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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 10.2Hemoglobin Transports Oxygen Efficiently by Binding Oxygen Cooperatively

Allostery is a property not limited to enzymes. The basic principles of allostery are also well illustrated by the oxygen-transport protein hemoglobin (Section 7.2). The binding of oxygen to hemoglobin isolated from red blood cells displays marked sigmoidal behavior (similar to that observed for the activity of ATCase, as a function of substrate concentration), which is indicative of cooperation between subunits (Figure 10.17). What is the physiological significance of the cooperative binding of oxygen by hemoglobin? Oxygen must be transported in the blood from the lungs, where the partial pressure of oxygen (pO2) is relatively high (approximately 100 torr), to the tissues, where the partial pressure of oxygen is much lower (typically 20 torr). Let us consider how the cooperative behavior represented by the sigmoidal curve leads to efficient oxygen transport. In the lungs, hemoglobin becomes nearly saturated with oxygen such that 98% of the oxygen-binding sites are occupied. When hemoglobin moves to the tissues, the saturation level drops to 32%. Thus, a total of 98 - 32 = 66% of the potential oxygen-binding sites contribute to oxygen transport. In comparison, for a hypothetical noncooperative transport protein, the most oxygen that can be transported from a region in which pO2 is 100 torr to one in which it is 20 torr is 63 - 25 = 38% (see Figure 10.17). Thus, the cooperative binding of oxygen by hemoglobin enables it to deliver 1.7 times as much oxygen as it would if the sites were independent. The homotropic regulation of hemoglobin by its ligand oxygen dramatically increases its physiological oxygen-carrying capacity.

Torr-

A unit of pressure equal to that exerted by a column of mercury 1 mm high at 0°C and standard gravity (1 mm Hg). Named after Evangelista Torricelli (1608–1647), inventor of the mercury barometer.

Figure 10.17. Cooperativity Enhances Oxygen Delivery by Hemoglobin.

Figure 10.17

Cooperativity Enhances Oxygen Delivery by Hemoglobin. Because of cooperativity between O2-binding sites, hemoglobin delivers more O2 to tissues than would a noncooperative protein (pO2, partial pressure of oxygen.)

10.2.1. Oxygen Binding Induces Substantial Structural Changes at the Iron Sites in Hemoglobin

The results of structural studies pioneered by Max Perutz revealed the structure of hemoglobin in various forms. Human hemoglobin A, present in adults, consists of four subunits: two α subunits and two β subunits. The α and β subunits are homologous and have similar three-dimensional structures (Section 7.4). The capacity of hemoglobin to bind oxygen depends on the presence of a bound prosthetic group called heme. The heme group is responsible for the distinctive red color of blood. The heme group consists of an organic component and a central iron atom. The organic component, called protoporphyrin, is made up of four pyrrole rings linked by methene bridges to form a tetrapyrrole ring. Four methyl groups, two vinyl groups, and two propionate side chains are attached.

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The iron atom lies in the center of the protoporphyrin, bonded to the four pyrrole nitrogen atoms. Under normal conditions, the iron is in the ferrous (Fe2+) oxidation state. The iron ion can form two additional bonds, one on each side of the heme plane. These binding sites are called the fifth and sixth coordination sites. In hemoglobin, the fifth coordination site is occupied by the imidazole ring of a histidine residue from the protein. In deoxyhemoglobin, the sixth coordination site remains unoccupied. The iron ion lies approximately 0.4 Å outside the porphyrin plane because iron, in this form, is slightly too large to fit into the well-defined hole within the porphyrin ring (Figure 10.18).

Figure 10.18. Position of Iron in Deoxyhemoglobin.

Figure 10.18

Position of Iron in Deoxyhemoglobin. The iron ion lies slightly outside the plane of the porphyrin in heme.

The binding of the oxygen molecule at the sixth coordination site of the iron ion substantially rearranges the electrons within the iron so that the ion becomes effectively smaller, allowing it to move into the plane of the porphyrin (Figure 10.19). This change in electronic structure is paralleled by changes in the magnetic properties of hemoglobin, which are the basis for functional magnetic resonance imaging (fMRI; Section 32.1.3). Indeed, the structural changes that take place on oxygen binding were anticipated by Linus Pauling, based on magnetic measurements in 1936, nearly 25 years before the three-dimensional structure of hemoglobin was elucidated.

Figure 10.19. Oxygen Binding Initiates Structural Changes.

Figure 10.19

Oxygen Binding Initiates Structural Changes. The iron ion moves into the plane of the heme on oxygenation. The proximal histidine is pulled along with the iron ion.

10.2.2. Oxygen Binding Markedly Changes the Quaternary Structure of Hemoglobin

The three-dimensional structure of hemoglobin is best described as a pair of identical αβ dimers (α1β1 and α2β2) that associate to form the hemoglobin tetramer (Figure 10.20). In deoxyhemoglobin, these αβ dimers are linked by an extensive interface, which includes, among other regions, the carboxyl terminus of each chain. The heme groups are well separated in the tetramer with iron-iron distances ranging from 24 to 40 Å. The deoxy form corresponds to the T state in the context of either the concerted or the sequential model for hemoglobin cooperativity. On oxygen binding, there are substantial changes in quaternary structure that correspond to the T-to-R state transition (Figure 10.21).

Figure 10.20. Quaternary Structure of Hemoglobin.

Figure 10.20

Quaternary Structure of Hemoglobin. Image mouse.jpg Hemoglobin, which is composed of two α chains and two β chains, functions as a pair of αβ dimers.

Figure 10.21. Transition from T to R State in Hemoglobin.

Figure 10.21

Transition from T to R State in Hemoglobin. Image mouse.jpg On oxygenation, one pair of αβ subunits shifts with respect to the other by a rotation of 15 degrees.

The α1β1 and α2β2 dimers rotate approximately 15 degrees with respect to one another. The dimers themselves are relatively unchanged, although localized conformational shifts do occur. Thus, the interface between the α1β1 and α2β2 dimers is most effected by this structural transition.

How does oxygen binding lead to the structural transition from the T state to the R state? When the iron ion moves into the plane of the porphyrin, the histidine residue bound in the fifth coordination site moves with it. This histidine residue is part of an α helix, which also moves (Figure 10.22). The carboxyl terminal end of this α helix lies in the interface between the two αβ dimers. Consequently, the structural transition at the iron ion is directly transmitted to the other subunits. The rearrangement of the dimer interface provides a pathway for communication between subunits, enabling the cooperative binding of oxygen.

Figure 10.22. Conformational Changes in Hemoglobin.

Figure 10.22

Conformational Changes in Hemoglobin. The movement of the iron ion on oxygenation brings the iron-associated histidine residue toward the porphyrin ring. The corresponding movement of the histidine-containing α helix alters the interface between (more...)

Is the cooperative binding of oxygen by hemoglobin best described by the concerted or the sequential model? Neither model in its pure form fully accounts for the behavior of hemoglobin. Instead, a combined model is required. Hemoglobin behavior is concerted in that hemoglobin with three sites occupied by oxygen is in the quaternary structure associated with the R state. The remaining open binding site has an affinity for oxygen more than 20-fold as great as that of fully deoxygenated hemoglobin binding its first oxygen. However, the behavior is not fully concerted, because hemoglobin with oxygen bound to only one of four sites remains in the T-state quaternary structure. Yet, this molecule binds oxygen 3 times as strongly as does fully deoxygenated hemoglobin, an observation consistent only with a sequential model. These results highlight the fact that the concerted and sequential models represent idealized limiting cases, which real systems may approach but rarely attain.

10.2.3. Tuning the Oxygen Affinity of Hemoglobin: The Effect of 2,3-Bisphosphoglycerate

Examination of the oxygen binding of hemoglobin fully purified from red blood cells revealed that the oxygen affinity of purified hemoglobin is much greater than that for hemoglobin within red blood cells. This dramatic difference is due to the presence within these cells of 2,3-bisphosphoglycerate (2,3-BPG) (also known as 2,3-diphosphoglycerate or 2,3-DPG). This highly anionic compound is present in red blood cells at approximately the same concentration as that of hemoglobin (~ 2 mM). Without 2,3-BPG, hemoglobin would be an extremely inefficient oxygen transporter, releasing only 8% of its cargo in the tissues.

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How does 2,3-BPG affect oxygen affinity so significantly? Examination of the crystal structure of deoxyhemoglobin in the presence of 2,3-BPG reveals that a single molecule of 2,3-BPG binds in a pocket, present only in the T form, in the center of the hemoglobin tetramer (Figure 10.23). On T-to-R transition, this pocket collapses. Thus, 2,3-BPG binds preferentially to deoxyhemoglobin and stabilizes it, effectively reducing the oxygen affinity. In order for the structural transition from T to R to take place, the bonds between hemoglobin and 2,3-BPG must be broken and 2,3-BPG must be expelled.

Figure 10.23. Mode of Binding of 2,3-BPG to Human Deoxyhemoglobin.

Figure 10.23

Mode of Binding of 2,3-BPG to Human Deoxyhemoglobin. Image mouse.jpg 2,3-BPG binds in the central cavity of deoxyhemoglobin. There, it interacts with three positively charged groups on each β chain.

Image tree.jpg 2,3-BPG binding to hemoglobin has other crucial physiological consequences. The globin gene expressed by fetuses differs from that expressed by human adults; fetal hemoglobin tetramers include two α chains and two γ chains. The γ chain, a result of another gene duplication, is 72% identical in amino acid sequence with the β chain. One noteworthy change is the substitution of a serine residue for His 143 in the β chain of the 2,3-BPG-binding site. This change removes two positive charges from the 2,3-BPG-binding site (one from each chain) and reduces the affinity of 2,3-BPG for fetal hemoglobin, thereby increasing the oxygen-binding affinity of fetal hemoglobin relative to that of maternal (adult) hemoglobin (Figure 10.24). This difference in oxygen affinity allows oxygen to be effectively transferred from maternal to fetal red cells. We see again an example of where gene duplication and specialization produced a ready solution to a biological challenge—in this case, the transport of oxygen from mother to fetus.

Figure 10.24. Oxygen Affinity of Fetal Red Blood Cells.

Figure 10.24

Oxygen Affinity of Fetal Red Blood Cells. Fetal red blood cells have a higher oxygen affinity than that of maternal red blood cells because fetal hemoglobin does not bind 2,3-BPG as well as maternal hemoglobin does.

10.2.4. The Bohr Effect: Hydrogen Ions and Carbon Dioxide Promote the Release of Oxygen

Rapidly metabolizing tissues, such as contracting muscle, have a high need for oxygen and generate large amounts of hydrogen ions and carbon dioxide as well (Sections 16.1.9 and 17.1). Both of these species are heterotropic effectors of hemoglobin that enhance oxygen release. The oxygen affinity of hemoglobin decreases as pH decreases from the value of 7.4 found in the lungs (Figure 10.25). Thus, as hemoglobin moves into a region of low pH, its tendency to release oxygen increases. For example, transport from the lungs, with pH 7.4 and an oxygen partial pressure of 100 torr, to active muscle, with a pH of 7.2 and an oxygen partial pressure of 20 torr, results in a release of oxygen amounting to 77% of total carrying capacity. Recall that only 66% of the oxygen would be released in the absence of any change in pH. In addition, hemoglobin responds to carbon dioxide with a decrease in oxygen affinity, thus facilitating the release of oxygen in tissues with a high carbon dioxide concentration. In the presence of carbon dioxide at a partial pressure of 40 torr, the amount of oxygen released approaches 90% of the maximum carrying capacity. Thus, the heterotropic regulation of hemoglobin by hydrogen ions and carbon dioxide further increases the oxygen-transporting efficiency of this magnificent allosteric protein.

Figure 10.25. Effect of pH and CO2 concentration on the oxygen affinity of hemoglobin.

Figure 10.25

Effect of pH and CO2 concentration on the oxygen affinity of hemoglobin. Lowering the pH from 7.4 (red curve) to 7.2 (blue curve) results in the release of O2 from oxyhemoglobin. Raising the CO2 partial pressure from 0 to 40 torr (purple curve) also favors (more...)

The regulation of oxygen binding by hydrogen ions and carbon dioxide is called the Bohr effect after Christian Bohr, who described this phenomenon in 1904. The results of structural and chemical studies have revealed much about the chemical basis of the Bohr effect. At least two sets of chemical groups are responsible for the effect of protons: the amino termini and the side chains of histidines β146 and α122, which have pKa values near pH 7. Consider histidine β146. In deoxyhemoglobin, the terminal carboxylate group of β146 forms a salt bridge with a lysine residue in the α subunit of the other αβ dimer. This interaction locks the side chain of histidine β146 in a position where it can participate in a salt bridge with negatively charged aspartate 94 in the same chain, provided that the imidazole group of the histidine residue is protonated (Figure 10.26). At high pH, the side chain of histidine β146 is not protonated and the salt bridge does not form. As the pH drops, however, the side chain of histidine β146 becomes protonated, the salt bridge with aspartate β94 forms, and the quaternary structure characteristic of deoxyhemoglobin is stabilized, leading to a greater tendency for oxygen to be released at actively metabolizing tissues. No significant change takes place in oxyhemoglobin over the same pH range.

Figure 10.26. Chemical Basis of the Bohr Effect.

Figure 10.26

Chemical Basis of the Bohr Effect. In deoxyhemoglobin, shown here, three amino acid residues form two salt bridges that stabilize the T quaternary structure. The formation of one of the salt bridges depends on the presence of an added proton on histidine (more...)

Carbon dioxide also stabilizes deoxyhemoglobin by reacting with the terminal amino groups to form carbamate groups, which are negatively charged, in contrast with the neutral or positive charges on the free amino groups. The amino termini lie at the interface between the αβ dimers, and these negatively charged carbamate groups participate in salt-bridge interactions, characteristic of the T-state structure, which stabilize deoxyhemoglobin's structure and favor the release of oxygen.

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Hemoglobin with bound carbon dioxide and hydrogen ions is carried in the blood back to the lungs, where it releases the hydrogen ions and carbon dioxide and rebinds oxygen. Thus, hemoglobin helps to transport hydrogen ions and carbon dioxide in addition to transporting oxygen. However, transport by hemoglobin accounts for only about 14% of the total transport of these species; both hydrogen ions and carbon dioxide are also transported in the blood as bicarbonate (HCO3-) formed spontaneously or through the action of carbonic anhydrase (Section 9.2), an abundant enzyme in red blood cells.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22596

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