The quaternary structure of hemoglobin confers striking additional properties, absent from monomeric myoglobin, which adapts this tetrameric protein to its unique biologic roles in the reciprocal transport of O2 and CO2 between the lungs and peripheral tissues.

 Hemoglobin Is Tetrameric

Hemoglobins are tetramers composed of pairs of two similar, but distinct, polypeptide subunits (Figure 1). Greek letters are used to designate each subunit type. The subunit composition of the principal hemoglobins are α2 β2 (HbA; normal adult hemoglobin), α2 γ2 (HbF; fetal hemoglobin), α2 β^S₂ (HbS; sickle cell hemoglobin), and α2 δ2 (HbA2 ; a minor adult hemoglobin). The primary structures of the β, γ, and δ chains of human hemoglobin are highly conserved.

Fig1. Hemoglobin.Shown is the three-dimensional structure of deoxyhemoglobin with a molecule of 2,3-bisphosphoglycerate (dark blue) bound. The two α subunits are colored in the darker shades of green and blue, the two β subunits in the lighter shades of green and blue, and the heme prosthetic groups in red. (Adapted with permission from Protein Data Bank ID no. 1b86.)

Myoglobin & the β Subunits of Hemoglobin Share Almost Identical Secondary & Tertiary Structures

Despite differences in the kind and number of amino acids present, myoglobin and the β polypeptide of hemoglobin A share almost identical secondary and tertiary structures. Similarities include the location of the heme and the helical regions, and the presence of amino acids with similar properties at comparable locations. Although it possesses seven rather than eight helical regions, the α polypeptide of hemoglobin also closely resembles myoglobin.

Oxygenation of Hemoglobin Triggers Conformational Changes in the Apoprotein

Hemoglobins can bind up to four molecules of O2 per tetramer, one per heme. However, once the first molecule of O2 becomes bound, the affinity of the other three subunits increases . Termed cooperative binding, this behavior permits hemoglobin to maximize both the quantity of O2 loaded at the Po2 of the lungs and the quantity of O2 released at the Po2 of the peripheral tissues.

P₅₀ Expresses the Relative Affinities of Different Hemoglobins for Oxygen

The quantity P₅₀ , a measure of O₂ concentration, is the partial pressure of O₂ at which a given hemoglobin reaches half saturation. Depending on the organism, P₅₀ can vary widely, but in all instances, it exceeds the normal Po2 of the peripheral tissues. For example, the values of P₅₀ for HbA and HbF are 26 and 20 mm Hg, respectively. In the placenta, this difference enables HbF to extract oxygen from the HbA in the mother’s blood. However, HbF is suboptimal postpartum since its higher affinity for O2 limits the quantity of O2 delivered to the tissues.

The subunit composition of hemoglobin tetramers under goes complex changes during development. The human fetus initially synthesizes a ξ₂ ε₂ tetramer. By the end of the first trimester, ξ and ε subunits have been replaced by α and γ sub units, forming HbF (α₂ γ₂ ), the hemoglobin of late fetal life. While synthesis of β subunits begins in the third trimester, the replacement of γ subunits by β subunits to yield adult HbA (α₂ β₂ ) does not reach completion until several weeks postpartum (Figure2).

Fig2. Developmental pattern of the quaternary structure of fetal and newborn hemoglobins.(Reproduced with permission from Ganong WF:Review of Medical Physiology, 20th ed. New York, NY: McGraw Hill; 2001.)

Oxygenation of Hemoglobin Is Accompanied by Large Conformational Changes

 The binding of the first molecule of O2 to deoxyHb shifts the heme iron toward the plane of the heme ring (Figure 3). This motion is transmitted through the proximal (F8) histidine and the residues attached thereto to the entire tetramer, triggering the rupture of salt bridges formed by the carboxyl terminal residues of all four subunits. As a result, one pair of α/β subunits rotates 15° with respect to the other, compacting the tetramer (Figure 4) and triggering other, profound changes in secondary, tertiary, and quaternary structures that accompany the transition of hemoglobin from the low-affinity T state to the high-affinity R state. These changes significantly increase the affinity of the remaining unoxygenated hemes for O2 , as subsequent binding events require the rupture of fewer salt bridges (Figure 5). The cooperativity of hemoglobin constitutes a form of allosteric (Gk allos“other,” steros “space”) behavior , since binding of O2 to one heme affects the binding affinity of the others.

Fig3. On oxygenation of hemoglobin the iron atom moves into the plane of the heme. Histidine F8 and its associated aminoacyl residues are pulled along with the iron atom. For a representation of this motion, see https://pdb101.rcsb.org/learn/videos/ oxygen-binding-in-hemoglobin.

Fig4. During transition of the T form to the R form of hemoglobin, the α2 β2 pair of subunits (green) rotates through 15° relative to the pair of α1 β1 subunits (yellow).The axis of rotation is eccentric, and the α2 β2 pair also shifts toward the axis somewhat. In the representation, the tan α1 β1 pair is shown fixed while the green α2 β2 pair of subunits both shifts and rotates.

Fig5. Transition from the T structure to the R structure. In this model, salt bridges (red lines) linking the subunits in the T structure break progressively as oxygen is added, and even those salt bridges that have not yet ruptured are progressively weakened (wavy red lines). The transition from T to R does not take place after a fixed number of oxygen molecules have been bound but becomes more probable as each successive oxygen binds. The transition between the two structures is influenced by protons, carbon dioxide, chloride, and 2,3-bisphosphoglycerate (BPG); the higher their concentration, the more oxygen must be bound to trigger the transition. Fully oxygenated molecules in the T structure and fully deoxygenated molecules in the R structure are not shown because they are unstable. (Modified with permission from Perutz MF. Hemoglobin structure and respiratory transport. Sci Am. 1978;239(6):92-125.)

Hemoglobin Assists in the Transport of CO2 to the Lungs

For every molecule of O₂ consumed during the course of respiration, one molecule of CO₂ is produced. Consequently, red blood cells must be able to efficiently absorb CO₂ from respiring tissues and subsequently release it for disposal in the lungs. Two key contributors to this process are the enzyme carbonic anhydrase, which catalyzes the hydration of CO₂ to water soluble carbonic acid, H₂CO₃ , and the ability of hemoglobin to cooperatively transition between the T and R states.

Carbonic Anhydrase Converts CO₂ to Water-Soluble Carbonic Acid & Bicarbonate

 As is the case for O₂ , the limited water solubility of CO₂ at neutral pH renders simply dissolving this gas in the bloodstream grossly insufficient to meet the body’s transport needs. The enzyme carbonic anhydrase, present in large quantities in red blood cells, catalyzes the hydration of CO₂ molecules to form water soluble carbonic acid, H₂CO₃ .

H₂CO₃ is a weak acid that, in turn, can dissociate to form bicarbonate ion, HCO₃ ⁻, and a proton. Since the pK_a1 of H₂CO₃ , 6.35, falls below physiologic pH, in red blood cells the majority of absorbed CO₂ is carried as water-soluble bicarbonate.

Binding of Protons to T-State Hemoglobin Increases CO₂ Uptake From Respiring Tissues

When hemoglobin transitions from the R to the T state, con formational changes take place in each of the two β–chains that lead to the formation of salt bridges between the side chains of Asp 94 and His 146. Formation of each salt bridge requires T-state hemoglobin to bind a proton from the sur rounding environment. As R-state hemoglobin gives up its bound O₂ to respiring tissues and subsequently transitions to the T state, the absorption of these two protons both buffers the pH of the acidifying red blood cell and shifts or pulls the equilibrium between H₂CO₃ and HCO₃ ⁻ in favor of bicarbonate, further increasing the quantity of carbon dioxide absorbed by the red blood cells from peripheral tissues.

 T-state hemoglobin binds two protons per tetramer. In actively respiring tissues, the proton concentration inside the red blood cells will increase as H₂CO₃ accumulates. The greater availability of H⁺, in turn, favors the formation of T-state hemoglobin, thereby enhancing the release of O₂ .

Proton binding by T-state hemoglobin enables the high levels of CO₂ in actively respiring tissues to drive the release of O₂ from hemoglobin while concomitantly enhancing the quantity of CO₂ absorbed by promoting the conversion of carbonic acid to bicarbonate. As a result, transition to the T state helps buffer or moderate the CO₂-mediated decrease in the pH of the red blood cells in venous blood.

Proton Release From R-state Hemoglobin Enhances CO₂ Release in the Lungs

On reaching the lungs, the dramatic increase in the partial pressure of oxygen drives the binding of O₂ to deoxyhemoglobin. O₂ binding, in turn, triggers the transition of hemoglobin from the T to the R state. The resulting conformational change causes the rupture of salt bridges in the two β–chains and the release of the two protons formerly chelated between Asp 94 and His 146. Once freed, the protons combine with bicarbonate to increase the concentration of carbonic acid, which in turns favors the carbonic anhydrase catalyzed dehydration of H₂CO₃ to form CO₂ , which can then be disposed by exhalation (Figure 6). This proton-mediated coupling of the transition of hemoglobin between T and R states with the equilibria between CO₂ , H₂CO₃ , and HCO₃ ⁻ is known as the Bohr effect.

Fig6. The Bohr effect.Carbon dioxide generated in peripheral tissues combines with water to form carbonic acid, which dissociates into protons and bicarbonate ions. Deoxyhemoglobin acts as a buffer by binding protons and delivering them to the lungs. In the lungs, the uptake of oxygen by hemoglobin releases protons that combine with bicarbonate ion, forming carbonic acid, which when dehydrated by carbonic anhydrase becomes carbon dioxide, which then is exhaled.

Additional CO₂ Is Transported as Carbamates of Hemoglobin

 About 15% of the CO₂ in venous blood is carried by hemoglobin as carbamates formed with the amino terminal nitrogens of its polypeptide chains:

Carbamate formation changes the charge on amino terminals from positive to negative, favoring salt bridge formation between α and β chains. As part of the Bohr effect, proton binding by T-state hemoglobin favors carbamate formation while proton release on transition to the R state favors carbamate breakdown and release of CO₂ .

2,3-BPG Stabilizes the T Structure of Hemoglobin

 The transition of hemoglobin to the T state opens a central cavity at the interface of its four subunits capable of binding one molecule of 2,3-bisphosphoglycerate, 2,3-BPG (see Figure 1). Binding of 2,3-BPG thus favors the shift from the R to the T state and the further release of bound O₂ , as conversion back to the R state requires the rupture of the additional salt bridges formed between 2,3-BPG and Lys EF6, His H21, and the terminal amino groups of Val NA1 from both β chains (Figure 7).

Fig7. Mode of binding of 2,3-bisphosphoglycerate (BPG) to human deoxyhemoglobin.BPG interacts with three positively charged groups on each β chain. (Adapted with permission from Arnone A. X-ray diffraction study of binding of 2,3-diphosphoglycerate to human deoxyhaemoglobin. Nature. 1972;237(5351):146-149.)

 2,3-BPG is synthesized from the glycolytic intermediate 1,3-BPG, a reaction catalyzed by the bifunctional enzyme 2,3-bisphosphogylcerate synthase/2-phosphatase (BPGM). BPG is hydrolyzed to 3-phosphoglycerate by the 2-phosphatase activity of BPGM and to 2-phosphoglycerate by a second enzyme, multiple inositol polyphosphate phosphatase (MIPP). The activities of these enzymes, and hence the level of BPG in erythrocytes, are sensitive to pH. The CO₂-induced acidification of the red blood cells triggers production of 2,3-BPG, which in turn reinforces the impact of carbonic acid–derived protons in shifting the R-T equilibrium in favor of the T state, increasing the quantity of O₂ released in peripheral tissues.

In the fetal hemoglobin, residue H21 of the γ subunit is Ser rather than His. Since Ser cannot form a salt bridge, BPG binds more weakly to HbF than to HbA. The lower stabilization afforded to the T state by BPG helps account for HbF having a higher affinity for O₂ than HbA.

 Adaptation to High Altitude

 Physiologic changes that accompany prolonged exposure to high altitude include increases in the number of erythrocytes, the concentration of hemoglobin within them, and the synthesis of 2,3-BPG. Elevated 2,3-BPG lowers the affinity of HbA for O₂ (increases P₅₀ ), which enhances the release of O₂ at peripheral tissues.

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