Ligand-dependent inequivalence of the α and β subunits of ferric human hemoglobin bound to haptoglobin.
Haptoglobin (Hp) prevents extra-erythrocytic hemoglobin- (Hb-)mediated damage. Hp binds αβ dimers of Hb, displaying heme-based reactivity. Here, kinetics and thermodynamics of cyanide, thiocyanate and imidazole binding to ferric human Hb (Hb(III)), Hb(III) dimers complexed with the human Hp phenotypes 1-1 and 2-2 (Hp1-1:Hb(III) and Hp2-2:Hb(III), respectively), and α(III) and β(III) chains are reported and analyzed in parallel with fluoride and azide binding properties (at pH 7.0 and 20.0 °C). Cyanide and fluoride bind to Hb(III), Hp1-1:Hb(III), Hp2-2:Hb(III), α(III), and β(III) with a simple behavior. In contrast, azide, thiocyanate and imidazole binding to Hb(III), Hp1-1:Hb(III) and Hp2-2:Hb(III) follows a two-step process, whereas ligand binding to α(III) and β(III) chains follows a simple behavior. However, azide, thiocyanate and imidazole binding to Hb(III), Hp1-1:Hb(III) and Hp2-2:Hb(III) is characterized by a simple equilibrium, reflecting the compensation of kinetic parameters. The fast and the slow step of azide, thiocyanate and imidazole binding to Hb(III), Hp1-1:Hb(III) and Hp2-2:Hb(III) mirror the ligand binding properties of the β(III) and α(III) chains, respectively. Values of kinetic and thermodynamic parameters for binding of ferric ligands to Hp1-1:Hb(III) and Hp2-2:Hb(III) match very well with those obtained for ligation of Hb(III) and α(III) and β(III) chains, confirming the ligand-dependent kinetic inequivalence of α(III) and β(III) subunits. However, a variation between tetrameric Hb(III) on one side and Hp1-1:Hb(III), Hp2-2:Hb(III), α(III), and β(III) on the other side for the rate-limiting step (likely referable to the dissociation of heme-coordinated H2O from the heme-Fe(III) atom) suggests a structural change(s) upon dimers to tetramer assembly in Hb(III).