Thermodynamic studies on the equilibrium properties of a series of recombinant βW37 hemoglobin mutants

In human hemoglobin (Hb) the β37 tryptophan residue (βW37), located at the hinge region of the α¹β² interface, forms many contacts with α subunit residues of the opposite dimer, in both the T and R quaternary structures. We have carried out equilibrium O₂ binding studies on a series of recombinant Hbs that have mutations at this residue site: βW37Y, βW37A, βW37G, and βW37E. Binding isotherms measured at high concentrations of these mutants were found to be shifted toward increased affinity and decreased cooperativity from that of the normal HbA(O) tetramer. Analysis of these binding isotherms indicated that amino acid substitutions at the β37 position could both destabilize the tetrameric form of the mutants relative to their constituent dimers and also alter cooperativity of the intact tetrameric species. These alterations from wild-type function are dependent on the particular side chain substituted, with the magnitude of change increasing as Trp is substituted by Tyr, Ala, Gly, and Glu. The dimer to tetramer assembly free energy of deoxy-βW37E, the most perturbed mutant in the series, was measured using analytical gel chromatography to be 9 kcal/tetramer less favorable than that of deoxy HbA(0). Stabilizing the βW37E tetramer by addition of IHP, or by cross-linking at the αK99 positions, does not restore normal O₂ binding behavior. Thermodynamic parameters of all the mutants were found to correlate with their CO binding rates and with their high-resolution X-ray crystal structures (see accompanying papers: Kwiatkowski et al. (1998) Biochemistry 37, 4325-4335; Peterson and Friedman (1998) Biochemistry 37, 4346-4357; Kavanaugh et al. (1998) Biochemistry 37, 4358-4373].