Role of His(E7) in Regulating Ligand Binding to the Subunits of Human HbA

Abstract

To resolve previous discrepancies between structural and functional studies, the role of the distal histidine in HbA was re-evaluated by preparing Gly, Ala, Leu, Gln, Phe and Trp(E7) mutants and measuring the effects on O2, CO, and NO binding to mutant/wild type hybrid tetramers and isolated mutant subunits. Substituting His(E7) with apolar amino acids dramatically increases O2 dissociation (20-500-fold) in both subunits, suggesting equally strong hydrogen bonds between His(E7) and bound O2 (ΔGH-bond ≈ −5.6 kJ/mol). Increasing the size of the E7 residue from Gly to Phe results in monotonic decreases in the bimolecular rates of ligand binding to both subunits, supporting the E7 gate as the pathway for ligand entry in HbA. The results for the Trp(E7) mutants are more complex. Both fast (∼150-200 μM−1s−1) and one or more slow phases (1 to 0.1 μM−1s−1) are observed after photolysis of CO. The fraction of the fast phase decreases markedly when [CO] is lowered. In contrast, when isolated α and βTrp(E7) deoxyHb subunits are mixed with CO in stopped flow experiments, only slow phases are observed. Thus, after photolysis of the CO form of Trp(E7) mutants, there appears to be a competition between bimolecular ligand rebinding to an “open” conformation and the movement of the indole side chain back into the E7 channel forming an equilibrium “closed,” slowly reacting conformation. This mechanism is supported by the crystal structure of the CO form of α (wt)/βTrp(E7), in which the mutant indole side chain is in an open conformation exposed to solvent. In the deoxyHb crystal structure of αTrp(E7)/β (wt), the indole ring of Trp(E7) is in a closed conformation, blocking both the ligand binding site and the E7 channel for ligand entry.