Harper's Illustrated Biochemistry, Volume 1 - Murray R. 1993

Structure and Function of Proteins and Enzymes
Proteins: Myoglobin and Hemoglobin
Hemoglobins

Biological Function of Hemoglobins

Hemoglobins are structurally related Proteins found in the erythrocytes of vertebrates. They perform two essential biological Functions:

1) they transport O₂ from the Lungs to peripheral Tissues;

2) they transport CO₂ and protons from peripheral tissues to respiratory Organs for subsequent elimination from the Organism. The Comparative biochemistry of hemoglobins is fascinating in its own right; however, we will focus our attention here exclusively on human hemoglobins.

Introduction/19.html">Primary Structure of Hemoglobin A

Unlike Myoglobin, which lacks a quaternary structure, hemoglobins are tetrameric proteins whose molecules are composed of various types of polypeptide chains (designated as a, ß, y, δ, S, etc.). The molecule contains two chains of each of two different types. The lengths of the a- and ß-chains are approximately the same—the a-chain contains 141 residues, and the ß-chain 146; nevertheless, the a- and ß-Polypeptides of hemoglobin A (HbA) are encoded by different genes and possess distinct primary structures. At the same time, the primary structures of the ß-, y-, and δ-chains of human hemoglobin are largely conserved.

Secondary and Tertiary Structure of Hemoglobin A

Despite differences in chain length and Amino Acid Sequence between myoglobin and the ß-polypeptide of HbA, they share nearly identical secondary and tertiary structures. This striking resemblance, which extends to the arrangement of the heme group and the eight helical segments, is partly due to the fact that equivalent positions in the primary structures of myoglobin and the ß-subunit of HbA contain Amino Acids that, although differing, share similar properties. The a-polypeptide is also very similar to myoglobin, although it contains seven helices rather than eight. As in myoglobin, hydrophobic residues are located in the interior of the structure, while hydrophilic residues (again with the exception of two Histidine residues) reside On the surface; this characteristic is equally typical of both a- and ß-subunits.

Quaternary Structure of Hemoglobin A

The Properties of Individual hemoglobins are inextricably linked to their quaternary structures, as well as their secondary and tertiary structures. The most common hemoglobins exhibit the following tetrameric structures: HbA (normal adult hemoglobin) — a₂ß₂; HbF (fetal hemoglobin) — a₂y₂; HbS (sickle-Cell hemoglobin) — a₂S₂; HbA₂ (minor adult hemoglobin) — a₂δ₂. The quaternary structure confers upon hemoglobin additional vital features (absent in myoglobin) that facilitate its unique biological function and enable strict regulation of its properties. Hemoglobin exhibits allosteric properties (from the Greek allos, other, and stereos, space, solid), making it an excellent model for understanding The behavior of other allosteric proteins.

Kinetics of Hemoglobin Oxygenation

Hemoglobin binds four oxygen molecules per tetramer (one per heme in each subunit); a particularly crucial distinction from myoglobin is its characteristic oxygen saturation curve, which has a sigmoidal shape (Fig. 6.8). Thus, hemoglobin's ability to bind O₂ depends on whether other O₂ molecules are already bound within the given tetramer. If so, subsequent O₂ molecules bind more readily. Consequently, hemoglobin exhibits cooperative binding kinetics, enabling it to bind the maximum amount of O₂ in the lungs and release the maximum amount of O₂ at the P_O2 levels prevailing in peripheral tissues. Compare, for example, the amounts of oxygen bound by hemoglobin and myoglobin in the lungs at P_O2 = 100 mm Hg versus in the tissues at P_O2 = 20 mm Hg (Fig. 6.8).

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Fig. 6.8. Oxygen binding curves for hemoglobin and myoglobin. The partial pressure of oxygen is approximately 100 mm Hg in arterial Blood, about 40 mm Hg in venous blood, and about 20 mm Hg in the capillaries of active Muscle; the minimum pressure required for the functioning of the cytochrome system Enzymes is ~5 mm Hg. As seen from the figure, the association of chains into a tetrameric structure significantly enhances the efficiency of tissue oxygen supply compared to monomeric proteins. (Isolated hemoglobin chains possess approximately the same oxygen affinity as myoglobin and exhibit a similar hyperbolic saturation curve.) (Modified from Stanbury J. B., Wyngaarden J. B., Fredrickson D. S. (editors): The Metabolic Basis of Inherited Diseases. 4th ed. McGraw-Hill, 1978.)

The affinity of hemoglobins for O₂ is characterized by the P₅₀ value — the P_O2 at which hemoglobin is half-saturated with oxygen. The P₅₀ value varies significantly among different organisms, but in all cases, it exceeds the P_O2 in the peripheral Tissues of the respective organism. This is well illustrated by human fetal hemoglobin (HbF). For HbA, P₅₀ = 26 mm Hg, whereas for HbF, P₅₀ = 20 mm Hg. Because of this difference, hemoglobin F extracts oxygen from HbA present in placental blood. However, after birth, HbF loses its function; possessing a higher affinity for O₂, it releases a smaller amount of it in the tissues.

Oxygenation is Accompanied by Significant Conformational Changes in Hemoglobin

The binding of O₂ is accompanied by the rupture of salt bridges formed by the terminal carboxyl groups of the subunits (Fig. 6.9). This facilitates the binding of subsequent O₂ molecules, as fewer salt bridges need to be broken. These alterations noticeably affect the secondary, tertiary, and especially the quaternary structure of hemoglobin. In the process, one a/ß-subunit pair rotates relative to the other a/ß-pair, leading to compaction of the tetramer and an increase in the affinity of the Hemes for O₂ (Figs. 6.10 and 6.11).

The quaternary structure of partially oxygenated hemoglobin is described as the T-state (from taut); fully oxygenated hemoglobin (HbO₂) corresponds to the R-state (relaxed) (Fig. 6.12). The terms R- and T-states are used to characterize the quaternary structure of allosteric enzymes; the T-state possesses a lower substrate affinity.

Conformational Changes in the Heme Environment

The Oxygenation of hemoglobin, much like that of myoglobin, is accompanied by structural alterations in the environment of the heme group. Upon oxygenation, the iron atom, which protrudes 0.06 nm out of the plane of the heme ring in deoxyhemoglobin, is drawn into this plane (Fig. 6.13). Following the iron atom, the proximal histidine (F8) and its neighboring linked residues also shift closer to the heme.

Fig. 6.9. Salt bridges between subunits in deoxyhemoglobin. Upon oxygenation, these non-covalent bonds, maintained by Electrostatic Interactions, are disrupted. (Modified from Stryer L.: Biochemistry, 2nd ed., Freeman, 1981.)

Carbon dioxide transport

Hemoglobin not only transports oxygen from the lungs to peripheral tissues but also accelerates The transport of CO₂ from tissues to the lungs. Hemoglobin binds CO₂ immediately following the release of oxygen; approximately 15% of the CO₂ present in the blood is transported by hemoglobin molecules. Carbonic anhydrase located in erythrocytes catalyzes The conversion of tissue-derived CO₂ into carbonic acid (Fig. 6.14). Carbonic acid rapidly dissociates into bicarbonate ions and protons, with the equilibrium favoring dissociation. To prevent a dangerous increase in blood acidity, a buffering system capable of absorbing excess protons must be present. Hemoglobin binds two protons for every four released oxygen molecules and determines the buffering capacity of the blood (Fig. 6.15). In the lungs, the reverse process occurs: the binding of oxygen to deoxyhemoglobin is accompanied by the release of protons, which combine with bicarbonate ions to form carbonic acid. Subsequently, highly efficient carbonic anhydrase catalyzes the conversion of carbonic acid into carbon dioxide, which is exhaled from the lungs. Thus, oxygen binding is tightly coupled to the exhalation of CO₂. This reversible phenomenon is known as the Bohr effect. The Bohr effect is a property of tetrameric hemoglobin and is determined by heme-heme interaction, which underlies cooperative effects. Myoglobin exhibits no Bohr effect.

Fig. 6.10. The transition of hemoglobin from the T to the R state involves the rotation of one tightly coupled pair of subunits (a₂/ß₂) by 15° relative to the other identical pair (a₂/ß₂). The axis of rotation is eccentric, meaning that the (a₂/ß₂) dimer simultaneously shifts closer to the axis of the tetramer. This figure illustrates the rotation and Displacement of the shaded a₂/ß₂ pair relative to the unshaded a₁/ß₁ pair (the latter is treated as stationary).

Fig. 6.11. Changes occurring in the a₁/ß₂ contact region during oxygenation. The contact effectively "jumps" from one residue to another, replacing one Hydrogen bond with another. The remaining bonds are formed by nonpolar residues. (From Perutz M. F.: Molecular pathology of human hemoglobin. Stereochemical interpretation of abnormal oxygen affinities. Nature 1971:232:408, with kind permission.)

Fig. 6.13. Upon oxygenation, the diameter of the iron atom's coordination sphere decreases, drawing the atom into the heme plane. Histidine F8 shifts along with the iron atom. (From Stryer L: Biochemistry, 2nd ed., Freeman, 1981, with modifications.)

Fig. 6.12. The probability of the transition from the T state to the R state increases with the successive oxygenation of each of the four heme groups. In the model shown here, the salt bridges (solid lines) that link the subunits in the T conformation are broken as oxygen binds, and even the salt bridges that remain intact are progressively weakened (wavy lines). The transition from the T to the R state is not rigidly coupled to the binding of a specific number of oxygen molecules; however, the probability of this transition increases with the binding of each additional oxygen molecule. The interconversion between the two states is influenced by protons, carbon dioxide, chloride, and DPG. The higher their concentration, the greater the number of oxygen molecules that must bind to enable the transition. Fully oxygenated molecules in the T conformation and fully deoxygenated molecules in the R conformation are not shown, as they are too unstable to be present in appreciable amounts. (From Perutz M. F.: Hemoglobin structure and respiratory transport. Sci. Am. [Dec.] 1978:239:92, with modifications.)

Fig. 6.14. The formation of carbonic acid in the reaction catalyzed by erythrocyte carbonic anhydrase and its dissociation into a bicarbonate ion and a proton.

Fig. 6.15. The Bohr effect. Carbon dioxide produced in peripheral tissues reacts with Water to form carbonic acid, which dissociates into a bicarbonate ion and a proton. Deoxygenated hemoglobin acts as a buffer—it binds protons and transports them to the lungs. In the lungs, oxygen binding by hemoglobin is accompanied by the release of protons from hemoglobin. The protons combine with the bicarbonate ion to form carbonic acid, which, with the assistance of carbonic anhydrase, is converted into carbon dioxide and water. Carbon dioxide is eliminated from the lungs in exhaled air.

Molecular Basis of the Bohr Effect

The protons responsible for the Bohr effect are released As a result of the disruption of salt bridges that accompanies oxygen binding to the T structure; they dissociate from the nitrogen atoms of the histidine residues (146) in the ß-chains. These protons shift the equilibrium toward the formation of carbonic acid, which is cleaved by carbonic anhydrase to yield CO₂ (Fig. 6.15).

Conversely, when oxygen is released, the T structure with its characteristic salt bridges is re-formed, a process during which protons attach to the histidine residues in the ß-chains. Thus, in peripheral tissues, protons promote the formation of salt bridges via protonation (at the nitrogen atom) of the terminal histidine residues in the ß-subunits. The formation of salt bridges drives the release of oxygen from the oxygenated R form of hemoglobin. Consequently, an increase in proton concentration facilitates oxygen release, while an increase in oxygen concentration stimulates proton release. The former effect is manifested as a shift of the oxygen dissociation curve to the right upon an increase in hydrogen ion (proton) concentration.

Regulation by 2,3-Bisphosphoglycerate

Oxygen deficiency in peripheral tissues leads to the accumulation of 2,3-bisphosphoglycerate (diphosphoglycerate, DPG) (Fig. 6.16). This compound is formed from 1,3-bisphosphoglycerate, an intermediate of Glycolysis. The hemoglobin tetramer binds a single molecule of DPG, which fits into the central cavity lined by residues from all four subunits. This cavity is large enough to accommodate DPG only when the hemoglobin molecule is in the T state and a sufficiently wide opening is formed between the H-helices of the ß-chains. DPG binding is achieved through the formation of salt bridges between the oxygen atoms of DPG and groups belonging to both ß-chains: the terminal amino groups of Val NA1 residues, the amino groups of Lys EF6 residues, and the side chains of His H21 residues (Fig. 6.17). Thus, DPG stabilizes the deoxygenated T form of hemoglobin by establishing cross-links between the ß-chains—additional salt bridges that must be broken during the transition of hemoglobin from the T to the R state.

Fig. 6.16. Structure of 2,3-bisphosphoglycerate.

DPG binds less tightly to fetal hemoglobin than to adult hemoglobin because its ß-chain has a Ser residue instead of His at position H21, which is unable to participate in forming the salt bridges that anchor DPG in the central cavity. Therefore, DPG is less effective at stabilizing the T conformation of fetal hemoglobin, giving it a higher oxygen affinity compared to adult hemoglobin.

The trigger for the transition between the R and T forms of hemoglobin is the movement of the iron atom into or out of the porphyrin ring plane. Steric and electrostatic factors serve as the source of Free energy for these processes (approximately 3,000 cal/mol). Thus, a very small displacement of the Fe²⁺ atom relative to the porphyrin ring induces significant conformational changes in hemoglobin and crucially determines its response to environmental signals.

Fig. 6.17. Mechanism of DPG binding to human deoxyhemoglobin. DPG interacts with three positively charged groups in each of the ß-chains. (From Arnone A.: X-Ray Diffraction study of binding of 2,3-diphosphoglycerate to human deoxyhemoglobin. Nature 1972:237:146, with permission.)

Mutant Human Hemoglobins

Mutations in the genes encoding the a- and ß-chains can significantly impair their biological function. Several hundred mutant human hemoglobins are known (most of which are functionally active), and some of those featuring drastic alterations in biological function are discussed below. A pathological condition in which a mutation alters the biological function of hemoglobin is referred to as a hemoglobinopathy.

In the M family of hemoglobins, the proximal or distal histidine residues in the a- or ß-subunits are replaced by Tyrosine residues. The iron atom within the heme is in the Fe³⁺ state as a result of forming a stable ionic complex with the phenolate anion of tyrosine. This anomaly results in methemoglobinemia, because the ferric heme is incapable of binding O₂. In the a-chain of hemoglobin M, the R–T equilibrium is shifted toward the T state. Oxygen affinity is low, and the Bohr effect is absent. In the ß-chains of M hemoglobins, the transition between the R and T states can still occur, and consequently, the Bohr effect is observed.

Mutations leading to the preferential Formation of the R-form (such as hemoglobin Chesapeake) are characterized by an increased oxygen affinity in the corresponding hemoglobins. Such hemoglobins are unable to deliver a sufficient amount of oxygen to peripheral tissues. This results in tissue Hypoxia, which leads to polycythemia (an elevated red blood cell concentration).

Hemoglobin in Sickle-Cell Anemia

In hemoglobin S, the Glu A₂(6)ß residue is replaced by Val. The A₂ residue (Glu or Val) is located on The surface of the hemoglobin molecule and is in contact with water; the substitution of the polar Glu residue with the nonpolar Val leads to the appearance of a "sticky patch" on the surface of the ß-subunit. This sticky patch is present in both oxygenated and deoxygenated hemoglobin S (it is absent in hemoglobin A). A complementary site exists on the surface of deoxygenated hemoglobin that is capable of tightly binding to the sticky patch of the ß-subunit, whereas in oxygenated hemoglobin this site is masked by other groups (Fig. 6.18). When hemoglobin S transitions to the deoxygenated state, its sticky patch binds to the complementary site on another deoxygenated hemoglobin molecule. Polymerization of deoxyhemoglobin S occurs, leading to its precipitation into long fibers. Deoxyhemoglobin S fibers mechanically deform THE RED BLOOD cell, giving it a sickle shape, which results in cell lysis and numerous secondary clinical manifestations. Thus, if it were possible to maintain hemoglobin S in the oxygenated state or at least minimize the concentration of deoxygenated hemoglobin S, we would be able to prevent the polymerization of deoxygenated hemoglobin S and the formation of "sickle" Cells. Clearly, the T-form of hemoglobin S is susceptible to polymerization. It is interesting to note (although of minor practical significance) that the ferric ion of methemoglobin A remains in the plane of the porphyrin ring and thereby stabilizes the R-form of hemoglobin. The same applies to the hemoglobin in sickle-cell anemia: hemoglobin S in the ferric state (methemoglobin S) does not undergo polymerization because it is stabilized in the R-form.

Fig. 6.18. Diagram illustrating the interaction of the sticky patch of hemoglobin S (black triangle) with the sticky patch receptor (light triangle) of deoxyhemoglobin A and deoxyhemoglobin S. The presence of complementary sites on the surface of the deoxyhemoglobin S molecule promotes its polymerization with the formation of fibrous structures. In the presence of deoxyhemoglobin A, the polymerization process halts because this molecule lacks a sticky patch on its surface. (From Stryer L.: Biochemistry, 2nd ed., Freeman, 1981, with some modifications.)

Deoxyhemoglobin A also possesses a receptor site capable of interacting with the sticky patch of oxygenated or deoxygenated hemoglobin S (Fig. 6.18), but the attachment of the "sticky" hemoglobin S to deoxyhemoglobin A is not sufficient for polymer formation, since deoxyhemoglobin A itself does not contain a sticky patch and cannot bind the next hemoglobin molecule. Consequently, the binding of deoxyhemoglobin A to the R- or T-form of hemoglobin S interrupts polymerization.

The polymerization of deoxyhemoglobin S results in the formation of helical fibrillar structures, with each hemoglobin molecule contacting four neighboring molecules (Fig. 6.19). The formation of such tubular fibers is responsible for the mechanical damage sustained by the red blood cell containing them: it acquires a sickle shape (Fig. 6.20) and becomes susceptible to lysis as it passes through slits in the splenic sinusoids.

Fig. 6.19. Proposed helical structure of a fiber composed of aggregated deoxyhemoglobin S molecules. (From Maugh T. II: A new understanding of sickle cell emerges. Science 1981:211:265, with permission.)

Fig. 6.20. Cytology/cytology/93.html">ELECTRON MICROGRAPHS OF normal (A) and sickle (B) red Blood Cells. The alterations in the ß-globin molecule that lead to this change in cell shape are caused by a single base mutation in DNA (A instead of T), resulting in the substitution of glutamate for valine in the ß-globin chain (ch. 36).

Thalassemias

Another important group of disorders associated with hemoglobin abnormalities is the thalassemias. They are characterized by a reduced rate of synthesis of hemoglobin $\alpha$-chains ($\alpha$-thalassemia) or $\beta$-chains (ß-thalassemia). This leads to anemia, which can assume a very severe form. In recent years, tangible progress has been made in elucidating the molecular mechanisms responsible for The Development of thalassemia (see ch. 36).

References

Dean J., Schechter A. N. Sickle-cell anemia: MOLECULAR AND CELLULAR basis of therapeutic approaches. (3 parts). N. Engl. J. Med.. 1978. 299. 752, 804, 863.

Klotz I.M., Haney D.N., King L.C. Rational approaches to Chemotherapy: Antisickling agents. Science, 1981,213, 724.

Perutz M. F. Hemoglobin structure and respiratory transport. Sci. Am. (Dec.). 1978, 239, 92.

Perutz M. F. The regulation of oxygen-affinity of hemoglobin: Influence of structure of globin on heme iron, Annu. Rev. Biochem., 1979, 48. 327.

Stamatoyannopoulos G. The molecular basis of hemoglobin disease, Annu. Rev. Genet., 1972, 6, 47.

Winslow R. M., Anderson W. F. The hemoglobinopathies. Page 1666. In: The Metabolic Basis of Inherited Disease, 5th ed., Stanbury J. B. et al. (eds.), McGraw-Hill, 1983.