LOSS OF ALLOSTERIC BEHAVIOR IN RECOMBINANT HEMOGLOBIN ALPHA(2)BETA(2)92(F8)HIS-]ALA - RESTORATION UPON ADDITION OF STRONG EFFECTORS

In the stereochemical model proposed by P.erutz [1], the Fe-His(F8) bo nd plays a significant role in the allosteric transition in hemoglobin and the resulting cooperativity in ligand binding, When this bond is ruptured, there is a loss in the transmission of the information conce rning ligand binding; examples are Hb(NO)(4) in the presence of inosit ol hexakisphosphate (IHP), or nickel substituted Hb hybrids which, des pite being liganded, exhibit deoxy-like properties, To study the effec ts of the loss of the iron proximal histidine bond, we have engineered the alpha(2) beta(2)(F8)H92A recombinant Hb. The replacement of the h ighly conserved proximal histidine F8 residue by an alanine results in a low affinity for the heme group and a loss of the allosteric proper ties; kinetics of CO recombination after photodissociation show only t he rapid bimolecular phase, characteristic of the high affinity R-stat e, However, a significant amount of deoxy (T-state) kinetics are obser ved after addition of external effecters such as IHP. The iron-histidi ne bond is apparently crucial for the heme-heme interaction, but the a llosteric equilibrium may still be influenced by external constraints.