Kinetics of cyanide and carbon monoxide dissociation from ferrous human haptoglobin:hemoglobin(II) complexes.

Haptoglobin (Hp) counterbalances the adverse effects of extra-erythrocytic hemoglobin (Hb) trapping the αß dimers of Hb. In turn, the Hp:Hb complexes display heme-based reactivity. Here, the kinetics of cyanide and carbon monoxide dissociation from ferrous-ligated Hp:Hb complexes are reported at pH 7.0 and 20.0 °C. Cyanide dissociation from Hp1-1:Hb(II)-CN- and Hp2-2:Hb-CN- has been followed upon the dithionite-mediated conversion of ferric to ferrous-ligated Hp:Hb complexes. Values of kon for the dithionite-mediated reduction of Hp1-1:Hb(III)-CN- and Hp2-2:Hb(III)-CN- are (7.3 ± 1.1) × 106 M-1 s-1 and (6.2 ± 1.0) × 106 M-1 s-1, respectively. Values of the first-order rate constant (i.e., h) for cyanide dissociation from Hp1-1:Hb(II)-CN- and Hp2-2:Hb(II)-CN- are (1.2 ± 0.2) × 10-1 s-1 and (1.3 ± 0.2) × 10-1 s-1, respectively. CO dissociation from Hp:Hb(II)-CO complexes has been followed by replacing CO with NO. Values of the first-order rate constant (i.e., l) for CO dissociation from Hp1-1:Hb(II)-CO are (1.4 ± 0.2) × 10-2 s-1 and (6.2 ± 0.8) × 10-3 s-1, and those from Hp2-2:Hb(II)-CO are (1.3 ± 0.2) × 10-2 s-1 and (7.3 ± 0.9) × 10-3 s-1. Values of kon, h, and l correspond to those reported for the R-state of tetrameric Hb and isolated α and ß chains. This highlights the view that the conformation of the Hb αß-dimers bound to Hp1-1 and Hp2-2 matches that of the R-state of the Hb tetramer. Furthermore, unlike ferric Hb(III), ligated ferrous Hb(II) does not show an assembly-linked structural change.

Fluoride and azide binding to ferric human hemoglobin:haptoglobin complexes highlights the ligand-dependent inequivalence of the α and ß hemoglobin chains.

Haptoglobin (Hp) binds human hemoglobin (Hb), contributing to prevent extra-erythrocytic Hb-induced damage. Hp forms preferentially complexes with αß dimers, displaying heme-based reactivity. Here, kinetics and thermodynamics of fluoride and azide binding to ferric human Hb (Hb(III)) complexed with the human Hp phenotypes 1-1 and 2-2 (Hp1-1:Hb(III) and Hp2-2:Hb(III), respectively) are reported (pH 7.0 and 20.0 °C). Fluoride binds to Hp1-1:Hb(III) and Hp2-2:Hb(III) with a one-step kinetic and equilibrium behavior. In contrast, kinetics of azide binding to and dissociation from Hp1-1:Hb(III)(-N3-) and Hp2-2:Hb(III)(-N3-) follow a two-step process. However, azide binding to 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 binding to Hp1-1:Hb(III) and Hp2-2:Hb(III) should reflect azide binding to the ferric ß and α chains, respectively, as also proposed for the similar behavior observed in Hb(III). Present results highlight the ligand-dependent kinetic inequivalence of Hb subunits in the ferric form, reflecting structural differences between the two subunits in the interaction with some ferric ligands.

Kinetic inequivalence between α and ß subunits of ligand dissociation from ferrous nitrosylated human haptoglobin:hemoglobin complexes. A comparison with O2 and CO dissociation.

Haptoglobin (Hp) counterbalances the adverse effects of extra-erythrocytic hemoglobin (Hb) by trapping the αß dimers of Hb in the bloodstream. In turn, the Hp:Hb complexes display Hb-like reactivity. Here, the kinetics of NO dissociation from ferrous nitrosylated Hp:Hb complexes (i.e., Hp1-1:Hb(II)-NO and Hp2-2:Hb(II)-NO, respectively) are reported at pH 7.0 and 20.0 °C. NO dissociation from Hp:Hb(II)-NO complexes has been followed by replacing NO with CO. Denitrosylation kinetics of Hp1-1:Hb(II)-NO and Hp2-2:Hb(II)-NO are biphasic, the relative amplitude of the fast and slow phase being 0.495 ± 0.015 and 0.485 ± 0.025, respectively. Values of koff(NO)1 and koff(NO)2 (i.e., (6.4 ± 0.8) × 10-5 s-1 and (3.6 ± 0.6) × 10-5 s-1 for Hp1-1:Hb(II)-NO and (5.8 ± 0.8) × 10-5 s-1 and (3.1 ± 0.6) × 10-5 s-1 for Hp2-2:Hb(II)-NO) are unaffected by allosteric effectors and correspond to those reported for the α and ß subunits of tetrameric Hb(II)-NO and isolated α(II)-NO and ß(II)-NO chains, respectively. This highlights the view that the conformation of the Hb α1ß1 and α2ß2 dimers matches that of the Hb high affinity conformation. Moreover, the observed functional heterogeneity reflects the variation of energy barriers for the ligand detachment and exit pathway(s) associated to the different structural arrangement of the two subunits in the nitrosylated R-state. Noteworthy, the extent of the inequivalence of α and ß chains is closely similar for the O2, NO and CO dissociation in the R-state, suggesting that it is solely determined by the structural difference between the two subunits.