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Review
. 2013 Mar 1;3(3):a011858.
doi: 10.1101/cshperspect.a011858.

Hemoglobin variants: biochemical properties and clinical correlates

Christopher S Thom  1 , Claire F Dickson, David A Gell, Mitchell J Weiss
Affiliations
Review

Hemoglobin variants: biochemical properties and clinical correlates

Christopher S Thom et al. Cold Spring Harb Perspect Med. .

Abstract

Diseases affecting hemoglobin synthesis and function are extremely common worldwide. More than 1000 naturally occurring human hemoglobin variants with single amino acid substitutions throughout the molecule have been discovered, mainly through their clinical and/or laboratory manifestations. These variants alter hemoglobin structure and biochemical properties with physiological effects ranging from insignificant to severe. Studies of these mutations in patients and in the laboratory have produced a wealth of information on hemoglobin biochemistry and biology with significant implications for hematology practice. More generally, landmark studies of hemoglobin performed over the past 60 years have established important paradigms for the disciplines of structural biology, genetics, biochemistry, and medicine. Here we review the major classes of hemoglobin variants, emphasizing general concepts and illustrative examples.

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Figures

Figure 1.
Figure 1.
The structure of Hb. (A) The α (pink) and β (red) Hb subunits have conserved α-helical folds. Helices are labeled A–H from the amino terminus. The α subunit lacks helix D. (B) The high O2 affinity R state quaternary structure of Hb with O2 (red spheres) bound at all four heme sites (protoporphyrin-IX as yellow sticks, with central iron atom as orange sphere). (C) Stereo (wall-eye) diagram of the heme pocket of β showing the proximal (F8) and distal (E7) histidines and selected residues in the distal heme pocket that influence ligand binding and autoxidation. (D) Hb tetramer is assembled from two identical αβ dimers (shown in red and gray for clarity). In the tetramer, each subunit makes contact with the unlike chain through a high affinity dimerization α1β1 interface and a lower affinity α1β2 dimer–tetramer interface (cyan).
Figure 2.
Figure 2.
Hb variants with altered subunit interactions. (A) Conversion from the low O2 affinity (deoxy, T state) to high O2 affinity (oxy, R state) involves a relative rotation of the α1β1 and α2β2 dimers, with changes in contacts across the α1β2 (and α2β1) interface (cyan). In this cartoon, the α1β1 dimer is held stationary to reveal the relative motion of the α2β2 dimer in going from the deoxy (orange) to the oxy (red) states. (B) The sigmoidal shape of the Hb-O2 saturation curve shows allosteric regulation by changes in pH, temperature, and 2,3DPG. These regulators, as well as Hb variants, influence the shape of the curve. High oxygen affinity variants, high pH, low 2,3DPG, or low temperature induce a “left shift” in the saturation curve (red line). Conversely, low oxygen affinity variants, low pH, high 2,3DPG, or high temperature induce a “right shift” (blue line). (C) Hb sequence variants at the allosteric α1β2 interface (cyan spheres) show an impaired response to O2 binding. Some sequence variants disrupt binding to other allosteric regulators, e.g., substitutions at βK82 (green) disrupt interactions with 2,3DPG that normally stabilize the low O2 affinity T state. Mutations that disrupt α1β1 (and α2β2) dimerization (blue spheres) increase the concentration of free monomers, which are unstable. (D) Some α mutations that disrupt binding to β may also disturb binding to the chaperone, AHSP. Other α variants, such as Turriff and Beziers (pink sphere) may inhibit only AHSP binding.
Figure 3.
Figure 3.
Hb variants that affect allosteric regulation. (A) The α2 and β2 subunits indicated on the right side of the figure show an overlay of the R-state (red/pink) and T-state (orange/light orange) quaternary structures of Hb. The allosteric α1β2 interface is boxed. (B) Detail of the α1β2 interface in the deoxy T state (β2 chain in orange, PDB 2DN2) and oxy R state (β2 chain in red, PDB 2DN1) showing selected H-bonding interactions. Note that the two H-bonding networks use nonoverlapping sets of side chains, hence mutations in these residues affect only one state. Mutation of Asp99 to Asn (Hb Kempsey) compromises electrostatic interactions in the deoxygenated state, thereby favoring the R state and causing impaired O2 release. Mutation of Asn102 to Thr (Hb Kansas) abrogates interactions with Asp94, favoring the T state and O2 binding inhibition.
Figure 4.
Figure 4.
Examples of M-type hemoglobins. (A) The heme pocket of wild-type Mb (gray) and the F8 His > Tyr mutation (orange, PDB 1HRM), which serves as a model for Hb M-Iwate. Effects on the protein fold are to increase the distance from the heme to the F helix, recapitulating features of the deoxy-α (T state) structure. (B) In Hb M-Iwate, α 87 Tyr F8 is deprotonated and favors Fe3+ oxidation state, resulting in rapid autoxidation. This ferric form is not reduced by met-Hb reductase. (C) With the normal His F8 present, ferric heme is readily reduced. (D) Substituting the distal His E7 side chain in Mb for a larger Tyr E7 (orange, PDB 1MGN), as also occurs in Hb Saskatoon, brings the Tyr hydroxyl group within binding distance of the iron, forming a hexacoordinate iron site. (E) In Hb Saskatoon the hexacoordinate ferric iron in the effected β chains can be reduced by met-Hb reductase, possibly owing to a transient protonation of Tyr E7. Note that Mb is used as a model for Hb subunits in A and D, whereas B and E are based on Hb spectroscopy.
Figure 5.
Figure 5.
Hb variants with amino acid changes in the heme pocket. (A) Stereo diagram of a model of the deoxy Hb Zurich β heme pocket (blue) overlaid with the wild-type β heme pocket (black, PDB 2DN2). This model of Hb Zurich is based on the structures solved by Phillips et al. (1981) and Tucker et al. (1978). It was generated with the macromolecular modeling program Coot (Emsley et al. 2010) by mutating the distal His of deoxy-β (PDB 2DN2) to Arg. The distal Arg E7 (red) is oriented toward the CD corner, disturbing the position of Phe CD1 and Phe CD4 (orange). The heme pocket entrance is much wider allowing increased access to ligands. However, unlike normal His E7, mutant Arg is unable to stabilize bound O2 via hydrogen bonding. (B) Stereo diagram showing the structural changes associated with substitution of β Val E11. The wild-type structure carrying the branched hydrophobic side chain Val (black bonds, PDB 2DN2) is overlaid with structures carrying the largest aromatic side chain Trp E11 (orange, PDB 101K) or a polar side chain Thr E11 (green PDB, 1HDB). It is clear that no major backbone or side chain repacking occurs. Thus, mutations in this position are likely to have specific effects in changing the volume of the heme pocket accessible to solvent or diatomic ligands and the electrostatic properties of the pocket. These changes will manifest as differences in O2 binding, ligand selectivity, autoxidation, and heme loss.
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