Mutations of the βN102 residue of HbA not only inhibit the ligand-linked T to R^e state transition, but also profoundly affect the properties of the T state itself

The properties of three HbA variants with different mutations at the β102 position, βN102Q, βN102T, and βN102A, have been examined. All three are inhibited in their ligand-linked transition from the low affinity T quaternary state to the high affinity R^e quaternary state. In the presence of inositol hexaphosphate, IHP, none of them exhibits cooperativity in the binding of oxygen. This is consistent with the destabilization of the Re state as a result of the disruption of the hydrogen bond that normally forms between the β102 asparagine residue and the α94 aspartate residue in the R^e state. However, these three substitutions also alter the properties of the T state of the hemoglobin tetramer. In the presence of IHP, the first two substitutions result in large increases in the ligand affinities of the β-subunits within the T state structure. The βN102A variant, however, greatly reduces the pH dependencies of the affinities of the α and β subunits, K₁(α) and K₁(β), respectively, for the binding of the first oxygen molecule in the absence of IHP. In the presence of IHP, the T state of this variant is strikingly similar to that of HbA under the same conditions. For both hemoglobins, K₁(α) and K₁(β) exhibit only small Bohr effects. In the absence of IHP, the affinities of the α and β subunits of HbA for the first oxygen are increased, and both exhibit greatly increased Bohr effects. However, in contrast to the behavior of HbA, the ligand-binding properties of the T state tetramer of the βN102A variant are little affected by the addition or removal of IHP. It appears that along with its effect on the stability of the liganded R^e state, this mutation has an effect on the T state that mimics the effect of adding IHP to HbA. It inhibits the set of conformational changes, which are coupled to the K₁ Bohr effects and normally accompany the binding of the first ligand to the HbA tetramer in the absence of organic phosphates.