Normal and Abnormal Hemoglobins

Studies of hemoglobin (Hb) structure, function, and expression over more than 50 years have produced many paradigms in biology and medicine. The first part of this chapter describes the structural and biochemical properties of Hb, emphasizing those that underlie its physiological roles. The conserved globin protein fold with the heme prosthetic group confers the basic function of reversible oxygen (O 2 ) binding, which is common to myoglobin (Mb), Hb, and numerous other recently discovered proteins such as neuroglobin and cytoglobin. The tetrameric quaternary structure of Hb allows for cooperative O 2 binding to achieve efficient loading of O 2 in lung capillaries at high partial pressure of O 2 (P O2 ≈100 mm Hg), and effective O 2 delivery to peripheral tissues at low P O2 (≈40 mm Hg). Several small molecules, including 2,3-diphosphoglycerate (2,3-DPG), chloride anion (Cl ), and carbon dioxide (CO 2 )/bicarbonate dynamically regulate O 2 binding to Hb in response to varying physiological conditions. Hb also facilitates transport of CO 2 in the form of bicarbonate anion and participates in oxidation reactions, both beneficial and deleterious. The second part of this chapter reviews the biochemistry and pathophysiology of abnormal Hbs that give rise to human diseases. Globin gene mutations that alter the properties of Hbs are common, affecting about 7% of the world’s population, and present a significant global health problem.

This chapter reviews Hb structure, biochemistry, and mutations that produce abnormal globin proteins. These “Hb variants” are usually caused by single amino acid substitutions, but also by amino acid insertions and/or deletions, antitermination mutations and altered posttranslational processing. The most common pathological Hb variants, HbS and HbC are reviewed in Chapter 20 . Other variants, discussed in the second part of this chapter, number more than a thousand and are rare individually but common collectively. Globin gene mutations that impair protein subunit production (i.e., thalassemias) are discussed in Chapter 21 . Several excellent references review the concepts discussed in this chapter.

Hemoglobin Structure and Function

The study of Hb and related proteins has contributed immensely to the understanding of protein function in general. In 1957, Kendrew and colleagues used X-ray diffraction to solve the structure of Mb at 6-Å resolution and a few years later refined this structure to 2 Å, making it possible to build models at near atomic resolution. These remarkable achievements showed for the first time what a protein actually “looks like.” Almost simultaneously the structure of Hb was determined at 5.5-Å resolution by Perutz and colleagues. Since then numerous biophysical studies have further refined the structures of normal and mutant Hbs to resolutions approaching 1 Å.

HbA, the major adult form of Hb, is a tetramer of two α polypeptide chains and two β polypeptide chains (α 2 β 2 ), each bound to a heme prosthetic group ( Box 19-1 ). The α and β globins are believed to have arisen from the duplication of a common ancestral gene approximately 450 million years ago, around the time that the lineages of cartilaginous fish and bony fish diverged. Subsequent gene duplications produced additional globin genes that are expressed at different times during ontogeny. During early human embryogenesis, the α-like (ζ) and β-like (ε and γ) chains produce embryonic Hbs (ζ 2 ε 2 , ζ 2 γ 2 , and α 2 ε 2 ). During midgestation, fetal Hb (HbF; α 2 γ 2 ) is predominantly expressed. From birth until around postnatal week 12, β-globin expression gradually increases and γ globin decreases as HbA replaces HbF. Another β-like globin (δ) is also expressed postnatally, and normal hemolysates contain approximately 2% HbA 2 2 δ 2 ). The properties of embryonic and fetal globins are tuned to the physiological conditions experienced in utero, particularly low O 2 tension, although the functions of all human Hb isoforms can be understood as variations on the mechanism of adult HbA. The onset of symptoms caused by deleterious globin gene mutations varies according to the developmental expression pattern of each particular gene, as discussed later in this chapter.

Box 19-1

Hemoglobin Structure

  • The globin fold is an evolutionary conserved structure made up of seven or eight α helices folded into a globular shape.

  • HbA is a tetramer made up of two α- and two β-globin subunits, each linked to a heme moiety that binds O 2 reversibly via a central iron molecule. Only reduced iron (Fe[II]) can bind O 2 .

  • Hb can bind other diatomic ligands via heme iron, including CO and NO.

  • The α1β1 (and α2β2) dimer is the basic structural unit of hemoglobin.

  • Various α- and β-like globins are expressed during human ontogeny. These include:

    • Embryonic: Gower 1 (ζ 2 ε 2 ), Gower 2 (α 2 ε 2 ), Hb Portland I (ζ 2 γ 2 ), Hb Portland II (ζ 2 β 2 )

    • Fetal: HbF (α 2 γ 2 )

    • Adult: HbA (α 2 β 2 ), ~95%; HbA 2 2 δ 2 ), 1.5%-3.5%; HbF (α 2 γ 2 ), 1%-2%.

CO, Carbon monoxide; Hb, hemoglobin; NO, nitric oxide; O 2 , oxygen.

Hemoglobin Primary Structure

Primary structure refers to the linear sequence of amino acids within a protein. The primary structures of the α-like and β-like globin chains are shown in Table 19-1 . Translation of the polypeptide chains on the ribosome is initiated with methionine, which is subsequently removed by methionine aminopeptidase before assembly of mature Hb tetramer. The α (141 amino acid residues) and β (146 amino acid residues) chains share only 41% amino acid sequence identity, yet they adopt very similar three-dimensional (3D; tertiary) structures. Numerous naturally occurring mutations alter the primary sequence of globin proteins (see the second part of this chapter).

TABLE 19-1

Primary Amino Acid Structure of Hemoglobin *

Helix α ζ Helix β δ γ ε
NA1 1 Val Ser NA1
1 Val
2 His
NA2 2 Leu Leu NA3 3 Leu Leu Phe Phe
A1 3 Ser Thr A1 4 Thr Thr Thr Thr
A2 4 Pro Lys A2 5 Pro Pro Glu Ala
A3 5 Ala Thr A3 6 Glu Glu Glu Glu
A4 6 Asp Glu A4 7 Glu Glu Asp Glu
A5 7 Lys Arg A5 8 Lys Lys Lys Lys
A6 8 Thr Thr A6 9 Ser Thr Ala Ala
A7 9 Asn Ile A7 10 Ala Ala Thr Ala
A8 10 Val Ile A8 11 Val Val Ile Val
A9 11 Lys Val A9 12 Thr Asn Thr Thr
A10 12 Ala Ser A10 13 Ala Ala Ser Ser
A11 13 Ala Met A11 14 Leu Leu Leu Leu
A12 14 Trp Trp A12 15 Trp Trp Trp Trp
A13 15 Gly Ala A13 16 Gly Gly Gly Ser
A14 16 Lys Lys A14 17 Lys Lys Lys Lys
A15 17 Val Ile A15 18 Val Val Val Met
A16 18 Gly Ser
AB1 19 Ala Thr
B1 20 His Gln B1 19 Asn Asn Asn Asn
B2 21 Ala Ala B2 20 Val Val Val Val
B3 22 Gly Asp B3 21 Asp Asp Glu Glu
B4 23 Glu Thr B4 22 Glu Ala Asp Glu
B5 24 Tyr Ile B5 23 Val Val Ala Ala
B6 25 Gly Gly B6 24 Gly Gly Gly Gly
B7 26 Ala Thr B7 25 Gly Gly Gly Gly
B8 27 Glu Glu B8 26 Glu Glu Glu Glu
B9 28 Ala Thr B9 27 Ala Ala Thr Ala
B10 29 Leu Leu B10 28 Leu Leu Leu Leu
B11 30 Glu Glu B11 29 Gly Gly Gly Gly
B12 31 Arg Arg B12 30 Arg Arg Arg Arg
B13 32 Met Leu B13 31 Leu Leu Leu Leu
B14 33 Phe Phe B14 32 Leu Leu Leu Leu
B15 34 Leu Leu B15 33 Val Val Val Val
B16 35 Ser Ser B16 34 Val Val Val Val
C1 36 Phe His C1 35 Tyr Tyr Tyr Tyr
C2 37 Pro Pro C2 36 Pro Pro Pro Pro
C3 38 Thr Gln C3 37 Trp Trp Trp Trp
C4 39 Thr Thr C4 38 Thr Thr Thr Thr
C5 40 Lys Lys C5 39 Gln Gln Gln Gln
C6 41 Thr Thr C6 40 Arg Arg Arg Arg
C7 42 Tyr Tyr C7 41 Phe Phe Phe Phe
CE1 43 Phe Phe CD1 42 Phe Phe Phe Phe
CE2 44 Pro Pro CD2 43 Glu Glu Asp Asp
CE3 45 His His CD3 44 Ser Ser Ser Ser
CE4 46 Phe Phe CD4 45 Phe Phe Phe Phe
CD5 46 Gly Gly Gly Gly
CE5 47 Asp Asp CD6 47 Asp Asp Asn Asn
CE6 48 Leu Leu CD7 48 Leu Leu Leu Leu
CE7 49 Ser His CD8 49 Ser Ser Ser Ser
CE8 50 His Pro D1 50 Thr Ser Ser Ser
D2 51 Pro Pro Ala Pro
D3 52 Asp Asp Ser Ser
D4 53 Ala Ala Ala Ala
D5 54 Val Val Ile Ile
D6 55 Met Met Met Leu
CE9 51 Gly Gly D7 56 Gly Gly Gly Gly
E1 52 Ser Ser E1 57 Asn Asn Asn Asn
E2 53 Ala Ala E2 58 Pro Pro Pro Pro
E3 54 Gln Gln E3 59 Lys Lys Lys Lys
E4 55 Val Leu E4 60 Val Val Val Val
E5 56 Lys Arg E5 61 Lys Lys Lys Lys
E6 57 Gly Ala E6 62 Ala Ala Ala Ala
E7 58 His His E7 63 His His His His
E8 59 Gly Gly E8 64 Gly Gly Gly Gly
E9 60 Lys Ser E9 65 Lys Lys Lys Lys
E10 61 Lys Lys E10 66 Lys Lys Lys Lys
E11 62 Val Val E11 67 Val Val Val Val
E12 63 Ala Val E12 68 Leu Leu Leu Leu
E13 64 Asp Ser E13 69 Gly Gly Thr Thr
E14 65 Ala Ala E14 70 Ala Ala Ser Ser
E15 66 Leu Val E15 71 Phe Phe Leu Phe
E16 67 Thr Gly E16 72 Ser Ser Gly Gly
E17 68 Asn Asp E17 73 Asp Asp Asp Asp
E18 69 Ala Ala E18 74 Gly Gly Ala Ala
E19 70 Val Val E19 75 Leu Leu Ile, Thr Ile
E20 71 Ala Lys E20 76 Ala Ala Lys Lys
EF1 72 His Ser EF1 77 His His His Asn
EF2 73 Val Ile EF2 78 Leu Leu Leu Met
EF3 74 Asp Asp EF3 79 Asp Asp Asp Asp
EF4 75 Asp Asp EF4 80 Asn Asn Asp Asn
EF5 76 Met Ile EF5 81 Leu Leu Leu Leu
EF6 77 Pro Gly EF6 82 Lys Lys Lys Lys
EF7 78 Asn Gly EF7 83 Gly Gly Gly Pro
EF8 79 Ala Ala EF8 84 Thr Thr Thr Ala
F1 80 Leu Leu F1 85 Phe Phe Phe Phe
F2 81 Ser Ser F2 86 Ala Ser Ala Ala
F3 82 Ala Lys F3 87 Thr Gln Gln Lys
F4 83 Leu Leu F4 88 Leu Leu Leu Leu
F5 84 Ser Ser F5 89 Ser Ser Ser Ser
F6 85 Asp Glu F6 90 Glu Glu Glu Glu
F7 86 Leu Leu F7 91 Leu Leu Leu Leu
F8 87 His His F8 92 His His His His
F9 88 Ala Ala F9 93 Cys Cys Cys Cys
FG1 89 His Tyr FG1 94 Asp Asp Asp Asp
FG2 90 Lys Ile FG2 95 Lys Lys Lys Lys
FG3 91 Leu Leu FG3 96 Leu Leu Leu Leu
FG4 92 Arg Arg FG4 97 His His His His
FG5 93 Val Val FG5 98 Val Val Val Val
G1 94 Asp Asp G1 99 Asp Asp Asp Asp
G2 95 Pro Pro G2 100 Pro Pro Pro Pro
G3 96 Val Val G3 101 Glu Glu Glu Glu
G4 97 Asn Asn G4 102 Asn Asn Asn Asn
G5 98 Phe Phe G5 103 Phe Phe Phe Phe
G6 99 Lys Lys G6 104 Arg Arg Lys Lys
G7 100 Leu Leu G7 105 Leu Leu Leu Leu
G8 101 Leu Leu G8 106 Leu Leu Leu Leu
G9 102 Ser Ser G9 107 Gly Gly Gly Gly
G10 103 His His G10 108 Asn Asn Asn Asn
G11 104 Cys Cys G11 109 Val Val Val Val
G12 105 Leu Leu G12 110 Leu Leu Leu Met
G13 106 Leu Leu G13 111 Val Val Val Val
G14 107 Val Val G14 112 Cys Cys Thr Ile
G15 108 Thr Thr G15 113 Val Val Val Ile
G16 109 Leu Leu G16 114 Leu Leu Leu Leu
G17 110 Ala Ala G17 115 Ala Ala Ala Ala
G18 111 Ala Ala G18 116 His Arg Ile Thr
G19 112 His Arg G19 117 His Asn His His
GH1 113 Leu Phe GH1 118 Phe Phe Phe Phe
GH2 114 Pro Pro GH2 119 Gly Gly Gly Gly
GH3 115 Ala Ala GH3 120 Lys Lys Lys Lys
GH4 116 Glu Asp GH4 121 Glu Glu Glu Glu
GH5 117 Phe Phe GH5 122 Phe Phe Phe Phe
H1 118 Thr Thr H1 123 Thr Thr Thr Thr
H2 119 Pro Ala H2 124 Pro Pro Pro Pro
H3 120 Ala Glu H3 125 Pro Gln Glu Glu
H4 121 Val Ala H4 126 Val Met Val Val
H5 122 His His H5 127 Gln Gln Gln Gln
H6 123 Ala Ala H6 128 Ala Ala Ala Ala
H7 124 Ser Ala H7 129 Ala Ala Ser Ala
H8 125 Leu Trp H8 130 Tyr Tyr Trp Trp
H9 126 Asp Asp H9 131 Gln Gln Gln Gln
H10 127 Lys Lys H10 132 Lys Lys Lys Lys
H11 128 Phe Phe H11 133 Val Val Met Leu
H12 129 Leu Leu H12 134 Val Val Val Val
H13 130 Ala Ser H13 135 Ala Ala Thr Ser
H14 131 Ser Val H14 136 Gly Gly Gly, Ala Ala
H15 132 Val Val H15 137 Val Val Val Val
H16 133 Ser Ser H16 138 Ala Ala Ala Ala
H17 134 Thr Ser H17 139 Asn Asn Ser Ile
H18 135 Val Val H18 140 Ala Ala Ala Ala
H19 136 Leu Leu H19 141 Leu Leu Leu Leu
H20 137 Thr Thr H20 142 Ala Ala Ser Ala
H21 138 Ser Glu H21 143 His His Ser His
HC1 139 Lys Lys HC1 144 Lys Lys Arg Lys
HC2 140 Tyr Tyr HC2 145 Tyr Tyr Tyr Tyr
HC3 141 Arg Arg HC3 146 His His His His

From Bunn HF, Nagel RL: Hemoglobins: normal and abnormal. In Nathan DG, Orkin SH, Ginsburg D, et al, editors: Nathan and Oski’s hematology of infancy and childhood , vol 7, Philadelphia, 2009, Saunders, p. 911–948; and modified from Bunn HF, Forget BG: Hemoglobin: molecular, genetic, and clinical aspects , Philadelphia, 1986, Saunders.

* The α and α-related (ζ) subunits are shown at the left. The β and β-related (δ, γ, and ε) chains are at the right. The amino acids’ relationship to the eight globin helices (A to H) is also shown. Thus A16 is the sixteenth amino acid in the A helix. Interhelical elbows are named for the two adjacent helices (e.g., AB1 is the first amino acid between helices A and B). The N- and C-terminal residues are labeled NA and HC, respectively. The residues are aligned to maximize the homology between subunits, which causes some gaps.

Hemoglobin Secondary Structure

Secondary structure refers to the organization of primary structure into sections of regular polypeptide backbone geometry. Hb chains are comprised primarily of α-helical secondary structure joined by turns or short sections with irregular polypeptide backbone conformation. The geometry of regular secondary structure is dictated by steric constraints (clashes between atoms in neighboring residues) and maximization of hydrogen bonding between amide and carbonyl groups in the backbone. Although hydrogen bonds are relatively weak individually, their repeating pattern in α helices has a cooperative effect to make the structure comparatively rigid. Through this property, α helices transmit forces in proteins and protein complexes to alter their structure and modulate function, as occurs for Hb. Most globin chains contain seven or eight α helices labeled A through H (from amino to carboxyl terminus), with the intervening links referred to as corners or loops. The position of each residue is described with reference to these structural elements. For example, the heme-coordinating histidine occurs at position F8 (residue 8 in helix F) in all globins (see Table 19-1 ).

Hemoglobin Tertiary Structure

Tertiary structure refers to the 3D structure of a polypeptide that is formed by packing secondary structural elements together. In general, hydrophobic side chains occupy the interior of a protein or protein domain, and polar and charged residues are arranged on the exterior ( Fig. 19-1, A and B ). Thus mutations converting a nonpolar interior amino acid to a polar one commonly produce an unstable protein (see the second part of this chapter). The α-helical segments of globin chains fold together into a globular shape with a cavity that is occupied by the O 2 -binding heme prosthetic group (see Fig. 19-1, B -D ). The basic tertiary structure, called the globin fold, occurs in a large, ancient protein family with members expressed in animals, plants, and bacteria. Figure 19-2 shows the tertiary structures of α and β globin with conserved α-helical arrangements and amino acid residues that mediate Hb function. Importantly, these structures are not static or rigid. Rather the tertiary structures of α and β globins (and the quaternary structure of the tetramer) change according to binding of O 2 or other ligands and also through interactions with allosteric effectors. These dynamic structural properties underlie the physiological function of Hb as a gas transporter and the pathologic features of many Hb variants.

Figure 19-1

The secondary structure and amino acid composition of the β chain of Hb.

A , Charged side chains ( blue : Lys, Arg, Glu, Asp) are all located on the surface of the structure exposed to the polar solvent (water). His side chains can be charged or uncharged depending on solvent and local protein environment and are not shown. B , Hydrophobic (nonpolar) side chains ( pale green : Ala, Val, Leu, Ile, Pro, Met, Phe, Trp) are clustered in the center of the structure, forming a hydrophobic core. Other polar, uncharged residues are not shown. C , A ribbon diagram showing the eight α helices, the location of the heme group (yellow/red spheres) , heme-coordinating proximal His (green) , and bound O 2 (magenta) . D , All atoms showing how O 2 (magenta) is buried in the heme pocket and stabilized by distal His E7. Rotation of the distal His E7 side chain is required to allow O 2 entry/exit from the distal heme pocket.

Figure 19-2

Structural features of the globin fold.

Ribbon representations of the α and β Hb chains in the O 2 -bound form, with the α helices A–H in the β chain and A–C and E–H in the α chain labeled. Note the conserved tertiary structure of the two globin subunits. Conserved residues in α and β are shown in stick representation, and carbon atoms are colored according to their function as follows. The proximal His F8 (green) coordinates the heme iron. The distal His E7 (cyan) makes electrostatic interactions that stabilize bound O 2 (red spheres) . Hydrophobic side chains in the distal heme pocket (yellow) make steric interactions with O 2 and other diatomic ligands. Hydrophobic Leu F4 prevents water from entering the proximal heme pocket and disrupting the His F8–Fe bond. Hydrophobic αPhe CE1/βPhe CD1 make important stabilizing interactions with the heme, help orient bound O 2 , and together with αPhe CE4/βPhe CD4 help position the distal His E7. Basic side chains (orange) interact with the negatively charged propionates of the heme group. The small side chains of Gly E8 and Gly B6 (red) are required to allow close packing of the E and B helices. Upon O 2 binding, tertiary structural changes in the globin chains are most significant in helices E and F and in the FG corner, which together with the heme group form the “allosteric core” (pale green) .

The Prosthetic Heme Group

Each α and β chain binds one heme b group ( Fig. 19-3 ), which forms the binding site for O 2 (see Fig. 19-1, C ). Heme is an amphipathic molecule that is highly insoluble in water, so it must be bound to protein carriers or protein active sites under physiologic conditions. Biosynthesis of protoporphyrin IX occurs through eight well-characterized steps that take place either in the cytosol or the intermembrane space of mitochondria. The final step, insertion of an iron ion, is catalyzed by the enzyme ferrochelatase.

Figure 19-3

The heme group.

The chemical structure of heme b, Fe(II)–protoporphyrin IX. The organic component, protoporphyrin IX, is a conjugated system of four pyrrole rings joined by methene bridges, with four methyl, two vinyl, and two propionate side chains. A central iron, the O 2 -binding molecule in Hb, is coordinated in a square-planar arrangement by lone pairs from the four pyrrole nitrogens.

(From Bunn HF, Nagel RL: Hemoglobins: normal and abnormal. In Nathan DG, Orkin SH, Ginsburg D, et al, editors: Nathan and Oski’s hematology of infancy and childhood , vol 7, Philadelphia, 2009, Saunders, p. 911–948.)

In the folded α and β chains, heme is bound into a deep pocket that is lined with hydrophobic residues (see Fig. 19-1, B and D ). Interactions between the protein and heme are critical for stabilizing the native globin fold. Thus mutations that destabilize heme–protein interactions lead to increased globin precipitation and the release of free heme, which has pathologic consequences (see the second part of this chapter). A coordinate covalent bond between the heme iron and the Nε atom of a histidine residue in the co-factor binding pocket, termed the proximal histidine (F8) , is of primary importance for globin structure and O 2 binding function (see Fig. 19-1, C and Residues of the Heme Pocket). A sixth iron–coordination site on the opposite (distal) side of the planar heme is available for reversible O 2 binding (see Fig. 19-1, C ). In addition to direct iron coordination to the proximal histidine, the following heme–globin interactions are important for maintaining the Hb tertiary structure: 1) hydrophobic interactions between nonpolar residues in the pocket and the porphyrin ring; 2) hydrogen bonding between residues in the distal heme pocket and diatomic ligands; and 3) electrostatic interactions between the heme propionates and amino acids on the surface of the protein. An asymmetric distribution of methyl and vinyl side groups on the heme ring defines a stereospecific orientation of heme binding. Heme iron can exist in a range of oxidation states in vivo, most commonly as Fe(II) or Fe(III), but the ferryl Fe(IV) is also biologically relevant (see Production of Ferryl Heme and Hemoglobin Protein Radicals ). Importantly, only Fe(II) (ferrous) heme can bind O 2 . The distal histidine (E7) of globin chains typically stabilizes bound O 2 , but under certain circumstances can also bind the sixth coordinate position of heme iron to form structures termed hemichromes or hemochromes when the iron is oxidized or reduced, respectively.

Hemoglobin Quaternary Structure

Quaternary structure refers to the packing of protein subunits into a multimeric complex. During Hb assembly, α and β monomers join to form a very high-affinity αβ dimer (association equilibrium constant [ K A ] >10 10 M −1 ) ( Fig. 19-4, A ). Two identical α1β1 and α2β2 dimers interact to form a tetramer, in which the constituent dimers are related by a two-fold rotational (C2) symmetry (see Fig. 19-4, B ). This interaction between dimers is coupled to O 2 ligand binding, with deoxygenated dimers having a higher affinity for self-association to form tetramers ( K A ≈4 × 10 10 M −1 ) than fully oxygenated dimers ( K A ≈10 6 M −1 ). The major dimer–dimer contacts involve nonidentical chains at the α1β2 and α2β1 interfaces. Changes in the interactions between α1β1 and α2β2 dimers are central to the mechanism of cooperative O 2 binding (see Fig. 19-4, C ).

Figure 19-4

Quaternary structure of Hb.

A , Schematic of the α1β1 dimer with helices shown as solid cylinders and the heme groups in space-fill representation. B , The Hb tetramer is comprised of two αβ dimers, related through a twofold rotational (C2 symmetry) axis (y) . A pseudo twofold axis (x) perpendicular to y , relates the positions of the α and β chains in each dimer. C , Looking down the pseudo twofold x axis, the van der Waals surface of the α2β2 dimer is shown. The position of the α1β1 dimer in the R (oxy) quaternary state is shown as gray/orange cylinders and yellow hemes . The position of the same α1β2 dimer in the T (deoxy) quaternary state is shown in blue. The α1β1 dimer undergoes a rotation of 15 degrees with respect to α2β2 about an axis that passes through both α chains.

Free globin subunits, particularly the apo forms lacking heme, are unstable and toxic as monomers and must be assembled rapidly and efficiently into Hb tetramers during erythropoiesis. The order of globin subunit assembly into mature Hb tetramers is unknown, but probably involves parallel pathways. For example, heme may be incorporated into apo α or β globins before or after their assembly into αβ dimers. In mammals, nascent apo or met α globins may be bound and stabilized by the molecular chaperone alpha Hb stabilizing protein (AHSP) during the early steps of Hb assembly in vivo.

Dioxygen Binding

The Oxygen Carrying Capacity of Hemoglobin

The solubility of O 2 in water (≈0.3 mM at 20° C and 1 atm) is insufficient to achieve full tissue oxygenation in most animals. Consequently animals have evolved to use heme or other respiratory pigments to transport O 2 . However, O 2 rapidly oxidizes free heme from the Fe(II) to Fe(III) state, which cannot bind O 2 (see Redox Reactions of Hemoglobin ). To overcome this potential limitation, globin proteins protect the heme group from oxidation and fine tune ligand binding to allow for reversible O 2 uptake and delivery. Hb carries O 2 from the lungs to peripheral tissue capillaries and CO 2 in the reverse direction. The concentration of Hb tetramers in red blood cells (RBCs) is typically about 5.2 mM (equivalent to ≈21 mM heme), and RBCs typically account for 45% of blood volume, giving a theoretical O 2 concentration of approximately 9 mM. O 2 is 21% by volume in air, which corresponds to roughly 8.75 mM in the gas phase at 1 atm and 20° C (1 mol gas under these conditions occupies 24 L by the ideal gas law). Hence Hb raises the O 2 concentration in blood to the same level as in air.

Residues of the Heme Pocket

Conserved, functionally important residues in the heme pockets of α and β globin are shown in Figure 19-2 . The proximal His-F8 stabilizes an axial iron ligand, which is essential for establishing the octahedral metal coordination geometry that is necessary for O 2 binding. Electrostatic interactions between bound O 2 and the distal His-E7 are of primary importance for ligand selectivity ( Fig. 19-5 ) and inhibiting heme oxidation (autoxidation). In both α and β globin, hydrogen bonding between His-E7 and O 2 has been identified through high resolution (1.25 Å) x-ray crystal structures, nuclear magnetic resonance, electron paramagnetic resonance (EPR), and site-directed mutagenesis. This interaction contributes around 1.9 kcal mol −1 to O 2 binding affinity. The importance of this interaction is illustrated by an approximate thousandfold increase in the O 2 dissociation rate from Mb when the distal His is replaced with a nonpolar amino acid. A number of nonpolar side chains in the proximal heme pocket make important steric interactions with bound O 2 (see Fig. 19-2 , yellow ). In particular, αPhe CE1/βPhe CD1, which is highly conserved in all globins, stabilizes the heme group and helps orient bound O 2 and His-E7 for efficient hydrogen bonding. βPhe-CD4 (αPhe-CE4) also plays a role in positioning of the distal His side chain to enable O 2 binding (see Fig. 19-2 ). In the proximal heme pocket, Leu F4 prevents water from entering and disrupting the His F8–iron bond.

Figure 19-5

Binding of O 2 in the heme pocket.

Stereo (cross-eye) diagram of the heme pocket of Hb β subunit. The heme Fe(II) (orange sphere) is coordinated by Nε of the proximal His F8. The iron−O 2 complex is polar, with net transfer of negative charge to O 2 (red spheres) . The polar Fe–O 2 interaction is stabilized by hydrogen bonding (magenta) with the distal His E7. Phe CE1 helps hold the heme in place and orients both bound O 2 and His E7 for more efficient hydrogen bonding. Val E11 contacts the bound O 2 and helps restrict solvent access to the distal pocket.

The heme pocket of globins is relatively enclosed (see Fig 19-1, D ), and multiple routes of O 2 entry have proposed, based on binding studies of xenon gas into nonpolar cavities of Mb and Hb. X-ray crystallographic and kinetic analyses of Mb and Hb indicate that O 2 enters via a channel caused by outward rotation of the distal His-E7 (see Figs. 19-1, D , and 19-2 , cyan ), which allows direct entry into the pocket rather than migration through other cavities in the protein.

In addition to O 2 binding, ferrous Fe(II) Hb also binds carbon monoxide (CO) and nitric oxide (NO) (see Nitric Oxide–Hemoglobin Reactions ). Studies with model porphyrin systems show that the Fe–CO bond is intrinsically stronger than the Fe–O 2 bond by a factor of 2 × 10 4 . If this ratio of affinities were true in the context of an Hb tetramer, then humans would be rapidly poisoned by endogenously produced CO, which is generated by heme catabolism, neuronal function, and cell signaling in the vasculature. High-level environmental CO exposure can also occur from cigarette smoke and internal combustion engines. Thankfully the relative affinity of CO and O 2 is reduced from 2 × 10 4 in free porphyrins to about 220 in Hb, largely because of the different electrostatic properties of the Fe–CO and Fe–O 2 complexes. The Fe–O 2 complex is highly polar and makes strong hydrogen bonding interactions with the distal His E7 (see Fig. 19-5 ). In contrast, the nonpolar Fe–CO complex interacts only weakly with the distal His.

Cooperative Oxygen Binding

Binding of O 2 to Mb can be described by a simple one–binding-site model that yields a hyperbolic curve ( Box 19-2 and Fig. 19-6 ):

<SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='Y=PO2/(P50+PO2)’>Y=PO2/(P50+PO2)Y=PO2/(P50+PO2)
Y = P O 2 / ( P 50 + P O 2 )

Box 19-2

Cooperative Binding of Oxygen to Hemoglobin

  • Cooperative O 2 binding results when the binding of O 2 to one site in Hb increases the O 2 affinity of the remaining sites. Similarly, release of O 2 from one site increases the probability that O 2 will be released from the remaining unbound sites. The overall effect enhances the amount of O 2 that can be bound and released over the narrow range of P O2 values that characterize arterial and venous blood (see Fig. 19-6 ).

  • Hb tetramers can bind four O 2 molecules (one per subunit). Binding of several O 2 molecules to deoxy-Hb causes a switch to a high affinity conformation. The high–O 2 affinity Hb quaternary structure is referred to as relaxed (R), and the low-affinity Hb structure is referred to as tense (T).

  • O 2 binding to the heme iron of one Hb subunit alters quaternary structure at the α1β2 and α2β1 interfaces of the tetramer, leading to structural changes in the heme pockets of unliganded Hb subunits to enhance their O 2 affinity. As a result, some mutations that affect the α1β2 and α2β1 interfaces alter the O 2 equilibrium curve, causing right or left shifts and different amounts of O 2 transport in vivo.

deoxy-Hb, Deoxygenated hemoglobin; Hb, hemoglobin; O 2 , oxygen; P O2 , partial pressure of oxygen.

Figure 19-6

O 2 equilibrium curves of sperm whale Mb and Hb in human red blood cells.

Myoglobin binds O 2 noncooperatively as a monomer with high affinity. The sigmoidal shape of the O 2 equilibrium curves reflect cooperative O 2 binding. In arterial blood (pH 7.4, 37° C, P O2 100 mm Hg), O 2 saturation is 98%. In mixed venous blood (pH 7.2, 37° C, P O2 40 mm Hg) O 2 saturation is 75%. The HbO 2 saturation difference between arterial and venous blood reflects the extent of O 2 delivery to peripheral tissues.

(From Imai K: Allosteric effects of haemoglobin, Cambridge, 1982, Cambridge University Press.)

In this equation, Y is the fractional O 2 saturation, P O2 is the partial pressure of O 2 , and P 50 is the value of P O2 at which 50% of the O 2 -binding sites are occupied. For the simple one–binding-site equilibrium, the dissociation constant, K d , is equal to P 50 . In contrast, the O 2 equilibrium curve for Hb is sigmoidal (see Fig. 19-6 ). The initial portion of the curve has a small slope, indicating that fully deoxygenated Hb (deoxy-Hb) has a relatively low affinity for O 2 . As O 2 loading proceeds, the slope of the curve steepens, reflecting increasing Hb affinity for O 2 . Ultimately, the slope levels off again as Hb approaches O 2 saturation. The shape of this curve indicates that O 2 binding to one or more sites causes an increase in the O 2 affinity of the remaining sites. This property, called positive cooperativity, is essential for Hb function.

Conversely, deoxygenation at one or more Hb sites decreases O 2 affinity for the remaining sites, favoring its release in peripheral tissues where O 2 concentration is relatively low. In this way, approximately 1.7 times more O 2 can be delivered to the tissues than would be possible with a hypothetical noncooperative O 2 binding protein with the same intrinsic O 2 affinity. An empirical relationship that described cooperative O 2 binding activity was proposed by A.V. Hill in 1910.

<SPAN role=presentation tabIndex=0 id=MathJax-Element-2-Frame class=MathJax style="POSITION: relative" data-mathml='Y=(PO2)n/(P50)n+(PO2)n’>Y=(PO2)n/(P50)n+(PO2)nY=(PO2)n/(P50)n+(PO2)n
Y = ( P O 2 ) n / ( P 50 ) n + ( P O 2 ) n
The exponent, n, termed Hill coefficient , measures cooperativity. A value of n > 1 represents positive cooperativity. A value of n = 1 yields no cooperativity. A value of n < 1 results in negative cooperativity. The Hill coefficient for HbA is approximately 2.8. Note that for the cooperative binding reaction, the apparent K d is equal to (P 50 ) n and is not the ligand concentration at half saturation.

Although the Hill equation reproduces the observed O 2 binding of Hb reasonably well, it implies simultaneous binding of multiple O 2 ligands. G.S. Adair determined that Hb contained four binding sites for O 2 and formulated a sequential binding model in which each O 2 binds with progressively higher affinity to each of the four sites. Adair’s equation gives an accurate fit to the experimental O 2 equilibrium curve and uses a general and physically plausible binding model. However, the generality of the model means that it does not provide any mechanistic insight into Hb cooperativity. In fact, cooperative binding implies some communication between the heme molecules in each globin chain. Before the structure of Hb (or any protein) was determined, Linus Pauling formulated a model based on a direct chemical interaction between heme groups and showed that O 2 binding characteristics could be reproduced, assuming that subunits of Hb were symmetrically arranged such that O 2 at one heme could influence binding at two adjacent sites. Given the distant spacing between the heme groups, communication must be mediated by the Hb protein matrix, with binding at one site sterically affecting O 2 binding to a heme group at another (“allo-”) site on an adjacent subunit.

Allostery and the Perutz–Monod, Wyman, Changeux Model of Hemoglobin Function

The term allosteric was first coined in 1961 by Monod and Jacob to described how regulatory ligands could influence the activity of enzymes by binding to distinct sites that did not overlap with the substrate binding site. Allostery, an extremely important concept in biochemistry, has been described as “the second secret of life,” second only to the genetic code. The Monod, Wyman, Changeux (MWC) model, developed in the early 1960s, still forms a theoretical basis for understanding cooperative O 2 binding to Hb. The theory itself was influenced by the structural view of Hb provided by Perutz. A key finding from Perutz and colleagues was the elucidation of the human deoxy-Hb structure at 5.5 Å, which provided an opportunity for comparison with the earlier structure of horse oxygenated Hb (HbO 2 ), also at 5.5 Å. At this resolution no difference in the structures of the individual subunits could be clearly identified. However, there was a clear difference in the quaternary structure. Perutz and colleagues noted that, “the two β chains in human reduced (deoxy-)Hb have moved apart, increasing the distance between their two haem groups 7 Å compared with that in horse oxyhaemoglobin.” This quaternary structural rearrangement of Hb in response to O 2 binding is at the heart of the MWC model.

The MWC model is shown in Figure 19-7, A . Its basic principle is that quaternary structural changes in Hb regulate the O 2 -binding affinity of the subunit chains. The MWC theory describes Hb as a symmetrical oligomer that can adopt two distinct quaternary structures, termed the tense (T) and relaxed (R) states or conformations. The T and R states are in equilibrium, and in the absence of O 2 , the T state is much more stable than the R state (see Fig. 19-7, A, top row ). The equilibrium between the T and R states of unliganded Hb is defined by the MWC parameter, L = [T 0 ]/[R 0 ], where [T 0 ] and [R 0 ] are the molar concentrations of the zero-liganded T and R states. In the T state, all the subunits have a tertiary conformation with low–O 2 affinity (small association equilibrium constant K T or high P 50 ), whereas in the R state, all subunits have a tertiary conformation with high–O 2 affinity (large association equilibrium constant K R or low P 50 ). Note that O 2 binding to the individual T (or R) state tetramers does not increase the affinity of the T (or R) state for binding additional O 2 molecules, so there is no cooperativity within the T (or R) quaternary state. However, because O 2 preferentially binds to empty sites in the R quaternary state rather than the T state ( K R > K T ), the T→R equilibrium must shift towards R with the addition of O 2 , according to the Le Châtelier principle. The basis for cooperativity is that the T→R quaternary switch involves a concerted switch of all subunits from the low–O 2 affinity tertiary structure to the high–O 2 affinity structure ( right-to-left shifts in Fig. 19-7, A ). The level of cooperativity is determined by the parameters L and c = K T / K R . The MWC model provides a good fit to experimental equilibrium O 2 -binding data, yielding typical parameters of L ≈ 5×10 6 and c ≈ 1.5 ×10 −3 and 1/ K T ≈ 80 torr for pH 7.4, 0.15 M NaCl, 21.5° C. These parameters are significantly modulated by pH, salt, and organic phosphate concentration (see Heterotropic Effectors of Oxygen Binding to Hemoglobin ). The parameter c reveals that the quaternary T→R switch corresponds to a 500- to 1000-fold increase in O 2 affinity. The MWC model neglects differences between α and β affinities, but these are likely to differ by less that twofold. Many excellent reviews of the MWC model with expanded and refined models are available.

Figure 19-7

Model for cooperative O 2 binding.

A, A schematic of the MWC-Perutz model based on Eaton, et al. The four subunits of the R or T quaternary structure are indicated as four circles or four squares , respectively. A solid black sphere represents an O 2 molecule bound to one subunit. The affinity constant for O 2 binding to any one subunit in the R or T state is K R or K T , respectively, hence there is no cooperativity within the R or T quaternary state. With no O 2 bound, the ratio of the T and R state is L = [T 0 ]/[R 0 ] ≈ 10 6 . Specific molecular interactions (e.g., salt bridges) between T state subunits (represented by joining lines ) are broken upon conversion to the R state. Binding of each O 2 molecule weakens these T state quaternary interactions by a factor c = K T / K R ≈ 10 −3 , shifting equilibrium towards the R state. The highly populated quaternary states at each oxygenation level (accounting for >90% of Hb molecules) are green ; the minor quaternary species are pale blue . B, The free energy change for successive O 2 binding events, obtained by fitting O 2 equilibration data obtained under standard conditions (21.5 °C, pH 7.4, 0.18 M Cl −1 ) to the four Adair binding constants, after adjustment for dimer-tetramer equilibrium. Note that binding becomes more favorable ( ΔG more negative) as O 2 saturation increases.

In order to move from the unliganded T 0 state to the fully O 2 -liganded R 4 state, the bonding and nonbonding interactions that stabilize the T 0 relative to R 0 quaternary structures (see lines joining T-state subunits in Fig. 19-7, A ) must be broken using free energy provided by O 2 binding. Consequently the free energy for binding of each successive O 2 becomes more favorable (see Fig. 19-7, B ). In the unconstrained R state, each binding event would yield −8.35 kcal mol −1 . The difference between the free energy for binding four O 2 molecules in HbA (−27.1 kcal mol −1 ) compared with binding in the unconstrained R state (−33.4 kcal mol −1 ) is called the cooperative free energy of binding (ΔG c = +6.3 kcal mol −1 ). To provide a molecular explanation for how O 2 binding is coupled to changes in tertiary and quaternary structure, Perutz developed his stereochemical model for cooperative O 2 binding, which is described in the following sections.

The High- and Low-Oxygen Affinity Quaternary Structures of Hemoglobin

The stereochemical mechanism proposed by Perutz identifies the T and R states within the MWC model as two discrete quaternary structures that differ in the relative orientation of the α1β1 and α2β2 dimers within the context of the Hb tetramer. The T-to-R quaternary switch of the tetramer involves about a 15-degree rotation of the α1β1 dimer relative to α2β2 (see Fig. 19-4, C ). The rotation occurs around an axis perpendicular to the C2 symmetry axis and passes through both α subunits. The β chains experience greater relative movement, moving closer together in the R state (as originally observed by Perutz). At the same time, the central cavity between the four globin chains, particularly the cleft between the β subunits, narrows in the R state and inhibits binding of 2,3-DPG, an important modulator of O 2 affinity (see 2,3-Diphosphoglycerate ).

Interactions between α1β1 and α2β2 dimers (i.e., α1β2 interactions) are weaker in the R (oxy) state compared with the T (deoxy) state, which accounts for the observation that R-state tetramers dissociate into α1β1 dimers at low protein concentrations, whereas T state Hb remains a tetramer and emphasizes the role of α1β2 interactions in cooperative O 2 binding. In contrast there is little or no change in the α1β1- (or α2β2-) dimer interface between the T and R quaternary states. Consequently residues at these interfaces do not contribute significantly to allostery. Accordingly, free α and β monomers and α1β1 dimers bind O 2 noncooperatively with a high affinity similar to that of R-state tetramers. Conversely, when crystallized or embedded in a silica gel, T-state Hb cannot undergo a quaternary T→R transition and binds O 2 noncooperatively at low affinity. These latter findings demonstrate that interactions at the α1β2 interface reduce O 2 affinity in the T state and to a first approximation account for the tetramer’s cooperativity.

In tetrameric Hb, the α 1 chain contacts both the α 2 and β 2 chains. Each of these interfaces plays a role in the allosteric mechanism. Interactions between nonidentical chains (α1β2 and α2β1 contacts) account for the majority of the buried surface area. A more restricted set of α1α2 contacts are present in the T and R states. The β1β2 interface is present in the R state but is lost as the β chains move apart in the T structure.

Shown in Figure 19-8, A , is the α1β1 dimer with the contact face for the α2β2 dimer facing up out of the page (the α2β2 dimer has been removed to reveal the contact surface). The α1β2 and α2β1 interfaces include the C helix and the FG corner (residues between the F and the G α helices) in each chain. These elements are colored blue and magenta in the α 1 and β 1 subunits shown in Figure 19-8 . The C helix from each Hb chain contacts the FG corner from the nonidentical chain across the α1β2 (or α2β1) interface. This interaction creates four pairs of contacts, as shown in Figure 19-8, B . In this panel, the C and FG segments of the α 2 and β 2 chains are shown as “bent tubes” overlaying the α1β1 dimer, with the rest of the chain omitted to allow the interface to be seen. Notably, the behavior of the C helix in α and β chains is different throughout the T→R quaternary change and is indicated by their different coloring in Figure 19-8 . Specifically, the molecular contacts between the C helices of the β chains (βC) and the FG corners of the α chains (αFG) change very little during the T→R quaternary transition (see Fig. 19-8, B , dark blue ). Therefore the βC–αFG contacts are known as the pivot (or joint ) contacts. In contrast, the αC helix moves relative to βFG corner, experiencing a translation of approximately 6 Å in the direction of the helix axis, equivalent to approximately one helical turn (5.4 Å) relative to the βFG corner (see Fig. 19-8, B , magenta ). The two alternative positions of the βFG loop relative to the αC helix are dictated by different side-chain packing interactions. Hence βAsp-99 in the βFG corner fits between the side chains of αThr-41 and αTyr-42 from the αC helix in the T state ( Fig. 19-9, B ). In the R state, βFG is shifted such that the side chain of βHis-97 fits between αThr-38 and αThr-41 in the αC helix (see Fig. 19-9, A ). These two alternative positions of the αC–βFG contact have led to this contact being described as the “switch” region of the α1β2 interface. It is thought that steric hindrance makes intermediates between these two positions unstable, giving rise to the two-state TR quaternary switch. The α1β2 interface is also characterized by different inter–subunit-hydrogen bonding patterns in the T and R states ( Fig. 19-10 ). The intricate nature of the switch region allows mutations at these residues to significantly perturb the stability of the T state ( L value in the MWC model) and therefore Hb function, sometimes with clinical consequences (see the second part of this chapter).

Figure 19-8

The α1β2 interface: switch and pivot interactions.

A , The α1β1 dimer from R-state HbA (Protein Data Bank [PDB] 2DN2) is shown with the α1β2 dimer removed to reveal the interdimer interface, looking down the pseudo twofold axis, x (see Fig.19-4, A ). Major contacts between the dimers are mediated by the C helices and FG loops ( blue and magenta ). B , The α1β1 dimer from T-state HbA (PDB 2DN1) is shown (orange, dark gray), superposed with the R-state structure from A (light blue transparency). The positions of the α2β2 dimers has been fixed to reveal the rotation of α1β1 between T and R structures. Only the C and FG segments of the α2β2 chains are shown (flexible “tubes”). Contacts between αFG and βC (blue) experience little change upon conversion from T to R (“pivot” contacts). In contrast, the αC helix moves relative to βFG loop ( magenta ; “switch” contacts).

Figure 19-9

The α1β2 switch contact.

Side chains of the βFG loop (orange) pack into grooves that are defined by side chains on the αC helix (gray) . To switch from the R state (PDB 2DN1) to the T state (PDB 2DN2), the βFG loop must move between two “valleys” that are separated by a “hill,” which is αThr 41.

Figure 19-10

Detail of the α1β2 interface in the deoxy T state (PDB 2DN2) and oxy R state (PDB 2DN1).

Hydrogen bonding networks in each state employ nonoverlapping sets of side chains (intrachain hydrogen bonds, black ; interchain hydrogen bonds, magenta ). Hb variants at these residue positions can alter the T→R equilibrium. Thus β99 (G1) Asp→Asn (Hb Kempsey) compromises electrostatic interactions only in the T state, thereby favoring the R state and resulting in high–O 2 affinity. Conversely, β102 (G4) Asn→Thr (Hb Kansas) disrupts the R state leading to low–O 2 affinity.

Although the switch region helps define distinct T and R structures, a range of R quaternary structures have been observed. For example, the C2 symmetry axis in liganded Hb that is crystallized under low-salt conditions at pH 5.8 is distinct from the T or R state structures determined at a neutral or high pH. This additional quaternary structure, R2 HbO 2 has been proposed to be either an intermediate in the T→R transition or a true end-point for fully oxy-Hb. The current understanding is that in the thermodynamic R state (fully oxy-Hb) proposed by the MWC model, Hb exists in a range of conformations, rather than as a single rigid structure. Crystal structures of the R, R2, and other quaternary variations provide information about the range of structures that are energetically accessible to HbO 2 . In support of this interpretation, recent measurements of O 2 binding to crystallized R- and R2-state Hb reveal that these are thermodynamically equivalent states, and a nuclear magnetic resonance (NMR) study of Hb in solution suggests that the average structure of liganded Hb lies approximately halfway between the crystallographic R- and R2-states. Despite considerable variation in the C2 symmetry axis between different R-state crystal structures, a set of common interactions between residues at the α1β2, α1α2, and β1β2 interfaces can be used as structural markers that identify the thermodynamic R state.

Altered Oxygen Binding at the Heme Site in the T and R States

In the unliganded state, pentacoordinate heme iron (Fe[II]) makes four bonds to pyrrole nitrogens and one axial bond with proximal His F8 (α87 or β92). A water molecule sits in the distal pocket of the α chain but does not coordinate the heme iron. The distal pocket of β chains appears empty, or there is no discrete position for a water molecule. The heme site in T-state deoxy-Hb has a high-spin Fe(II) electronic configuration, and the iron is displaced 0.4 to 0.6 Å out of the plane of the four pyrrole nitrogen atoms in the direction of the proximal His F8 ligand. Studies on model porphyrins indicate that an out-of-plane iron is the preferred structure for high-spin Fe(II) porphyrin and hence the iron site in T-state deoxy-Hb is unstrained. Importantly, O 2 binding requires a change in electronic configuration to a low-spin, hexacoordinate, in-plane iron. This configuration is seen in R-state HbO 2 where the heme iron is only 0.09 and 0.06 Å out of the porphyrin plane in α and β chains, respectively. Hence the iron site in R-state HbO 2 is also unstrained. However, accommodating the O 2 -liganded heme in the T-quaternary structure introduces strain into the Hb structure because movement of iron into the porphyrin plane requires upward movement of the F-helix away from the α1β2 interface and consequently reduces the free energy of O 2 binding. Thus in effect, the low–O 2 affinity of the T state can be explained, because the O 2 -liganded heme is a poor fit in the T-state structure. In the R-state oxy-Hb, the His F8 imidazole ring is positioned over the heme such that the Fe–Nε bond is normal to the plane of the porphyrin ( Fig. 19-11, A , blue ). This geometry is important to minimize steric repulsion between the imidazole and porphyrin rings when iron is in the porphyrin plane. However, in the T-state structure, the His F8 imidazole is rotated relative to heme plane (see Fig. 19-11, A , orange ). In this position there is a greater energetic cost for moving iron into the heme plane because of steric repulsion between Cε of His F8 and the pyrrole nitrogens of the porphyrin.

Figure 19-11

O 2 binding is coupled to tertiary structural changes in globin chains.

A , Schematic of the heme and proximal His F8 in T-state deoxy-Hb (orange) and R-state oxy-Hb (blue-gray) , looking along the plane of the porphyrin. The central iron atom ( orange sphere in deoxy Hb) moves about 0.5 Å into the plane of the porphyrin ring upon O 2 ligation. Steric repulsion between His F8 Cε and heme pyrrole nitrogens (N P ) introduces an energetic penalty against iron moving into the heme plane in the T state. Atoms are not drawn to scale. B , The most significant tertiary structure changes between α-globin chains from T- and R-state Hb occur at the F helix and EF loop, with smaller changes in parts of the E, G, and H helices (T state, orange ; R state, blue ). Other regions are essentially unchanged (salmon) . C , Movements of the F helix and FG corner are linked to movement of the heme iron, coupled through the His F8 side chain. Combined motions of the α and β chains from a single α1β1 dimer results in the FG loops moving approximately 2.5 Å closer together (T state, orange ; R state, blue-gray ). D , The α1β1 dimer is the base structural unit of hemoglobin. The stable portion of the dimer, including the α1β1 interface (pink) , supports the allosteric core of each chain (blue) , which includes the heme group (sticks), EF corner, F helix, and FG corner. Changes in the allosteric core, including movement of the FG corners, couple O 2 binding to quaternary structural changes.

Determining how strain, introduced into T-state Hb by O 2 binding, is distributed across the tertiary and quaternary T-state structure is the subject of ongoing research. In Perutz’s stereochemical model, the strain energy is at least partly compensated (or “stored”) by salt bridges present only in the quaternary T state, as described in the following sections.

Quaternary Interactions Contribute to Cooperativity

Salt bridges are electrostatic interactions between oppositely charged groups. These can involve the side chains of basic amino acid residues (Arg or Lys), acidic residues (Glu and Asp), N-terminal amino groups, or C-terminal carboxyl groups of polypeptide chains. Perutz identified eight specific salt bridges present in the T state (but not the R state) that stabilize the low-affinity quaternary conformation. More recent, higher-resolution crystal structures of T-state (deoxy-Hb) and R-state (oxy- and carbonmonoxy-Hb) Hb suggest minor modifications of this set of interactions ( Table 19-2 ). In the R state, these interactions are largely replaced by weaker hydrogen bonding interactions between polar residues or to water molecules. For example, the C-terminal residues αArg 141 (HC3) and βHis 146 (HC3) participate in T-state–specific salt bridges ( Fig. 19-12 ). Removing both αArg 141 and βHis 146 with carboxypeptidase B increases O 2 affinity and reduces cooperativity for the resultant modified Hb molecule. Similar conclusions were obtained using T-state Hb trapped in gels. Removal of αArg 141 alone caused a 15-fold increase in O 2 affinity of the T state, compared with a threefold increase for the β146 His→Leu mutation.

TABLE 19-2

Salt bridges and Hydrogen Bonding Interactions of the T- and R-State Quaternary Structure

Donor Acceptor
T-State Quaternary Structure
α1 Lys127 (H10) ε-amino group (+) α2 Arg141 (HC3) carboxyl terminus (–) *
α1 Arg141 (HC3) guanidinium (+) α2 Asp126 (H9) side chain carboxyl (–) *
α1 Lys40 (C5) ε-amino group (+) β2 His146 (HC3) carboxyl terminus (–) *
β1 His146 (HC3) imidazolium (+) β1 Asp94 (FG1) side chain carboxyl (–) *
α1 Arg92 (FG4) guanidinium (+) β2 Glu43 (CD2) side chain carboxyl (–) *
α1 Asn97 (G4) side chain amine β2 Asp99 (G1) side chain carboxyl (–)
α1 Tyr42 (C7) phenol β2 Asp99 (G1) side chain carboxyl (–)
α1 Lys127 (H10) ε-amino group (+) α1 Val1 (NA1) backbone carbonyl
β1 Val1 (NA1) amino terminus (+) β1 Leu78 (EF2) backbone carbonyl
β1 Trp37 (C3) indole amino group α2 Asp94 (FG1) side chain carboxyl (–)
R-State Quaternary Structure
β1 Val1 (NA1) amino terminus (+) β2 His146 (HC3) carboxyl terminus (–) *
α1 Asn97 (G4) side chain amide α1 Tyr42 (C7) phenol
α1 Val1 (NA1) amino terminus (+) α2 Ser138 (H21) backbone carbonyl
β1 Trp37 (C3) indole amino group β1 Asn102 (G4) side chain carbonyl
β1 Asn102 (G4) side chain amine α2 Asp94 (FG1) side chain carboxyl (–)
β1 Arg40 (C6) guanidinium (+) α2 Thr41 (C6) hydroxyl
β1 Arg40 (C6) guanidinium (+) α2 Leu91 (FG3) backbone carbonyl

* Rows containing asterisks indicate salt bridges. No asterisk indicates hydrogen bonding interactions.

Figure 19-12

Electrostatic interactions at the HbA dimer–dimer interfaces.

α1β1 (light gray, gold) and α2β2 (dark gray, orange) dimers make different sets of interactions in the T (left) and R (right) quaternary structures. These interactions are summarized in Table 19-2 and depicted in the lower panels of this figure. Note that the central cavity of the Hb tetramer widens in the T state (top left) allowing 2,3-DPG to bind at the β1β2 interface (bottom panel) .

Beginning with Szabo and Karplus, several groups created mathematical models suggesting that T-state–specific salt bridges could account for the free energy of cooperativity. Consistent with experimental evidence, some models suggested that ligand binding in the T state strains but does not break the salt bridges. Importantly, it seems that the lower O 2 binding affinity of T-state Hb is the result of effects from salt bridges and other interactions at the α1β2 interface, which transmits ligand-induced strain in the allosteric core (heme, proximal His, F helix and FG corner). Crystallized T-state deoxyHb can be oxygenated without switching to the R structure. This procedure has revealed changes in the stereochemistry of the heme, shifts in the FG corners towards the R-state quaternary structure, and lengthening (i.e., weakening) of the T-state–specific salt bridges. For example, the C-terminal salt bridge between β 1 His 146 (HC3) and α 2 Lys 40 (C5) (see Fig. 19-12 ) is lengthened by 0.9 Å in the oxy T state compared to the deoxy T state. However, more recent computational approaches conclude that the free energy of cooperativity is also stored across many other molecular interactions.

Hydrogen bonds at the α1β2 interface may also be strained when the T state is liganded. For example, Hb variants with nonconservative switch-region substitutions at βAsp 99 or αTyr 42 show increased O 2 affinity and reduced cooperativity (see Fig. 19-10 and discussion of Hb Kempsey in High–Oxygen affinity Hemoglobin Variants). In contrast, conservative substitutions of Tyr or Asp gave less marked effects. Similarly, at the α1β2 hinge region, the side chain of βTrp37 makes an inter-subunit hydrogen bond in the T state that stabilizes the α1β2 interface in the T relative to the R state, and mutations of βTrp 37 decrease cooperativity.

Coupling of Oxygen Binding to Structural Changes in Hemoglobin

Superposition of the α1β1 dimers from T- and R-state Hb reveals a set of residues that have invariant tertiary structure. A group of 74 residues are used as a reference frame to describe structural changes in the α and β chains, as well as the α1β1 and α2β2 dimers, during T→R shifts. These include α-chain residues 23 through 42 (helices B and C), α 57 through 63 (helix E), α 101 through 111 (helix G), and α 118 through 125 (helix H); and β-chain residues β 51 through 57 (helix D), β 110 through 116 (helix G), and β 119 through 132 (helix H). These reference-frame residues predominantly map to the α1β1 interface, reinforcing the concepts that relative orientation of the α1β1 and α2β2 dimers represents the major quaternary rearrangement associated with cooperative O 2 binding.

The concerted changes in iron coordination and globin tertiary structure are shown diagrammatically in Figure 19-11 . During the T-to-R switch, helix F moves closer to the center of the globin, the gap between helices E and F narrows, and the heme iron moves toward the porphyrin ring (see Fig. 19-11, A -C ). In addition (not shown in Fig. 19-11 ) the α heme group moves about 0.5 Å towards the back of the heme pocket, and the β heme undergoes a larger translation of 1.5 Å and a 9-degree rotation. Motion of the heme iron in and out of the porphyrin plane upon O 2 binding and/or dissociation is transmitted through the proximal His, causing the F helix to pivot. The relatively rigid α-helical structure allows a small movement of the heme iron in or out of the porphyrin (≈0.5 Å) to be amplified, resulting in relatively large motions at the FG corners. The combined tertiary structural changes in the α and β subunits from the same high- affinity (α1β1 or α2β2) dimer bring the αFG and βFG corners approximately 2.5 Å closer together in the R state (see Fig. 19-11, D , blue ). In contrast, the relative positions of the αC and βC helices are little changed (see Fig. 19-11, D , pink ). The net result of contracting the distance between the FG corners, while keeping the distance between the C helices fixed, is that some of the C-to-FG contacts across the α1β2 interface must altered. As described previously, the α 1 C-to-β 2 FG contacts break and reform (switch), whereas the α 1 FG-to-β 2 C contacts pivot, resulting in the 15-degree rotation of α1β1 relative to α2β2 (see Fig. 19-4, C ). Although the αC helix (hinge region) does not undergo substantial conformation changes, it is believed to store significant strain upon T-state oxygenation.

Several lines of evidence confirm that the bond between iron and the proximal His-F8 plays key roles in transmitting the effect of O 2 binding to the F helix and in promoting structural rearrangements at the tertiary and quaternary level. For example, when deoxy-Hb was crystallized in the T-quaternary state and the crystals were oxidized and soaked with sodium cyanide (CN), the binding of the strong CN ligand to the T-state α chains forced the iron-His–F8 bond to rupture, indicating that introducing ligands into the T-state structure places the iron-His–F8 bond under strain. In another experiment, Ho, Barrick, and colleagues specifically ablated the iron-His–F8 bond using an F8 His-to-Gly mutation and introduced free imidazole, which bound into the proximal heme pocket and coordinated the heme iron. They found that this complex exhibited increased O 2 affinity and reduced cooperativity. This experiment suggested that the proximal heme ligand in wild-type Hb transmits approximately two-thirds of the cooperative free energy of O 2 binding. Steric interactions between the pyrrole rings and heme pocket residues are also involved.

Changes in Tertiary Structure Precede Quaternary Switching

Gibson, Eaton, and their colleagues employed time-resolved spectroscopy to study internal ligand movement and conformational events that occur after laser photolysis of the Fe–CO bond but before bimolecular rebinding of the ligand from solvent. This approach has shown that tertiary structural changes after ligand binding or dissociation occur within a much shorter time frame (hundreds of nanoseconds [ns] to 1-2 microseconds [µs]) compared with quaternary structural changes (on the order of milliseconds [ms]). Computational methods indicate that fast tertiary T→R structural transitions precede slow quaternary switching and that tertiary structural changes induced by oxygen binding to multiple subunits increases the probability that Hb will make the T→R switch. These studies show that the “concerted” quaternary switch, integral to the MWC model, is more accurately viewed as a coupling between rapid tertiary and slow quaternary events. Efforts to develop quantitative biophysical models to fully explain the coupling of tertiary and quaternary structural changes to ligand binding, incorporating all equilibrium O 2 binding and kinetic data, are ongoing.

Heterotropic Effectors of Oxygen Binding to Hemoglobin

In addition to O 2 , other small molecules and ions known as heterotropic allosteric effectors bind with different affinities to T- and R-state Hb and as a result impact cooperative O 2 binding ( Box 19-3 ). The physiologically important species are CO 2 , hydrogen cation (H + ), Cl and 2,3-DPG. In general, these molecules bind more tightly to the T state, thereby reducing the O 2 binding affinity of Hb. This binding causes a “right shift” in the O 2 saturation curve ( Fig. 19-13 ). Heterotropic effectors have their most significant effect at low P O2 values, because they augment O 2 release in the tissues. In the lungs, at relatively high P O2 (≈95 mm Hg), the slightly decreased O 2 affinity caused by allosteric effectors has little effect on O 2 uptake.

Box 19-3

Allosteric Regulators of Hemoglobin

  • Allosteric regulation refers to changes in the structure and activity of a protein that results from binding of regulatory substances outside of the protein’s active site.

  • Allosteric regulators of Hb modulate O 2 binding by affecting the T-to-R transition.

  • Cooperative O 2 binding is a form of allosteric regulation.

  • Other allosteric regulators of Hb include H + , Cl , and 2,3-DPG, which bind outside of the heme pocket to alter Hb O 2 affinity.

  • CO 2 produced in peripheral tissues generates H + , which binds Hb to reduce its O 2 affinity, facilitating tissue oxygenation. This is the alkaline Bohr effect.

  • 2,3-DPG, a by-product of glycolysis, binds Hb to reduce its O 2 affinity. Hypoxia stimulates 2,3-DPG production to enhance O 2 delivery to tissues.

  • Fetal Hb (α 2 γ 2 ) exhibits low affinity for 2,3-DPG, which facilitates transfer of O 2 from mother to fetus.

2,3-DPG, 2,3-Diphosphoglycerate; Cl , chloride anion; CO 2 , carbon dioxide; H + , hydrogen cation; Hb, hemoglobin; O 2 , oxygen.

Figure 19-13

Heterotropic effectors alter the Hb-O 2 equilibrium curve.

The O 2 equilibrium curve for HbA in cells (black line) was simulated using P 50 = 20 mm Hg and n Hill = 2.7. High pH (low H + ), low 2,3-DPG, low temperature, and high–O 2 affinity Hb variants all cause a “left shift” in the HbO 2 saturation curve ( red line ; simulated with P 50 = 13 mm Hg, n Hill = 2.7). Low pH, high 2,3-DPG, high temperature, and low–O 2 affinity Hb variants cause a “right shift” ( blue line ; simulated with P 50 = 28 mm Hg, n Hill = 2.7). All curves are sigmoidal due to the enhanced affinity of Hb for O 2 upon binding successive O 2 molecules (see Box 19-2 ).

(Modified from Thom CS, Dickson CF, Gell DA, Weiss MJ: Hemoglobin variants: biochemical properties and clinical correlates. Cold Spring Harb Perspect Med 3:a011858, 2013.)

Carbon Dioxide Transport and the Bohr Effect

In 1904 the physiologist Christian Bohr showed that CO 2 reduced the O 2 affinity of Hb. Inside RBCs, carbonic anhydrase converts dissolved CO 2 to carbonic acid, which dissociates to bicarbonate and H + (i.e., CO 2 + H 2 O ⇌ H 2 CO 3 ⇌ HCO 3 + H + ). Upon conversion from the R to the T state, ionizable chemical groups within Hb bind H + ion. Hence increasing [H + ] or decreasing pH shifts equilibrium in favor of the low-affinity T state. The decreased O 2 affinity as pH changes from about 7.4 in the lungs to 7.2 or less in tissue capillary beds is known as the alkaline (or physiological ) Bohr effect. Below pH 6.0, the affinity for O 2 increases again. This is called the acid Bohr effect, which does not occur in vivo. The physiologic importance of the Bohr effect is that increased pCO 2 (acidification) occurs at sites of high metabolic activity, promoting O 2 release from Hb where it is most required. For example, pCO 2 rises from approximately 35 torr in lung alveoli to about 50 torr in exercising muscles, causing the pH of blood to fall from about 7.4 to 7.2 or lower. This translates into a right-shifted O 2 saturation curve (see Fig. 19-13 ) with a 25% increase in the P 50 for O 2 by the alkaline Bohr effect (∂ logP 50 /∂ pH = −0.5 in 0.1 M Cl , pH 7.4).

Hb facilitates the transport and disposal of CO 2 in several ways. As O 2 is released in actively respiring tissue capillaries, T-state Hb takes up H + , which promotes conversion of more CO 2 to bicarbonate via deprotonation of carbonic acid in red cells, thereby increasing the CO 2 carrying capacity of blood. Approximately 70% to 80% of CO 2 transport occurs as bicarbonate. In the lungs, the formation of R-state Hb releases bound H + , converting bicarbonate back to carbonic acid and CO 2 , which exchanges with air in the alveoli. An additional 15% to 20% of CO 2 is carried as carbamino-Hb, which is formed by reaction of CO 2 with the terminal amine groups of α and β chains (CO 2 + Hb-NH 2 ⇌ Hb-NH-COO + H + ). Finally, about 7% of CO 2 gas is simply dissolved in the blood.

The Bohr effect is largely attributed to formation of specific salt bridges involving chemical groups that can bind and release H + in response to physiological changes in pH. His residues have basic side chains with pKa of about 6.5, meaning that they can adopt different charge states in the physiological pH range, depending on their chemical environment. In the equilibrium His + -to-His + H + reaction, equal amounts of the charged and neutral His side chains are present when pH equals pKa. When pH is greater than pKa, the neutral state predominates, and when pH is less than pKa, the positively charged state predominates. A key point is that local electrostatic interactions have a significant effect on the pKa value. Salt bridges in the T or low-affinity quaternary conformation stabilize the charged His + side chain, increasing its pKa. In other words, the affinity of these His side chains for H + is higher in the T state compared with the R state (the necessary condition for an allosteric effector). Then these salt bridges are broken during the transition to the R state when O 2 binds, causing a decrease on pKa and loss of the His protons.

The side chain that contributes most significantly to the alkaline Bohr effect is βHis-146 (HC3), which forms a salt bridge with βAsp-94 (FG1) in T-state Hb (see Fig. 19-12 ). Ho and colleagues measured proton dissociation by NMR to determine the pKa of all His side chains in T- and R-state Hb. They found that the pKa of βHis-146 increases from 6.4 in the R state to 7.9 in the T state and accounts for 60% of the Bohr effect (similar to the finding of Kilmartin et al ). Meanwhile, the sum of the contributions from all 13 surface histidyl residues in the αβ dimer accounted for 86% of the alkaline Bohr effect in 0.1 M Cl , pH 7.4, the remaining effect being the result of other basic groups including the N-terminal amines. A tertiary Bohr effect has also been described (independent of quaternary bonds), which may involve protonation of buried His side chains including βHis-103 and the distal His side chains in the α and β heme pockets.

Cl is another physiologic inhibitor of O 2 binding that plays a role in the Bohr effect. As bicarbonate ions move out of the RBC down a concentration gradient, they exchange for Cl , which is imported into the red cell to maintain charge balance. This exchange is called the Hamburger or chloride shift, and is mediated by the anion exchange protein Band 3 on the erythrocyte plasma membrane. The alkaline Bohr effect is reduced by approximately 50% in the absence of Cl . Virtually all of the chloride-independent contribution of the Bohr effect occurs through βHis-146. Multiple basic groups contribute to the chloride-dependent Bohr effect. The pKa of the amino terminal group of αVal-1 (NA1) and a number of His side chains show O 2 -linked pKa changes that are dependent on Cl concentration. Specific Cl binding sites may exist, but these have not been reliably identified through crystallographic studies. A more global network of electrostatic interactions of Cl with multiple His side chains, the amino termini, and the positively charged central channel may ultimately be involved.


2,3-DPG (also known as 2,3-bisphosphoglycerate or 2,3-BPG ) is produced from 1,3-DPG as a by-product of glycolysis in a reaction called the Rapoport-Luebering shunt. The concentration of 2,3-DPG in RBCs is about 5 mM, approximately equimolar with Hb tetramers. 2,3-DPG is an allosteric inhibitor of O 2 binding and increases the P 50 of HbA to achieve more efficient O 2 delivery. 2,3-DPG carries 5 negative charges that interact with cationic groups lining the entrance to the central Hb cavity between the amino termini of the β chains in the 2,3-DPG binding site (see Fig. 19-12 ). The 2.5 Å crystal structure of HbA containing bound 2,3-DPG reveals the major electrostatic interactions to be salt bridges between the βHis-2 and βLys-82 side chains and the carboxyl and phosphate groups of the effector (see Fig. 19-12 ). βHis-143 also lies in the binding pocket, but this side chain appears to neutral at pH 7 and may not be essential for 2,3-DPG binding. In the R-state structure, movement of the β chains closes the central channel and occludes that binding site for 2,3-DPG. Thus 2,3-DPG binding to Hb causes a large shift in equilibrium towards the T state (15- to 30-fold increase in the MWC parameter, L = [T 0 ]/[R 0 ], and a significant decrease in K T , see Allostery and the Perutz–Monod, Wyman, Changeux Model of Hemoglobin Function ). Kinetic and equilibrium measurements at O 2 binding suggest that up to 50% of the decrease in P 50 caused by 2,3-DPG is the result of changes in K T and perhaps even K R .

2,3-DPG provides a means for physiological regulation of Hb O 2 affinity under conditions of hypoxia that occur at high altitude. Similarly 2,3-DPG synthesis increases in anemia, favoring O 2 dissociation to counter decreased Hb concentration (see Fig. 19-13 ). In pregnancy 2,3-DPG can be increased by 30%. Fetal Hb (HbF, α 2 γ 2 ) shows weak binding to 2,3-DPG compared with HbA, which is presumably an adaptation to the P O2 gradient between the maternal and fetal tissues. A number of sequence changes in the N- and C-termini of γ globin, compared with β globin, have been investigated with the conclusion that the diminished 2,3-DPG binding is complex and cannot be attributed to single residue changes. HbF also shows limited CO 2 binding as carbamates but retains the chloride independent Bohr effect.

Glycolytic enzyme defects affect 2,3-DPG production and therefore HbO 2 dissociation. In general, glycolytic defects affecting enzymes downstream of 2,3-DPG such as pyruvate kinase result in a higher level of 2,3-DPG, causing a right-shifted O 2 dissociation curve with increased O 2 unloading by red cell HbA in peripheral tissues (see Fig. 19-13 ). Clinically this reduces the erythropoietic drive. In contrast, glycolytic defects upstream of 2,3-DPG such as hexokinase deficiency cause a reduction in 2,3-DPG concentration and lead to increased Hb O 2 affinity and increased erythropoietic drive. The physiologic differences exhibited in anemic glycolysis defective patients with right-shifted (pyruvate kinase deficiency) versus left-shifted (hexokinase deficiency) O 2 -saturation curves are described in a classic study from Oski et al. The patient with hexokinase deficiency (left-shifted curve) demonstrated rapidly decreased central venous P O2 tension and increased cardiac output in response to exercise. In contrast, the patient with pyruvate kinase deficiency (right-shifted curve) was able to offload more O 2 to tissues, reflected by a more gradually decreased central venous P O2 and a relatively slower rise in cardiac output. Thus at the same Hb levels a patient with pyruvate kinase deficiency (increased 2,3-DPG) has greater exercise tolerance than one with hexokinase deficiency, in which 2,3-DPG production is inhibited.

Redox Reactions of Hemoglobin

Physiologically, iron can exist in the II, III, and IV oxidation states. However, only Hb containing Fe(II) heme is capable of reversible binding to molecular O 2 . Oxidation of the heme-iron to the III and IV states can trigger a range of reactions that lead to Hb denaturation and the production of reactive oxygen species (ROS) that damage erythroid cells.


In the presence of O 2 , α and β chains can spontaneously oxidize in a process called autoxidation, generating Fe(III) methemoglobin (met-Hb) and superoxide. The resulting Fe(III) met-Hb is unable to bind to O 2 , exhibits ~60-fold decrease in resistance to denaturation, is prone to form toxic insoluble globin precipitates containing dissociated hemin, and contributes to iron redox cycling and the generation of ROS (see Section on Production of ferryl heme and Hb protein radicals ). To counter this, erythrocytes contain high levels of superoxide dismutase and catalase that convert superoxide to H 2 O and O 2 . Erythrocytes also have high levels of the reducing agents ascorbate and glutathione, as well as a soluble form of cytochrome b5 reductase (also known as met-Hb reductase) that converts met-Hb back to the Fe(II) state. Despite these mechanisms, approximately 1% of Hb exists in the non-functional, oxidized Fe(III) state at any time, probably due in part to reactions with NO. The mechanisms of spontaneous Hb autoxidation have been the subject of much investigation. Early studies suggested that an electron might is transferred from Fe(II) to bound O 2 , leading to direct dissociation of superoxide anion :

<SPAN role=presentation tabIndex=0 id=MathJax-Element-3-Frame class=MathJax style="POSITION: relative" data-mathml='Hb−Fe(II)−O2→Hb−Fe(III)++O2·−’>HbFe(II)O2HbFe(III)++O2·Hb−Fe(II)−O2→Hb−Fe(III)++O2·−
Hb − Fe ( II ) − O 2 → Hb − Fe ( III ) + + O 2 · −

The normal Fe(II)–O 2 bond involves partial transfer of electron density from the iron to the O 2 , such that the HbO 2 can be described as . This charge transfer is reversed when molecular O 2 dissociates. The activation barrier for dissociation of negatively charged O 2 ·− from the positively charged iron center makes the barrier to direct superoxide anion dissociation high. An alternative reaction is an outer sphere electron transfer from the Fe(II)–porphyrin to an O 2 molecule that is not coordinated to the iron:

<SPAN role=presentation tabIndex=0 id=MathJax-Element-4-Frame class=MathJax style="POSITION: relative" data-mathml='Hb−Fe(II)+O2→Hb−Fe(III)++O2·−’>HbFe(II)+O2HbFe(III)++O2·Hb−Fe(II)+O2→Hb−Fe(III)++O2·−
Hb − Fe ( II ) + O 2 → Hb − Fe ( III ) + + O 2 · −

However, these reactions by themselves do not explain the dependence of autoxidation on O 2 concentration. It can be shown that both reactions cause autoxidation rate to increase up to a maximum asymptotic value with increasing [O 2 ]. However, a plot of autoxidation rate against O 2 concentration is bell-shaped, with the maximum rate occurring at approximately P 50 and an asymptotic value at very high O 2 concentrations. Work with various globins and model heme compounds suggested that electron transfer can occur by an outer sphere reaction for hexacoordinate Fe(II)-porphyrin that has a solvent molecule or other anionic ligand (L) at the sixth coordination site to facilitate formation of stable ferric iron complex :

<SPAN role=presentation tabIndex=0 id=MathJax-Element-5-Frame class=MathJax style="POSITION: relative" data-mathml='Hb−Fe(II)−O2+L⇌Hb−Fe(II)+L+O2⇌Hb−Fe(II)−L+O2→Hb−Fe(III)+−L+O2·−’>HbFe(II)O2+LHbFe(II)+L+O2HbFe(II)L+O2HbFe(III)+L+O2·Hb−Fe(II)−O2+L⇌Hb−Fe(II)+L+O2⇌Hb−Fe(II)−L+O2→Hb−Fe(III)+−L+O2·−
Hb − Fe ( II ) − O 2 + L ⇌ Hb − Fe ( II ) + L + O 2 ⇌ Hb − Fe ( II ) − L + O 2 → Hb − Fe ( III ) + − L + O 2 · −

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Apr 1, 2019 | Posted by in HEMATOLOGY | Comments Off on Normal and Abnormal Hemoglobins
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