An investigation of the distal histidyl hydrogen bonds in oxyhemoglobin: effects of temperature, pH, and inositol hexaphosphate.

On the basis of X-ray crystal structures and electron paramagnetic resonance (EPR) measurements, it has been inferred that the O(2) binding to hemoglobin is stabilized by the hydrogen bonds between the oxygen ligands and the distal histidines. Our previous study by multinuclear nuclear magnetic resonance (NMR) spectroscopy has provided the first direct evidence of such H-bonds in human normal adult oxyhemoglobin (HbO(2) A) in solution. Here, the NMR spectra of uniformly (15)N-labeled recombinant human Hb A (rHb A) and five mutant rHbs in the oxy form have been studied under various experimental conditions of pH and temperature and also in the presence of an organic phosphate, inositol hexaphosphate (IHP). We have found significant effects of pH and temperature on the strength of the H-bond markers, i.e., the cross-peaks for the side chains of the two distal histidyl residues, α58His and β63His, which form H-bonds with the O(2) ligands. At lower pH and/or higher temperature, the side chains of the distal histidines appear to be more mobile, and the exchange with water molecules in the distal heme pockets is faster. These changes in the stability of the H-bonds with pH and temperature are consistent with the changes in the O(2) affinity of Hb as a function of pH and temperature and are clearly illustrated by our NMR experiments. Our NMR results have also confirmed that this H-bond in the β-chain is weaker than that in the α-chain and is more sensitive to changes in pH and temperature. IHP has only a minor effect on these H-bond markers compared to the effects of pH and temperature. These H-bonds are sensitive to mutations in the distal heme pockets but not affected directly by the mutations in the quaternary interfaces, i.e., α(1)β(1) and/or α(1)β(2) subunit interface. These findings provide new insights regarding the roles of temperature, hydrogen ion, and organic phosphate in modulating the structure and function of hemoglobin in solution.</p

NMR investigation of the dynamics of tryptophan side-chains in hemoglobins.

NMR relaxation measurements of 15N spin-lattice relaxation rate (R(1)), spin-spin relaxation rate (R(2)), and heteronuclear nuclear Overhauser effect (NOE) have been carried out at 11.7T and 14.1T as a function of temperature for the side-chains of the tryptophan residues of 15N-labeled and/or (2H,15N)-labeled recombinant human normal adult hemoglobin (Hb A) and three recombinant mutant hemoglobins, rHb Kempsey (betaD99N), rHb (alphaY42D/betaD99N), and rHb (alphaV96W), in the carbonmonoxy and the deoxy forms as well as in the presence and in the absence of an allosteric effector, inositol hexaphosphate (IHP). There are three Trp residues (alpha14, beta15, and beta37) in Hb A for each alphabeta dimer. These Trp residues are located in important regions of the Hb molecule, i.e. alpha14Trp and beta15Trp are located in the alpha(1)beta(1) subunit interface and beta37Trp is located in the alpha(1)beta(2) subunit interface. The relaxation experiments show that amino acid substitutions in the alpha(1)beta(2) subunit interface can alter the dynamics of beta37Trp. The transverse relaxation rate (R(2)) for beta37Trp can serve as a marker for the dynamics of the alpha(1)beta(2) subunit interface. The relaxation parameters of deoxy-rHb Kemspey (betaD99N), which is a naturally occurring abnormal human hemoglobin with high oxygen affinity and very low cooperativity, are quite different from those of deoxy-Hb A, even in the presence of IHP. The relaxation parameters for rHb (alphaY42D/betaD99N), which is a compensatory mutant of rHb Kempsey, are more similar to those of Hb A. In addition, TROSY-CPMG experiments have been used to investigate conformational exchange in the Trp residues of Hb A and the three mutant rHbs. Experimental results indicate that the side-chain of beta37Trp is involved in a relatively slow conformational exchange on the micro- to millisecond time-scale under certain experimental conditions. The present results provide new dynamic insights into the structure-function relationship in hemoglobin.</p

Role of β/δ101Gln in regulating the effect of temperature and allosteric effectors on oxygen affinity in woolly mammoth hemoglobin.

<p>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.</p

A comparative NMR study of the polypeptide backbone dynamics of hemoglobin in the deoxy and carbonmonoxy forms.

Model-free-based NMR dynamics studies have been undertaken for polypeptide backbone amide N-H bond vectors for both the deoxy and carbonmonoxy forms of chain-specific, isotopically (15N and 2H) labeled tetrameric hemoglobin (Hb) using 15N-relaxation parameters [longitudinal relaxation rate (R1), transverse relaxation rate (R2), and heteronuclear nuclear Overhauser effect (NOE)] measured at two temperatures (29 and 34 degrees C) and two magnetic field strengths (11.7 and 14.1 T). In both deoxy and carbonmonoxy forms of human normal adult hemoglobin (Hb A), the amide N-H bonds of most amino acid residues are rigid on the fast time scale (nanosecond to picosecond), except for the loop regions and certain helix-helix connections. Although rigid in deoxy-Hb A, beta146His has been found to be free from restriction of its backbone motions in the CO form, presumably due to the rupture of its hydrogen bond/salt bridge network. We now have direct dynamics evidence for this structural transition of Hb in solution. While remarkably flexible in the deoxy state, alpha31Arg and beta123Thr, neighbors in the intradimer (alpha1beta1) interface, exhibit stiffening upon CO binding. These findings imply a role for alpha31Arg and beta123Thr in the intradimer communication but contradict the results from X-ray crystallography. We have also found that there is considerable flexibility in the intradimer (alpha1beta1) interface (i.e., B, G, and H helices and the GH corner) and possible involvement of several amino acid residues (e.g., alpha31Arg, beta3Leu, beta41Phe, beta123Thr, and beta146His) in the allosteric pathway. Several amino acid residues at the intradimer interfaces, such as beta109Val, appear to be involved in possible conformational exchange processes. The dynamic picture derived from the present study provides new insights into the traditional description of the stereochemical mechanism for the cooperative oxygenation of Hb A based on X-ray crystallographic results.</p