BIOCHEMISTRY - L. Stryer - 1984

VOLUME 1

PART I. CONFORMATION AND DYNAMICS

CHAPTER 4. HEMOGLOBIN: AN ALLOSTERIC PROTEIN

4.10. Salt Bridges Between Individual Chains Rigidify the Deoxyhemoglobin Structure

In oxyhemoglobin, the C-terminal residues in all four chains possess almost complete rotational freedom. In deoxyhemoglobin, by contrast, these terminal groups are anchored (Fig. 4.14). This occurs because, first, the carboxyl terminus of the α-chain interacts with the amino terminus of the α₂-chain. Second, the Arginine side chain at this C-terminus forms a bond with the aspartate of the α₂-chain. Third, the carboxyl terminus of the β₁-chain is linked to a Lysine side chain belonging to the α₂-chain. Finally, the imidazole side chain of the C-terminal residue of the β₁-chain interacts with an aspartate side chain also belonging to the β₁-chain. These pairs of amino acid residues form so-called salt bridges, i.e., noncovalent electrostatic bonds between oppositely charged groups. Due to The formation of these eight salt bridges, deoxyhemoglobin possesses a much more rigid, strained Structure than oxyhemoglobin.

Class="center">Fig. 4.14. Cross-linking between subunits in deoxyhemoglobin. Upon oxygenation, these noncovalent electrostatic bonds are readily disrupted

The quaternary structure of deoxyhemoglobin is designated as the T state (from tense), whereas the Quaternary Structure of oxyhemoglobin is designated as the R state (from relaxed). The designations T and R are commonly used to describe alternative quaternary structures of allosteric Proteins, with the T state always exhibiting a lower substrate affinity.

4.11. Upon Oxygenation, the Iron Atom Moves into the Porphyrin Plane

We have previously discussed the conformational changes occurring at some distance from the heme. Let us now examine what happens to the heme itself upon oxygenation. In deoxyhemoglobin, the iron atom protrudes by approximately 0.6 Å out of the heme plane due to steric repulsion arising between the proximal Histidine and the nitrogen atoms of the porphyrin ring (Fig. 4.15). Upon oxygenation, the iron atom moves into the porphyrin plane, forming a strong bond with O₂. The gain in energy resulting from the formation of this bond more than tenfold outweighs the energy cost of pulling the proximal histidine closer to the heme plane. Structural studies of A large number of synthetic iron Porphyrins have shown that in compounds where 5 coordination positions are occupied, the iron atom lies out of the heme plane, whereas if all 6 coordination positions are occupied, the iron atom resides in or very nearly in the heme plane. The localization of the iron atom also depends on its electron spin; specifically, the high-spin state favors an out-of-plane position.

Fig. 4.15. Upon oxygenation, the iron atom moves into the heme plane. The proximal histidine (F8) is pulled along with the iron atom and becomes less tilted. The iron atom does not initially lie in the heme plane due to steric repulsion between a carbon atom of the imidazole ring and a nitrogen atom of the heme

4.12. The Motion of the Iron Atom Is Transmitted to Other Subunits via the Proximal Histidine

How does the movement of the iron atom into the heme plane help trigger the transition of the quaternary structure from the T state to the R state? The key element in transmitting structural changes from the heme of one subunit to another subunit is the side chain of the proximal histidine, which is dragged along by the shifting iron atom (Fig. 4.16). The heme and proximal histidine form tight contacts with the side chains of 15 amino acid residues, and consequently, oxygenation alters the structures of the F helix and the EF and FG corners. These shifts are then transmitted to the subunit-subunit contact regions. Tyrosine HC2, which was nestled in the pocket between the F and H helices, is pushed outward, leading to the rupture of salt bridges between the chains. As a result, the equilibrium between the two quaternary structures shifts toward the R state upon oxygenation. Thus, a structural change within a single subunit (oxygenation) induces a structural change at the subunit interfaces. In this manner, oxygen binding to one heme is communicated to distant PARTS OF THE molecule.

Fig. 4.16. Conformational changes induced by the movement of the iron atom during oxygenation. The proximal histidine is attracted by the iron atom and becomes less tilted. The oxygenated structure is shown in red, the deoxygenated in blue

4.13. Mechanism of Cooperative Oxygen Binding

Why does the fourth O₂ molecule bind to Hemoglobin approximately 300 times more tightly than the first? This phenomenon can be understood through the analogy illustrated in Fig. 4.17. Deoxyhemoglobin has a rigid structure reinforced by eight salt bridges between the four subunits. Oxygen binding can occur only after the rupture of some of these bonds allows the iron atom to move into the heme plane. How many salt bridges must break for an O₂ molecule to bind? This depends on which molecule in succession is binding: the first, second, third, or fourth. Binding the first O₂ molecule requires breaking more bonds than binding subsequent ones. Because breaking salt bridges requires energy, binding the first O₂ molecule is energetically less favorable than binding the rest. Meanwhile, the binding of the second and third O₂ molecules occupies an intermediate position in terms of Energy Expenditure between the first and the fourth (Fig. 4.18). This gradual increase in oxygen affinity imparts the classically observed sigmoidal shape to the O₂ binding curve.

Fig. 4.17. Explanation of the course of hemoglobin oxygenation using postage stamps as an analogy. To tear away one stamp out of four, two perforated sides must be ruptured. To detach the second stamp, a break is needed on only one side; to detach the third, on one side as well. Afterward, the fourth stamp remains free

Fig. 4.18. Free energy of binding for successive O₂ molecules (cf. the model described in Fig. 4.17). Binding affinity (-∆G°) increases because fewer cross-links must be broken to attach the fourth O₂ molecule than the first

This allosteric mechanism can also be described at a finer structural level. Almost all deoxyhemoglobin molecules exist in the T state. As O₂ molecules bind sequentially, the probability of transitioning to the R state increases. The O₂ affinity of the T state is 300-fold lower than that of the R state because the movement of the proximal histidine into the heme plane is more constrained in the T state. Specifically, in the T state, the proximal histidine is more tilted relative to the heme plane (see Fig. 4.16), thereby increasing the steric repulsion that hinders its displacement. The more symmetrical position of the proximal histidine in the R state facilitates its approach to the heme. Furthermore, in the T state, the O₂ binding site in the β subunit is sterically blocked by valine E11 to a greater extent than in the R state.