Determination of Pathways for Oxygen Binding to Human Hemoglobin A

[摘要] The role of His(E7) in ligand binding to HbA was re-examined in view ofdiscrepancies between early kinetic studies and recent structure determinations.Replacing His(E7) with Gly, Leu, Phe, and Trp causes 20- to 500-fold increases in therates of O2 dissociation from both subunits, and FfIR spectra reveal a shift in the C-Ostretching frequency peak from 1950 cm-1 to -1970 cm-1 for apolar mutations, indicatingloss of a positive electrostatic field next to bound ligands. Thus, the native His(E7) sidechain 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 therate constants for CO, 02 and NO association to HbA. Substituting His(E7) with Trp alsoslows down opening and closing of the E7 gate. Ligand binding to the Trp(E7) subunits ismarkedly biphasic due to a competition between very fast recombination to an openconformer and relaxation of the indole side chain to closed or blocked forms. Crystalstructures of Hb and Mb Trp(E7) mutants provide structural models for these closed andblocked conformers. In the closed state, the indole side chain fills both the E7 channeland 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. Thebimolecular 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 wildtypeparameter (-7 IlM-1s-1). Filling potential alternative ligand pathways with xenondoes 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 andGS positions dramatically affects both geminate recombination and bimolecular ligandbinding. Taken together, these results demonstrate that the E7 channel is the majorpathway for ligand entry and escape in HbA and that previously proposed ligandmigration routes involving Xe cavities are not functionally significant.

[发布机构] Rice University

[效力级别] Cellular