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

Haptoglobin (Hp) counterbalances the adverse effects of extra-erythrocytic hemoglobin (Hb) by trapping the αβ 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 k and k (i.e., (6.4 ± 0.8) × 10 s and (3.6 ± 0.6) × 10 s for Hp1-1:Hb(II)-NO and (5.8 ± 0.8) × 10 s and (3.1 ± 0.6) × 10 s for Hp2-2:Hb(II)-NO) are unaffected by allosteric effectors and correspond to those reported for the α and β subunits of tetrameric Hb(II)-NO and isolated α(II)-NO and β(II)-NO chains, respectively. This highlights the view that the conformation of the Hb αβ and αβ 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 β chains is closely similar for the O, NO and CO dissociation in the R-state, suggesting that it is solely determined by the structural difference between the two subunits.