The properties of individual hemoglobins are consequences of their quaternary as well as of their secondary and tertiary structures. The quaternary structure of hemoglobin confers striking additional properties, absent from monomeric myoglobin, which adapts it to its unique biologic roles. The allosteric (Gk allos “other,” steros “space”) properties of hemoglobin provide, in addition, a model for understanding other allosteric proteins (see Chapter 17).
Hemoglobins are tetramers composed of pairs of two different polypeptide subunits (Figure 6–6). Greek letters are used to designate each subunit type. The subunit composition of the principal hemoglobins are α2β2 (HbA; normal adult hemoglobin), α2γ2 (HbF; fetal hemoglobin), α2βS2 (HbS; sickle cell hemoglobin), and α2δ2 (HbA2; a minor adult hemoglobin). The primary structures of the β, γ, and δ chains of human hemoglobin are highly conserved.
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 from Protein Data Bank ID no. 1b86.)
Myoglobin & the β Subunits of Hemoglobin Share Almost Identical Secondary and 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 bind four molecules of O2 per tetramer, one per heme. A molecule of O2 binds to a hemoglobin tetramer more readily if other O2 molecules are already bound (Figure 6–5). Termed cooperative binding, this phenomenon 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. Cooperative interactions, an exclusive property of multimeric proteins, are critically important to aerobic life.
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 will exceed the 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 undergoes complex changes during development. The human fetus initially synthesizes a ξ2ε2 tetramer. By the end of the first trimester, ξ and ε subunits have been replaced by α and γ subunits, forming HbF (α2γ2), 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 (α2β2) does not reach completion until some weeks postpartum (Figure 6–7).
Developmental pattern of the quaternary structure of fetal and newborn hemoglobins. (Reproduced, with permission, from Ganong WF: Review of Medical Physiology, 20th ed. McGraw-Hill, 2001.)
Oxygenation of Hemoglobin Is Accompanied by Large Conformational Changes
The binding of the first O2 molecule to deoxyHb shifts the heme iron toward the plane of the heme ring from a position about 0.04 nm beyond it (Figure 6–8). This motion is transmitted to the proximal (F8) histidine and to the residues attached thereto, which in turn causes the rupture of salt bridges between 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 6–9). Profound changes in secondary, tertiary, and quaternary structures accompany the O2-induced transition of hemoglobin from the low-affinity T (taut) state to the high-affinity R (relaxed) 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 6–10). The terms T and R also are used to refer to the low-affinity and high-affinity conformations of allosteric enzymes, respectively.
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 http://www.rcsb.org/pdb/101/motm.do?momID=41.
During transition of the T form to the R form of hemoglobin, the α2β2 pair of subunits (green) rotates through 15ç relative to the pair of α1β1 subunits (yellow). The axis of rotation is eccentric, and the α2β2 pair also shifts toward the axis somewhat. In the representation, the tan α1β1 pair is shown fixed while the green α2β2 pair of subunits both shifts and rotates.
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 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 and redrawn, with permission, from Perutz MF: Hemoglobin structure and respiratory transport. Sci Am [Dec] 1978;239:92.)
After Releasing O2 at the Tissues, Hemoglobin Transports CO2 & Protons to the Lungs
In addition to transporting O2 from the lungs to peripheral tissues, hemoglobin transports CO2, the byproduct of respiration, and protons from peripheral tissues to the lungs. Hemoglobin carries CO2 as carbamates formed with the amino terminal nitrogens of the polypeptide chains:
Carbamate formation changes the charge on amino terminals from positive to negative, favoring salt bridge formation between α and β chains.
Hemoglobin carbamates account for about 15% of the CO2 in venous blood. Much of the remaining CO2 is carried as bicarbonate, which is formed in erythrocytes by the hydration of CO2 to carbonic acid (H2CO3), a process catalyzed by carbonic anhydrase. At the pH of venous blood, H2CO3 dissociates into bicarbonate and a proton.
Deoxyhemoglobin binds one proton for every two O2 molecules released, contributing significantly to the buffering capacity of blood. The somewhat lower pH of peripheral tissues, aided by carbamation, stabilizes the T state and thus enhances the delivery of O2. In lungs, the process reverses. As O2 binds to deoxyhemoglobin, protons are released and combine with bicarbonate to form carbonic acid. Dehydration of H2CO3, catalyzed by carbonic anhydrase, forms CO2, which is exhaled. Binding of oxygen thus drives the exhalation of CO2 (Figure 6–11). This reciprocal coupling of proton and O2 binding is termed the Bohr effect. The Bohr effect is dependent upon cooperative interactions between the hemes of the hemoglobin tetramer. By contrast, the monomeric structure of myoglobin precludes it from exhibiting the Bohr effect.
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.
Protons Arise From Rupture of Salt Bridges When O2 Binds
Protons responsible for the Bohr effect arise from rupture of salt bridges during the binding of O2 to T-state hemoglobin. In the lungs, conversion to the oxygenated R state breaks salt bridges involving β chain residue His 146. The subsequent dissociation of protons from His 146 drives the conversion of bicarbonate to carbonic acid (Figure 6–11). Upon the release of O2, the T structure and its salt bridges re-form. This conformational change increases the pKa of the β chain His 146 residues, which bind protons. By facilitating the re-formation of salt bridges, an increase in proton concentration enhances the release of O2 from oxygenated (R-state) hemoglobin. Conversely, an increase in Po2 promotes proton release.
2,3-BPG Stabilizes the T Structure of Hemoglobin
A low Po2 in peripheral tissues promotes the synthesis of 2,3-bisphosphoglycerate (BPG) in erythrocytes. The hemoglobin tetramer binds one molecule of BPG in the central cavity formed by its four subunits (Figure 6–6). However, the space between the H helices of the β chains lining the cavity is sufficiently wide to accommodate BPG only when hemoglobin is in the T state. BPG forms salt bridges with the terminal amino groups of both β chains via Val NA1 and with Lys EF6 and His H21 (Figure 6–12). BPG therefore stabilizes deoxygenated (T-state) hemoglobin by forming additional salt bridges that must be broken prior to conversion to the R state.
Mode of binding of 2,3-bisphosphoglycerate (BPG) to human deoxyhemoglobin. BPG interacts with three positively charged groups on each β chain. (Based on Arnone A: X-ray diffraction study of binding of 2,3-diphosphoglycerate to human deoxyhemoglobin. Nature 1972;237:146. Copyright © 1972. Adapted by permission from Macmillan Publishers Ltd.)
Synthesis of BPG from the glycolytic intermediate 1,3-bisphosphoglycerate is 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. As a consequence, BPG concentration and binding are influenced by and, reinforce the impact of, the Bohr effect on O2 binding and delivery by hemoglobin.
Residue H21 of the γ subunit of HbF 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 accounts 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 BPG. Elevated BPG lowers the affinity of HbA for O2 (increases P50), which enhances the release of O2 at peripheral tissues.