Dynamics of Allostery in Hemoglobin: Roles of the Penultimate Tyrosine H bonds

The tyrosine residues adjacent to the C termini of the hemoglobin (Hb) subunits, αY140 and βY145, are expected to play important structural roles, because the C termini are the loci of T-state quaternary salt-bridges, and because the tyrosine side-chains bridge the H and F helices via H bonds to the αV93 and βV98 carbonyl groups. These roles have been investigated via measurements of oxygen binding, 1H NMR spectra, resonance Raman (RR) spectra, and time-resolved resonance Raman (TR3) spectra on site mutants in which the H⋯F H bonds are eliminated by replacing the tyrosine residues with phenylalanine. The TR3 spectra confirm the hypothesis, based on TR3 studies of wild-type Hb, that the H⋯F H bonds break and then re-form during the sub-microsecond phase of the R–T quaternary transition. The TR3 spectra support the inference from other mutational studies that the αβ dimers act as single dynamic units in this early phase, motions of the E and F helices being coupled tightly across the dimer interface. Formation of T quaternary contacts occurs at about the same rate in the mutants as in HbA. However, these contacts are weakened substantially by the Y/F substitutions. Equilibrium perturbations are apparent also, especially for the α-subunits, in which relaxation of the Fe–His bond, strengthening of the A⋯E interhelical H bond, and weakening of the “switch” quaternary contact in deoxyHb are all apparent. Structural effects are less marked for the β-chain Y/F replacement, but the Bohr effect is reduced by 25%, indicating that the salt-bridge and H bond interactions of the adjacent C terminus are loosened. The α-chain replacement reduces the Bohr effect much more, consistent with the global perturbations detected by the structure probes.