Splitting of the Allosteric Function in Human Hemoglobin with an Altered Alpha1Beta1 Interface

Abstract

Previously, it was shown that human semihemoglobins, i.e., hemoglobin dimers of the form (alpha)(beta) in which only one subunit, either alpha or beta, contained a functional heme group while the complementary apo subunit lacked heme, were sensitive to allosteric effectors, which caused modulation of their affinity for oxygen (Tsuneshige, A. et al. (2004) J. Biol. Chem. 279, 48959-48967). The presented evidences contradicted the classic tenet of the “two-state” model of allostery in which modulation of the affinity for oxygen could only be achieved by a tetrameric hemoglobin adopting the high-affinity “R” or the low-affinity “T” conformations in a reversible fashion.For the present study, we have prepared a hemoglobin molecule in which the residues alpha104Cys and beta112Cys were chemically modified following a reaction of the sulhydryl groups with a thiopyridyl reagent. These two residues are present in the alpha1beta1 interface, thus we expected that their chemical modifications would impair drastically the intradimeric communication. Surprisingly, oxygenation curves at different pH values, or in the presence of the allosteric efector inositol hexakisphosphate (IHP) showed striking common characteristics: symmetric shape, presence of cooperative binding of oxygen, and a corresponding decrease in overall oxygen affinity in response to acidic conditions or the presence of IHP. Moreover, affinities for oxygen at low and high saturation levels were both affected in similar fashion under any solution condition. These results strongly suggest that the modified hemoglobin behave like dimers exhibiting allosteric properties.