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, alpha Y140 and beta Y145, 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 alpha V93 and beta V98 carbonyl groups. These roles have been investigated via measurements of oxygen binding, H-1 NMR spectra, resonance Raman (RR) spectra, and time-resolved resonance Raman (TR3) spectra on site mutants in which the (HF)-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 (HF)-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 alpha beta 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 alpha-subunits, in which relaxation of the Fe-H is 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 beta-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 alpha-chain replacement reduces the Bohr effect much more, consistent with the global perturbations detected by the structure probes. (c) 2005 Elsevier Ltd. All rights reserved.