ALLOSTERIC MECHANISM OF HEMOGLOBIN - RUPTURE OF SALT-BRIDGES RAISES THE OXYGEN-AFFINITY OF THE T-STRUCTURE

The T-structure of human haemoglobin is linked by salt-bridges between its four subunits, formed by the C-terminal arginine residues of the alpha-subunits and the C-terminal histidine residues of the beta-subun its. Ln the R-structure, these salt-bridges are absent. The oxygen aff inity of the T-structure is lower than that of the R-structure by the equivalent of 3.5 kcal/mol haem. This difference has been attributed t o the constraints imposed upon the T-structure by the salt-bridges, wh ich were thought to hinder the changes in tertiary structure needed fo r firm oxygen binding. We have subjected this postulate to a rigorous test by measuring the oxygen equilibria of T-state crystals of an abno rmal human haemoglobin in which the C-terminal histidine residues of t he beta-chains are replaced by leucine residues. This replacement remo ves the salt-bridges from the histidine imidazole groups to the neighb ouring aspartate residues. The crystals have an oxygen affinity about three times greater than that of crystals of normal haemoglobin. Hill' s coefficient is close to unity. The oxygen affinity is unaffected by pH, chloride or the allosteric effector bezafibrate. Equilibrium curve s determined by single crystal microspectrophometry using light polari sed parallel and normal to the crystallographic alpha-axis show no sig nificant difference between the oxygen affinities of alpha and beta-ha ems. Our results show that rupture of salt-bridges raises the oxygen a ffinity of the T-structure even when this is clamped firmly by the cry stal lattice. (C) 1998 Academic Press.