FUNCTIONAL CONSEQUENCES OF MUTATIONS AT THE ALLOSTERIC INTERFACE IN HETERO-HEMOGLOBIN AND HOMO-HEMOGLOBIN TETRAMERS

A seminal difference exists between the two types of chains that constitute the tetrameric hemoglobin in vertebrates. While alpha chains associate weakly into dimers, beta chains self-associate into tightly assembled tetramers. While heterotetramers bind ligands cooperatively with moderate affinity, homotetramers bind ligands with high affinity and without cooperativity. These characteristics lead to the conclusion that the beta4 tetramer is frozen in a quaternary R-state resembling that of liganded HbA. X-ray diffraction studies of the liganded beta4 tetramers and molecular modeling calculations revealed several differences relative to the native heterotetramer at the ''allosteric'' interface (alpha1beta2 in HbA) and possibly at the origin of a large instability of the hypothetical deoxy T-state of the beta4 tetramer. We have studied natural and artificial Hb mutants at different sites in the beta chains responsible for the T-state conformation in deoxy HbA with the view of restoring a low ligand affinity with heme-heme interaction in homotetramers. Functional studies have been performed for oxygen equilibrium binding and kinetics after flash photolysis of CO for both hetero- and homotetramers. Our conclusion is that the ''allosteric'' interface is so precisely tailored for maintaining the assembly between alphabeta dimers that any change in the side chains of beta40 (C6), beta99 (G1), and beta101 (G3) involved in the interface results in increased R-state behavior. In the homotetramer, the mutations at these sites lead to the destabilization of the beta4 hemoglobin and the formation of lower affinity noncooperative monomers.