Haemoglobin: Cooperativity in Protein–Ligand Interactions


Haemoglobin (Hb) is an essential component of the circulatory system of vertebrates. Its chief physiological function is to transport oxygen from the lungs to the tissues. Hb binds oxygen cooperatively, that is the affinity of the protein for the first oxygen molecule is less than that for subsequent oxygen molecules. Human adult Hb was among the first proteins whose complete three‐dimensional structure was determined by X‐ray crystallography and it has been used as a model for understanding allosteric proteins. Recent X‐ray crystallographic and multinuclear NMR (nuclear magnetic resonance) studies of both ligated and unligated forms of Hb have shown that many structures exist in crystalline and solution states. These results have challenged the classical two‐structure allosteric model of this protein. To understand Hb at the atomic level, we need to correlate the structural, dynamic and functional properties of hemoglobin in solution.

Key Concepts:

  • There are many T‐ and R‐types of crystal structures of unligated and ligated forms of haemoglobin, in contrast to the classical two‐structure model for haemoglobin allostery.

  • The structures of haemoglobin in both unligated and ligated forms in solution are different from those in crystals and exist as dynamic ensembles of various structures.

  • The ligand‐binding data for proximal histidyl‐detached recombinant haemoglobins show that Perutz's proximal histidyl coupling mechanism contributes approximately two‐thirds to the total interaction energy between haems and that there are alternative coupling pathways for the remaining third.

  • The Bohr effect, a heterotropic effect, is an excellent example of a global network of electrostatic interactions, rather than a few specific amino acid residues, that play a dominant role in an important physiological function of haemoglobin.

  • Recent experimental results strongly suggest that allosteric proteins, such as haemoglobin, may require multiple pathways for signal communication, as is illustrated in the cooperative oxygenation and in the Bohr effect.

  • Haemoglobin is a molecule with considerable plasticity.

  • The classical two‐structure mechanism for haemoglobin allostery cannot account for the structure, dynamics and function of the haemoglobin molecule and needs revision.

Keywords: cooperativity; allosteric interactions; Bohr effect; haemoglobin structure–dynamics–function relationship; crystal structures of haemoglobin determined by X‐ray crystallography; solution structures of haemoglobin determined by multinuclear NMR spectroscopy

Figure 1.

Illustrations of Hb A structure produced using the graphics program MOLMOL (Koradi et al., ) and PyMOL (DeLano, ). Coordinates of Hb A in the T (deoxy; Fermi et al., ), R (oxy; Shaanan, ), R2 (CO; Silva et al., ). RR2 (CO; Safo and Abraham, ) and R3 (CO, Safo and Abraham, ) conformations were obtained from Brookhaven Protein Databank files 2HHB, 1HHO, 1BBB, 1MKO and 1YZI, respectively. (a) Deoxy‐Hb A, viewed along the 2‐fold symmetry axis showing the water‐filled central cavity. The haems are shown in a space‐filling representation, whereas the polypeptide chains are represented by ribbons coloured green, yellow, cyan and orange for α1, β1, α2 and β2, respectively. (b) Backbone trace of oxy‐(R) and deoxy‐Hb (T), coloured red and blue, respectively. The α1β1 dimers of the two conformations, shown as thin coils towards the rear of the figure, have been superimposed at the α1β1 interface. Thicker, lighter‐coloured coils towards the front of the figure represent the α2β2 dimer and illustrate the repositioning of this dimer on quarternary structure rearrangement. (c) Individual α subunits of oxy‐ and deoxy‐Hb with haems superimposed. The haems are viewed edge‐on, in a ball‐and‐stick representation. The protein chain is shown as a smooth coil passing through the backbone Cα atoms, coloured red for oxy‐Hb and blue for deoxy‐Hb. Also shown are bonds of the proximal histidine and the distal ligand (oxygen). (d) Switch region of the α1β2 interface of Hb in the T, R, R2, RR2 and R3 conformations, shown in blue, red, green, yellow and light blue, respectively. Here, the backbone atoms of residues α1 38–44 (shown in darker colours) have been superimposed, to emphasize the relative motion of β2His97.

Figure 2.

Schematic illustration of the R and R2 crystal structures, together with the solution conformation (determined by RDC) of HbCO A. Helices are shown in cylinders. The α1β1 dimers of the two structures have been superimposed and are indistinguishable. The α2‐ and β2 chains of the R, solution and R2 structures are shown in dark, medium and light shades, respectively, of red and blue. The C2 symmetry axes of the R and R2 structures are shown as thin black and white rods, respectively. Insert gives the alignment frames of the best‐fit solution structure in bicelles (x, y, z) and Pf1 phage (x′, y′, z′), where the x and x′ axes coincide with the C2 axis. The R→R2 rotation axis is shown in orange. Reproduced with permission from Lukin et al. (; Figure 2). Copyright 2003 National Academy of Sciences, USA.

Figure 3.

Correlation between observed RDCs of HbCO A in solution and calculated RDCs based on the crystal structures R (1RD), R2 (1BBB), RR2 (1MKO) and R3 (1YZI). The quality factors (Q) are 14.6%, 15.2%, 17.9% and 26.4%, respectively. Reproduced from Gong et al. (; Figure 3), with permission from the American Chemical Society.

Figure 4.

Quality of fit results from the correlation between the observed RDC values of deoxy‐Hb in solution and the calculated RDC values based on the crystal structures 1A3N, 4HHB, 1HGA, 1KD2, 1RQ3, 1XXT, 1BZ0, 1YHR and 2DN2, in the absence and presence of IHP. The X‐ray crystal structure file names are shown along the x‐axis, and their corresponding reduced χ2 values in the absence (red) and presence (blue) of IHP are along the y‐axis. Reproduced from Sahu et al. (; Figure 2), with permission from the American Chemical Society.

Figure 5.

Measured oxygen‐binding curves of Hb A in 0.1 mol L−1 phosphate at 29°C at pH 6.69, 6.97 and 7.88, and at pH 7.05 in the presence of 2,3‐BPG. The inset shows Hill plots for the oxygenation data. We thank Ms Nancy T Ho for providing the oxygen‐binding data shown here.

Figure 6.

Bohr effect of Hb A in 2H2O in 0.1 M HEPES buffer plus 0.1 M NaCl at 29°C. The net Bohr effect of Hb A (green curve) is determined from oxygen dissociation measurements. The contributions of individual histidyl residues are calculated using pK values measured by 1H NMR spectroscopy (see Table ). The contributions of α20His, α112His and β117His are negligible and are not shown. The blue curve is the sum of the calculated contributions of all the individual histidyl residues. Reproduced from Lukin and Ho (; Figure 5), with permission from the American Chemical Society.



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Further Reading

Ackers GK (1998) Deciphering the molecular code of hemoglobin allostery. Advances in Protein Chemistry 51: 185–253.

Barrick D, Lukin JA, Simplaceanu V and Ho C (2004) Nuclear magnetic resonance spectroscopy in the study of hemoglobin cooperativity. Methods in Enzymology 379: 28–54.

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Ho C (1992) Proton nuclear magnetic resonance studies on hemoglobin: cooperative interactions and partially ligated intermediates. Advances in Protein Chemistry 43: 153–312.

Ho C, Eaton WA, Collman JP et al. (1982) Hemoglobin and Oxygen Binding. New York: Elsevier North Holland.

Ho C and Russu IM (1987) How much do we know about the Bohr effect of haemoglobin? Biochemistry 26: 6299–6305.

Shulman RG (2001) Spectroscopic contributions to the understanding of hemoglobin function: implications for structural biology. IUBMB Life 51: 351–357.

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Ho, Chien, and Yuan, Yue(Apr 2010) Haemoglobin: Cooperativity in Protein–Ligand Interactions. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001345.pub2]