Trpβ37 residues are located in helices C of the β-subunits and are involved in contacts with the segments FG of the α-subunits at the interdimeric α₁β₂ and α₂β₁ interfaces of the hemoglobin tetramer. In the quaternary structure change that accompanies ligand binding to hemoglobin, these contacts undergo minimal changes in relative orientation and in packing, thereby acting as hinges, or flexible joints. The exchange rates of the indole proton of Trpβ37(C3) were measured by nuclear magnetic resonance spectroscopy, in both deoxygenated and ligated hemoglobin. The results indicate that, at 15°C, the exchange rate is increased from 9.0.10^-6 to 3.3·10^-4 s^-1 upon ligand binding to hemoglobin. This change suggests that the structural units at the hinge regions of the α₁β₂/α₂β₁ interfaces containing Trpβ37(C3) are specifically stabilized in unligated hemoglobin, and experience a change in structural free energy of ∼4 kcal/(mol tetramer) upon ligand binding. Therefore, the hinge regions of the α₁β₂/α₂β₁ interfaces could play a role in the transmission of free energy through the hemoglobin molecule during its allosteric transition.