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 const itute the tetrameric hemoglobin in vertebrates. While alpha chains ass ociate weakly into dimers, beta chains self-associate into tightly ass embled tetramers. While heterotetramers bind ligands cooperatively wit h moderate affinity, homotetramers bind ligands with high affinity and without cooperativity. These characteristics lead to the conclusion t hat the beta4 tetramer is frozen in a quaternary R-state resembling th at of liganded HbA. X-ray diffraction studies of the liganded beta4 te tramers and molecular modeling calculations revealed several differenc es relative to the native heterotetramer at the ''allosteric'' interfa ce (alpha1beta2 in HbA) and possibly at the origin of a large instabil ity of the hypothetical deoxy T-state of the beta4 tetramer. We have s tudied natural and artificial Hb mutants at different sites in the bet a chains responsible for the T-state conformation in deoxy HbA with th e view of restoring a low ligand affinity with heme-heme interaction i n homotetramers. Functional studies have been performed for oxygen equ ilibrium binding and kinetics after flash photolysis of CO for both he tero- and homotetramers. Our conclusion is that the ''allosteric'' int erface is so precisely tailored for maintaining the assembly between a lphabeta dimers that any change in the side chains of beta40 (C6), bet a99 (G1), and beta101 (G3) involved in the interface results in increa sed R-state behavior. In the homotetramer, the mutations at these site s lead to the destabilization of the beta4 hemoglobin and the formatio n of lower affinity noncooperative monomers.