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

Abstract

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.

Figure 1

His and Trp side-chain regions of 600-MHz (¹H, ¹⁵N) HSQC spectra of fully ¹⁵N-labeled HbO₂ A in H₂O at pH 8.0 (upper row); 7.0 (middle row); and 6.5 (lower row) at 7, 29 and 37 °C, respectively. The cross-peaks for the relevant directly bonded ¹⁵N-¹H pairs are identified for each residue.

Figure 2

Horizontal 1D slices along the ¹H axis through the cross peak of side-chain of β63His extracted from 600-MHz (¹H, ¹⁵N) HSQC spectra of fully ¹⁵N-labeled rHbO₂ A in H₂O under various conditions of pH and temperature. The panels are marked with the same letter as the corresponding panels (pH, temperature) from Figure 1.

Figure 3

Oxygen-binding properties of Hb A as a function of pH at 29 °C (red), 11 °C (green), and 7 °C (magenta) in 0.1 M sodium phosphate buffer without IHP (filled triangles) and with 4 times molar concentration of IHP (open circles).

Figure 4

Ring-current-shifted proton resonances in ¹H NMR spectra (600 MHz) of rHbO₂ A in the absence and presence of IHP (A and B), and mutant rHbO₂ (αL29W) (C) in 95% H₂O, 5% D₂O, 0.1 M sodium phosphate buffer at different pH (8.0, 7.0 and 6.5) and temperatures (29 and 7 °C), respectively.

Figure 5

His and Trp side-chain regions of 600-MHz (¹H, ¹⁵N) HSQC spectra of fully ¹⁵N-labeled HbO₂ A in H₂O in the presence of IHP at pH 8.0 (upper row), pH 7.0 (middle row) and pH 6.5 (lower row); and by columns, at 7 °C (left) and 29 °C (right), respectively. The cross-peaks for the relevant directly bonded ¹⁵N-¹H pairs are labeled.

Figure 6

His and Trp side-chain regions of 600-MHz (¹H, ¹⁵N) HSQC spectra of fully ¹⁵N-labeled mutant rHb O₂ (αV96W) (A and B); rHb O₂ (βD99N) (C and D); rHb O₂ (αY42D/βD99N) (E and F); and rHb O₂ (αL29W) (G and H) in 95% H₂O, 5% D₂O, 0.1 M sodium phosphate buffer at pH 7.0, 7 °C (left column) and 29 °C (right column), respectively. The cross-peaks for directly bonded ¹⁵N-¹H of the relevant side-chains are labeled.

Figure 7

His and Trp side-chain regions of 600-MHz (¹H, ¹⁵N) HSQC spectra of fully ¹⁵N-labeled mutant rHb O₂ (αV96W/βN108K) in H₂O at pH 8.0, 7 °C (left) and 29 °C (right), respectively. The cross-peaks for directly bonded ¹⁵N-¹H of the relevant side-chains are labeled.

Figure 8

Individual heme pockets in the α- and β-subunits of oxy- CO- and deoxy-Hb A with hemes superimposed, colored red for HbO₂ A, blue for HbCO A, and grey for deoxy-Hb A, respectively. The Fe atom in the heme is colored green for HbO₂ A, yellow for HbCO A and blue for deoxy-Hb A, respectively. Illustrations of the Hb A structure produced by using the graphics program PyMOL (51). Coordinates of Hb A in the oxy-, CO and deoxy-forms were obtained from Brookhaven Protein Databank files 2DN1, 2DN3, and 2DN2, respectively.