Coupling of the oxygen-linked interaction energy for inositol hexakisphosphate and bezafibrate binding to human HbA0

The energetics of signal propagation between different functional domains (i.e, the binding sites for O-2, inositol hexakisphospate (IHP), and bezafibrate (BZF)) of human HbA(0) was analyzed at different heme ligation states and through the use of a stable, partially heme ligated intermediate. Present data allow three main conclusions to be drawn, and namely: (i) IHP and BZF enhance each others binding as the oxygenation proceeds, the coupling free energy going from close to zero in the deoxy state to -3.4 kJ/mol in the oxygenated form; (ii) the simultaneous presence of IHP and BZF stabilizes the hemoglobin T quaternary structure at very low O-2 pressures, but as oxygenation proceeds it does not impair the transition toward the R structure, which indeed occurs also under these conditions; (iii) under room air pressure (i.e. pO(2) = 150 torr), IHP and BZF together induce the formation of an asymmetric dioxygenated hemoglobin tetramer, whose features appear reminiscent of those suggested for transition state species (i.e. T- and R-like tertiary conformation(s) within a quaternary R-like structure).