Mutations of the betaN102 residue of HbA not only inhibit the ligand-linked T to Re state transition, but also profoundly affect the properties of the T state itself.

The properties of three HbA variants with different mutations at the beta102 position, betaN102Q, betaN102T, and betaN102A, have been examined. All three are inhibited in their ligand-linked transition from the low affinity T quaternary state to the high affinity Re 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 beta102 asparagine residue and the alpha94 aspartate residue in the Re 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 beta-subunits within the T state structure. The betaN102A variant, however, greatly reduces the pH dependencies of the affinities of the alpha and beta subunits, K1(alpha) and K1(beta), 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(alpha) and K1(beta) exhibit only small Bohr effects. In the absence of IHP, the affinities of the alpha and beta 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 betaN102A variant are little affected by the addition or removal of IHP. It appears that along with its effect on the stability of the liganded Re 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.

Intersubunit interactions associated with Tyr42 alpha stabilize the quaternary-T tetramer but are not major quaternary constraints in deoxyhemoglobin.

Previous mutational studies on Tyr42alpha variants as well as the current studies on the mutant hemoglobin alphaY42A show that the intersubunit interactions associated with Tyr42alpha significantly stabilize the alpha1beta2 interface of the quaternary-T deoxyhemoglobin tetramer. However, crystallographic studies, UV and visible resonance Raman spectroscopy, CO combination kinetic measurements, and oxygen binding measurements on alphaY42A show that the intersubunit interactions formed by Tyr42alpha have only a modest influence on the structural properties and ligand affinity of the deoxyhemoglobin tetramer. Therefore, the alpha1beta2 interface interactions associated with Tyr42alpha do not contribute significantly to the quaternary constraints that are responsible for the low oxygen affinity of deoxyhemoglobin. The slight increase in the ligand affinity of deoxy alphaY42A correlates with small, mutation-induced structural changes that perturb the environment of Trp37beta, a critical region of the quaternary-T alpha1beta2 interface that has been shown to be the major source of quaternary constraint in deoxyhemoglobin.

Ligand binding to symmetrical FeZn hybrids of variants of human HbA with modifications in the alpha1-beta2 interface.

The equilibria of oxygen binding to and kinetics of CO combination with the symmetrical iron-zinc hybrids of a series of variants of human adult hemoglobin A have been measured at pH 7 in the presence of inositol hexaphosphate (IHP). In addition, the kinetics of CO combination have also been measured in the absence of IHP. The hybrids have the heme groups of either the alpha or the beta subunits replaced by zinc protoporphyrin IX, which is unable to bind a ligand and is a good model for permanently deoxygenated heme. The variants examined involve residues located in the alpha1beta2 interface of the hemoglobin tetramer. Alterations of residues located in the hinge region of the interface are found to affect the properties of both the alpha and the beta subunits of the protein. In contrast, alterations of residues in the switch region of the interface have substantial effects only on the mutant subunit and are poorly communicated to the normal partner subunit. When the logarithms of the rate constants for the combination of the first CO molecule with a single subunit in the presence of IHP are analyzed as functions of the logarithms of the dissociation equilibrium constants for the binding of the first oxygen under the same conditions, a linear relationship is found. The relationship is somewhat different for the alpha and beta subunits, consistent with the well-known differences in the geometries of their ligand binding sites.

Effects of heterotropic allosteric effectors on the equilibrium and kinetics of the reaction of a single ligand molecule with an alpha or beta subunit of deoxygenated HbA.

Symmetrical FeZn hybrids of human HbA have been used to measure K(1)(alpha) and K(1)(beta), the dissociation constants for the binding of a single molecule of oxygen to unliganded HbA at an alpha subunit and at a beta subunit, respectively. The kinetic constants, l(1)'(alpha) and l(1)'(beta), for the combination of the first CO molecule to unliganded HbA at an alpha or a beta subunit, respectively, were also measured. Measurements were carried out between pH 6 and pH 8 in the presence and absence of inositol hexaphosphate (IHP). Both equilibrium constants exhibit a significant Bohr effect in the absence of IHP. The addition of IHP to a concentration of 0.1 mM increases both dissociation constants in a pH-dependent manner with the result that both Bohr effects are greatly reduced. These results require a negative thermodynamic linkage between the binding of a single oxygen at either an alpha or a beta subunit and the binding of IHP to the T quaternary structure of HbA. Although the beta hemes are relatively near the IHP binding site, a linkage between that site and the alpha hemes, such that the binding of a single oxygen molecule to the heme of one alpha subunit reduces the affinity of the T state for IHP, requires communication across the molecule. l(1)'(alpha) exhibits a very slight pH dependence, with a maximum variation of 20%, while l(1)'(beta) varies with pH three times as much. IHP has no effect on the pH dependence of either rate constant but reduces l(1)'(alpha) marginally, 20%, and l(1)'(beta) by 2-fold at all pH values.

Conformational substates of the oxyheme centers in alpha and beta subunits of hemoglobin as disclosed by EPR and ENDOR studies of cryoreduced protein.

Exposure of frozen solutions of oxyhemoglobin to gamma-irradiation at 77 K yields EPR- and ENDOR-active, one-electron-reduced oxyheme centers which retain the conformation of the diamagnetic precursor. EPR spectra have been collected for the centers produced in human HbO(2) and isolated alphaO(2) and betaO(2) chains, as well as alphaO(2)beta(Zn), alpha(Zn)betaO(2), and alphaO(2)beta(Fe(3+)) hybrids, each in frozen buffer and in frozen glasses that form in the presence of glycols and sugars and also in the presence of IHP. These reveal two spectroscopically distinct classes of such ferriheme centers (g(1)

Correlation of protein functional properties in the crystal and in solution: the case study of T-state hemoglobin.

The relevance of three-dimensional structures of proteins, determined by X-ray crystallography, is an important issue that is becoming even more critical in light of the Structural Genomics Initiative. As a case study, a detailed comparison of functional properties of the T quaternary states of genetically or chemically modified human hemoglobins (Hbs) in solution and in the crystal was performed. Oxygen affinities of Hbs in crystals correlate with the rate constants of their initial combination with carbon monoxide (CO) in solution, indicating that changes in ligand affinity caused by the modifications are similarly observed in both physical states.