O<sub>2</sub> and Water Migration Pathways between the Solvent and Heme Pockets of Hemoglobin with Open and Closed Conformations of the Distal HisE7

Hemoglobin transports O<sub>2</sub> by binding the gas at its four hemes. Hydrogen bonding between the distal histidine (HisE7) and heme-bound O<sub>2</sub> significantly increases the affinity of human hemoglobin (HbA) for this ligand. HisE7 is also proposed to regulate the release of O<sub>2</sub> to the solvent via a transient E7 channel. To reveal the O<sub>2</sub> escape routes controlled by HisE7 and to evaluate its role in gating heme access, we compare simulations of O<sub>2</sub> diffusion from the distal heme pockets of the T and R states of HbA performed with HisE7 in its open (protonated) and closed (neutral) conformations. Irrespective of HisE7’s conformation, we observe the same four or five escape routes leading directly from the α- or β-distal heme pockets to the solvent. Only 21–53% of O<sub>2</sub> escapes occur via these routes, with the remainder escaping through routes that encompass multiple internal cavities in HbA. The conformation of the distal HisE7 controls the escape of O<sub>2</sub> from the heme by altering the distal pocket architecture in a pH-dependent manner, not by gating the E7 channel. Removal of the HisE7 side chain in the GlyE7 variant exposes the distal pockets to the solvent, and the percentage of O<sub>2</sub> escapes to the solvent directly from the α- or β-distal pockets of the mutant increases to 70–88%. In contrast to O<sub>2</sub>, the dominant water route from the bulk solvent is gated by HisE7 because protonation and opening of this residue dramatically increase the rate of influx of water into the empty distal heme pockets. The occupancy of the distal heme site by a water molecule, which functions as an additional nonprotein barrier to binding of the ligand to the heme, is also controlled by HisE7. Overall, analysis of gas and water diffusion routes in the subunits of HbA and its GlyE7 variant sheds light on the contribution of distal HisE7 in controlling polar and nonpolar ligand movement between the solvent and the hemes.