The role of His(E7) in ligand binding to HbA was re-examined in view of discrepancies between early kinetic studies and recent structure determinations. Replacing His(E7) with Gly, Leu, Phe, and Trp causes 20- to 500-fold increases in the rates of O2 dissociation from both subunits, and FfIR spectra reveal a shift in the C-O stretching frequency peak from 1950 cm-1 to -1970 cm-1 for apolar mutations, indicating loss of a positive electrostatic field next to bound ligands. Thus, the native His(E7) side chain forms a strong hydrogen bond (- -8 kJ/mol) with bound 02 in both HbA subunits. Increasing the size of the E7 residue from Gly to Trp monotonically decreases the rate constants for CO, 02 and NO association to HbA. Substituting His(E7) with Trp also slows down opening and closing of the E7 gate. Ligand binding to the Trp(E7) subunits is markedly biphasic due to a competition between very fast recombination to an open conformer and relaxation of the indole side chain to closed or blocked forms. Crystal structures of Hb and Mb Trp(E7) mutants provide structural models for these closed and blocked conformers. In the closed state, the indole side chain fills both the E7 channel and the distal pocket, inhibiting binding to iron from any direction. In the blocked state, Trp(E7) is located in the solvent interface but still blocks entry into the E7 channel. The bimolecular rate constants for CO binding to the closed and blocked states are 0.08 IlMIS- l and 0.7IlM-1S-1, respectively, which are -100 and -lO-fold slower than average wildtype parameter (-7 IlM-1s-1). Filling potential alternative ligand pathways with xenon does not affect the rate or fraction of ligand escape from either HbA subunit. In contrast, reducing the volume of the distal pocket by space-filling mutations at the BlO, Ell and GS positions dramatically affects both geminate recombination and bimolecular ligand binding. Taken together, these results demonstrate that the E7 channel is the major pathway for ligand entry and escape in HbA and that previously proposed ligand migration routes involving Xe cavities are not functionally significant.