Distal Histidine Stabilizes Bound O2 and Acts as a Gate for Ligand Entry in Both Subunits of Adult Human Hemoglobin

The role of the distal histidine in regulating ligand binding to adult human hemoglobin (HbA) was re-examined systematically by preparing His(E7) to Gly, Ala, Leu, Gln, Phe, and Trp mutants of both Hb subunits. Rate constants for O2, CO, and NO binding were measured using rapid mixing and laser photolysis experiments designed to minimize autoxidation of the unstable apolar E7 mutants. Replacing His(E7) with Gly, Ala, Leu, or Phe causes 20–500-fold increases in the rates of O2 dissociation from either Hb subunit, demonstrating unambiguously that the native His(E7) imidazole side chain forms a strong hydrogen bond with bound O2 in both the α and β chains (ΔGHis(E7)H-bond ≈ −8 kJ/mol). As the size of the E7 amino acid is increased from Gly to Phe, decreases in kO2′, kNO′, and calculated bimolecular rates of CO entry (kentry′) are observed. Replacing His(E7) with Trp causes further decreases in kO2′, kNO′, and kentry′ to 1–2 μm−1 s−1 in β subunits, whereas ligand rebinding to αTrp(E7) subunits after photolysis is markedly biphasic, with fast kO2′, kCO′, and kNO′ values ≈150 μm−1 s−1 and slow rate constants ≈0.1 to 1 μm−1 s−1. Rapid bimolecular rebinding to an open α subunit conformation occurs immediately after photolysis of the αTrp(E7) mutant at high ligand concentrations. However, at equilibrium the closed αTrp(E7) side chain inhibits the rate of ligand binding >200-fold. These data suggest strongly that the E7 side chain functions as a gate for ligand entry in both HbA subunits.