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 alpha beta 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 alpha(III) and beta(III) chains are reported and analyzed in parallel with fluoride and azide binding properties (at pH 7.0 and 20.0 degrees C). Cyanide and fluoride bind to Hb(III), Hp1-1:Hb(III), Hp2-2:Hb(III), alpha(III), and beta(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 alpha(III) and beta(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 beta(III) and alpha(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 alpha(III) and beta(III) chains, confirming the ligand-dependent kinetic inequivalence of alpha(III) and beta(III) subunits. However, a variation between tetrameric Hb(III) on one side and Hp1-1:Hb(III), Hp2-2:Hb(III), alpha(III), and beta(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).