Distal histidine stabilizes bound O2 and acts as a gate for ligand entry in both subunits of adult human hemoglobin.

Ivan Birukou,John S Olson,Rachel L Schweers

doi:10.1074/jbc.m109.053934

Abstract

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 O(2), 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 O(2) dissociation from either Hb subunit, demonstrating unambiguously that the native His(E7) imidazole side chain forms a strong hydrogen bond with bound O(2) in both the alpha and beta chains (DeltaG(His(E7)H-bond) approximately -8 kJ/mol). As the size of the E7 amino acid is increased from Gly to Phe, decreases in k(O2)', k(NO)', and calculated bimolecular rates of CO entry (k(entry)') are observed. Replacing His(E7) with Trp causes further decreases in k(O2)', k(NO)', and k(entry)' to 1-2 microM(-1) s(-1) in beta subunits, whereas ligand rebinding to alphaTrp(E7) subunits after photolysis is markedly biphasic, with fast k(O2)', k(CO)', and k(NO)' values approximately 150 microM(-1) s(-1) and slow rate constants approximately 0.1 to 1 microM(-1) s(-1). Rapid bimolecular rebinding to an open alpha subunit conformation occurs immediately after photolysis of the alphaTrp(E7) mutant at high ligand concentrations. However, at equilibrium the closed alphaTrp(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.

Highlights

IntroductionNeither the dissociation nor association rate constants for O2 binding to the R state ␤Gly(E7) mutant of HbA appeared to increase significantly, implying no electrostatic stabilization of bound ligands by the native His(E7) in ␤ subunits and an already open gate or alternative pathway
This approach is difficult when examining distal histidine mutants because of the following: 1) the quantum yield for complete photodissociation of O2 into solvent is inherently small, Յ0.1, and decreases even further when His(E7) is replaced with an apolar amino acid; 2) the E7 apolar mutants autoxidize rapidly with half-lives ranging from a few minutes to less than 1 h; and 3) complete removal of CO from His(E7) mutants is very difficult because these variants have KCO/KO2 ratios in the range 4000 – 40,000
Time courses for bimolecular O2 binding to isolated subunits in photolysis experiments with HbCO are shown in Fig. 2 for the complete set of E7 mutations

Summary

Introduction

Neither the dissociation nor association rate constants for O2 binding to the R state ␤Gly(E7) mutant of HbA appeared to increase significantly, implying no electrostatic stabilization of bound ligands by the native His(E7) in ␤ subunits and an already open gate or alternative pathway. These surprising kinetic results were explained by the first high resolution structure of human oxyhemoglobin published by Shaanan [12], in which the N⑀H atoms of distal histidine in ␤ subunits seemed to be further away from the bound O2 atoms and pointing toward the heme plane. The experimental evidence has been weak, due in part to the technical difficulties of gener-

Results

Discussion

Conclusion