For many decades, hemoglobin has been the textbook example among allosteric proteins. Its structure has been depicted to great detail, and its function has been described according to several models, being the Two-State, T↔R Model of Monod, Wyman and Changeux, with the extension of the stereochemical model of Perutz, the best attempt to understand its allosteric behavior of ligand binding.
In the present work, we have attempted to reconstruct step- by-step the tetrameric human hemoglobin from its components, namely α and β subunits. Normally, β subunits assemble into tetramers of the form β4; however, alkylation of the βCys112 residue with either acetamide or N-ethyl succinamide produced monomers. Construction of αβ dimeric semihemoglobins in which only one type of the subunits contained heme while the complementary subunit remained heme-less, namely, in the apo form, revealed that their oxygen affinity lacked cooperativity and showed an affinity for oxygen that was intermediate to the first and last binding in native tetrameric hemoglobin. Strikingly, when the residue βCys112 was chemically modified with bulkier moieties, such as thiopyridyl, the binding of oxygen exhibited cooperativity that was compatible with a system with two oxygen binding sites. In other words, although semihemoglobins are known to be dimeric, the chemical modification of the α1β1 interface, wherein βCys112 is located, apparently produced the association of semihemoglobins into tetramers, and was responsible for the "cross-talk" between two heme-carrying β subunits, irrespective of the fact that α-subunits lacked heme. The binding for the first and second oxygen molecules showed affinities that were lower and higher than that of the monotonical unmodified dimeric semihemoglobin, respectively. This finding provides a clear evidence that the α1β1 interface, currently considered inert and playing no role in the cooperative oxygen binding, is pivotal and allosterically linked.