Recombinant hemoglobins with low oxygen affinity and high cooperativity.

By introducing an additional H-bond in the alpha(1)beta(2) subunit interface or altering the charge properties of the amino acid residues in the alpha(1)beta(1) subunit interface of the hemoglobin molecule, we have designed and expressed recombinant hemoglobins (rHbs) with low oxygen affinity and high cooperativity. Oxygen-binding measurements of these rHbs under various experimental conditions show interesting properties in response to pH (Bohr effect) and allosteric effectors. Proton nuclear magnetic resonance studies show that these rHbs can switch from the oxy (or CO) quaternary structure (R) to the deoxy quaternary structure (T) without changing their ligation states upon addition of an allosteric effector, inositol hexaphosphate, and/or reduction of the ambient temperature. These results indicate that if we can provide extra stability to the T state of the hemoglobin molecule without perturbing its R state, we can produce hemoglobins with low oxygen affinity and high cooperativity. Some of these rHbs are also quite stable against autoxidation compared to many of the known abnormal hemoglobins with altered oxygen affinity and cooperativity. These results have provided new insights into the structure-function relationship in hemoglobin.</p

Site mutations disrupt inter-helical H-bonds (alpha14W-alpha67T and beta15W-beta72S) involved in kinetic steps in the hemoglobin R-->T transition without altering the free energies of oxygenation.

Three recombinant mutant hemoglobins (rHbs) of human normal adult hemoglobin (Hb A), rHb (alphaT67V), rHb (betaS72A), and rHb (alphaT67V, betaS72A), have been constructed to test the role of the tertiary intra-subunit H-bonds between alpha67T and alpha14W and between beta72S and beta15W in the cooperative oxygenation of Hb A. Oxygen-binding studies in 0.1 M sodium phosphate buffer at 29 degrees C show that rHb (alphaT67V), rHb (betaS72A), and rHb (alphaT67V, betaS72A) exhibit oxygen-binding properties similar to those of Hb A. The binding of oxygen to these rHbs is highly cooperative, with a Hill coefficient of approximately 2.8, compared to approximately 3.1 for Hb A. Proton nuclear magnetic resonance (NMR) studies show that rHb (alphaT67V), rHb (betaS72A), rHb (alphaT67V, betaS72A), and Hb A have similar quaternary structures in the alpha(1)beta(2) subunit interfaces. In particular, the inter-subunit H-bonds between alpha42Tyr and beta99Asp and between beta37Trp and alpha94Asp are maintained in the mutants in the deoxy form. There are slight perturbations in the distal heme pocket region of the alpha- and beta-chains in the mutants. A comparison of the exchangeable 1H resonances of Hb A with those of these three rHbs suggests that alpha67T and beta72S are H-bonded to alpha14W and beta15W, respectively, in the CO and deoxy forms of Hb A. The absence of significant free energy changes for the oxygenation process of these three rHbs compared to those of Hb A, even though the inter-helical H-bonds are abolished, indicates that these two sets of H-bonds are of comparable strength in the ligated and unligated forms of Hb A. Thus, the mutations at alphaT67V and betaS72A do not affect the overall energetics of the oxygenation process. The preserved cooperativity in the binding of oxygen to these three mutants also implies that there are multiple interactions involved in the oxygenation process of Hb A.</p