BIOCHEMISTRY - L. Stryer - 1984

VOLUME 1

CHAPTER I. CONFORMATION AND DYNAMICS

CHAPTER 4. HEMOGLOBIN: AN ALLOSTERIC PROTEIN

4.14. Bisphosphoglycerate Lowers Oxygen Affinity by Cross-Linking Deoxyhemoglobin

BPF specifically binds to deoxyhemoglobin at a stoichiometry of 1 BPG per Hemoglobin tetramer. This is a remarkably interesting stoichiometry. One would expect a protein with an α₂β₂ Structure to have at least two binding sites for any small molecule. The existence of only a single binding site immediately suggests that BPG binds on the Symmetry axis of the hemoglobin molecule within the central cavity, where the four subunits converge (Fig. 4.19). The BPG-binding site is formed by positively charged residues belonging to both β chains, namely the α-amino group, Lysine EF6, and Histidine H21. Evidently, these groups interact readily with the negatively charged BPG, which carries nearly 4 negative charges at physiological pH. Stereochemically, BPG is complementary to the constellation of 6 positively charged groups of the β chains facing the inner cavity of the hemoglobin molecule (Fig. 4.20). The weaker binding of BPG by fetal hemoglobin compared with hemoglobin A is explained by the fact that fetal hemoglobin has a Serine instead of a histidine at the H21 position.

Class="center">Fig. 4.19. Bisphosphoglycerate binding site in the central cavity of the deoxyhemoglobin molecule

Fig. 4.20. Mode of BPG binding to human deoxyhemoglobin. Bisphosphoglycerate interacts with three positively charged groups in each β chain

Upon oxygenation, BPG is released because the central cavity becomes too small. Specifically, the gap between the H helices of the β chains narrows. Furthermore, the distance between the α-amino groups increases from 16 to 20 Å, abolishing the contact between them and the phosphate groups of BPG.

We can now understand why BPG lowers oxygen affinity. By cross-linking the β chains, BPG stabilizes The quaternary structure of deoxyhemoglobin; in other words, BPG shifts the equilibrium toward the T state. As already mentioned, deoxyhemoglobin contains 8 salt bridges that anchor its terminal carboxyl groups. For oxygenation to occur, these salt bridges must be broken. The binding of BPG creates additional salt bridges that must also be disrupted. As a result, the oxygen affinity of hemoglobin is reduced upon BPG binding.

4.15. CO ₂ Binds to the Terminal Amino Groups of Hemoglobin, Lowering Its Oxygen Affinity

In aerobic organisms, approximately 0.8 molecule of CO₂ is produced for every molecule of O₂ consumed. Most of the CO₂ is transported by the Blood as bicarbonate, which is formed in red Blood Cells through the action of Carbonic anhydrase:

CO₂ + H₂O ⇄ HCO₃^- + H⁺.

A significant portion of the H⁺ released in this reaction binds to deoxyhemoglobin, thereby participating in the Bohr effect. In addition, CO₂ is transported by hemoglobin in the form of carbamate. This process is based on a reversible reaction between the unprotonated α-amino groups of hemoglobin and CO₂:

R—NH₂ + CO₂ ⇄ R—NHCOO^- + H⁺

The bound carbamates form salt bridges that stabilize the T state. It follows that CO₂ binding decreases the oxygen affinity of hemoglobin. Conversely, CO₂ binds more tightly to deoxyhemoglobin than to oxyhemoglobin.

4.16. Mechanism of the Bohr Effect

How does hemoglobin bind protons during its transition from the oxy to the deoxy form? Clearly, this transition must be accompanied by an increase in the affinity of specific sites for H⁺. Specifically, the pK values of individual groups must increase during the transition from the oxy to the deoxy form, since an increase in pK implies stronger H⁺ binding. Hemoglobin binds about 0.5 H⁺ per O₂ molecule released. This H⁺ uptake helps maintain a constant pH in metabolically active Tissues.

Which specific groups experience an increase in their pK values? Let us first consider potential H⁺-binding sites in hemoglobin (Figs. 4.21 and Table 4.1) and then narrow down the choices. The pK values of the carboxyl groups in glutamate and aspartate side chains are typically ~4. It is unlikely that their pK values would rise to 7–8, a condition required to participate in the Bohr effect. Similarly, it is unlikely that the pK values of Tyrosine, lysine, and Arginine side chains could shift to a sufficient extent, since their pK values normally exceed 10. Consequently, the Bohr effect must be mediated by histidine, Cysteine, and the terminal amino group, as the normal pK values of these groups lie around 7.

Fig. 4.21. Typical pK values of acidic groups in Proteins. The microenvironment of an individual group can shift its actual pK value upward or downward

By combining chemical and X-ray crystallographic data, researchers have been able to identify the specific groups responsible for the Bohr effect. X-ray analysis pointed to the involvement of histidine residues located at the C-terminus of each hemoglobin β chain (histidine-146β). The validity of this hypothesis was tested in the following way: hemoglobin was prepared whose β chains lacked histidine-146. This was achieved using carboxypeptidase B, a specific proteolytic enzyme that cleaves the peptide bond formed by a basic C-terminal amino acid—that is, one containing a free carboxyl group—in certain polypeptide chains. This modification of hemoglobin resulted in a twofold reduction of the Bohr effect. Hence, histidine-146 of both β chains appears to make the major contribution to the Bohr effect.

Table 4.1. pK values of ionizable groups in proteins

The Role of hemoglobin terminal amino groups in the Bohr effect was convincingly demonstrated as follows. The terminal amino groups were modified by cyanate Treatment, which yields a carbamoyl derivative no longer capable of binding H⁺. When only the terminal amino groups of the β-chains were carbamoylated, the Bohr effect was fully preserved. However, when the terminal amino groups of the α-chains were also modified, the Bohr effect was significantly reduced.

X-ray crystallography provides a detailed picture of how these groups contribute to the Bohr effect. In oxyhemoglobin, histidine-146β rotates freely, whereas in deoxyhemoglobin, this terminal residue participates in a series of interactions. Of particular importance is the interaction between the imidazole ring of this histidine and the negatively charged aspartate-94 within the same β-chain. The close proximity of this negatively charged group increases the likelihood of proton binding by the histidine (Fig. 4.22). In other words, the proximity of aspartate-94 raises the pK of histidine-146. Thus, upon the transition from oxy- to deoxyhemoglobin, histidine-146 acquires a higher affinity for H⁺ due to the local change in charge within its immediate environment.

Similarly, the microenvironment of the terminal amino groups of the α-chains changes upon deoxygenation. In oxyhemoglobin, these groups are free. In deoxyhemoglobin, the terminal amino group of one α-chain interacts with the terminal carboxyl group of the other α-chain. The proximity of the negatively charged carboxylated residue in deoxyhemoglobin enhances the affinity of this terminal amino group for H⁺. X-Ray Diffraction data indicate that a third group also participates in the Bohr effect, namely histidine-122 in the α-chain.

Fig. 4.22. Aspartate-94 increases the pK of histidine-146 in deoxyhemoglobin, but not in oxyhemoglobin. The proximity of the negative charge on aspartate-94 promotes the protonation of histidine-146 in deoxyhemoglobin.

Thus, the microenvironment of the three pairs of proton-binding groups (the terminal amino group and two histidines) differs between oxy- and deoxyhemoglobin. In deoxyhemoglobin, this local environment is more negatively charged. Consequently, as oxygen is released, these groups bind H⁺.

4.17. Communication Within the Protein Molecule

As we have seen, the binding of O₂, H⁺, CO₂, and BPG by hemoglobin is functionally interrelated. These molecules bind to spatially distinct sites, and communication between these sites is mediated by conformational changes within the protein. The existence of separate binding sites for these ligands is due to the differences in their chemical structures. Interactions between distinct binding sites are mediated by shifts in quaternary structure. In essence, all known allosteric proteins consist of two or more polypeptide chains. The contact interface between two chains is capable of amplifying and transmitting conformational shifts from one subunit to another. An allosteric protein does not possess strictly fixed properties; rather, its functional behavior depends on the presence of specific molecules in its environment. It follows that allosteric interactions are of paramount importance for cellular function. Thus, the evolutionary transition from Myoglobin to hemoglobin gave rise to a structure capable of sensing and responding to environmental signals.