Role of β/δ101Gln in Regulating the Effect of Temperature and Allosteric Effectors on Oxygen Affinity in Woolly Mammoth Hemoglobin

The oxygen affinity of woolly mammoth hemoglobin (rHb WM) is less affected by temperature change than that of Asian elephant hemoglobin (rHb AE) or human normal adult hemoglobin (Hb A). We report here a biochemical–biophysical study of Hb A, rHb AE, rHb WM, and three rHb WM mutants with amino acid substitutions at β/δ101 (β/δ101Gln→Glu, Lys, or Asp) plus a double and a triple mutant, designed to clarify the role of the β/δ101 residue. The β/δ101Gln residue is important for responding to allosteric effectors, such as phosphate, inositol hexaphosphate (IHP), and chloride. The rHb WM mutants studied generally have higher affinity for oxygen under various conditions of pH, temperature, and salt concentration, and in the presence or absence of organic phosphate, than do rHb WM, rHb AE, and Hb A. Titrations for the O₂ affinity of these mutant rHbs as a function of chloride concentration indicate a lower heterotopic effect of this anion due to the replacement of β/δ101Gln in rHb WM. The alkaline Bohr effect of rHb WM and its mutants is reduced by 20–50% compared to that of Hb A and is independent of changes in temperature, in contrast to what has been observed in the hemoglobins of most mammalian species, including human. The results of our study on the temperature dependence of the O₂ affinity of rHb WM and its mutant rHbs illustrate the important role of β/δ101Gln in regulating the functional properties of these hemoglobins

Solution Structure and Dynamics of Human Hemoglobin in the Carbonmonoxy Form

The solution structure of human adult carbonmonoxy hemoglobin (HbCO A) was refined using stereospecifically assigned methyl groups and residual dipolar couplings based on our previous nuclear magnetic resonance structure. The tertiary structures of individual chains were found to be very similar to the X-ray structures, while the quaternary structures in solution at low salt concentrations resembled the X-ray R structure more than the R2 structure. On the basis of chemical shift perturbation by inositol hexaphosphate (IHP) titration and docking, we identified five possible IHP binding sites in HbCO A. Amide–water proton exchange experiments demonstrated that αThr38 located in the α1β2 interface and several loop regions in both α- and β-chains were dynamic on the subsecond time scale. Side chain methyl dynamics revealed that methyl groups in the α1β2 interface were dynamic, but those in the α1β1 interface were quite rigid on the nanosecond to picosecond and millisecond to microsecond time scales. All the data strongly suggest a dynamic α1β2 interface that allows conformational changes among different forms (like T, R, and R2) easily in solution. Binding of IHP to HbCO A induced small structural and dynamic changes in the α1β2 interface and the regions around the hemes but did not increase the conformational entropy of HbCO A. The binding also caused conformational changes on the millisecond time scale, very likely arising from the relative motion of the α1β1 dimer with respect to the α2β2 dimer. Heterotropic effectors like IHP may change the oxygen affinity of Hb through modulating the relative motion of the two dimers and then further altering the structure of heme binding regions