1. Lecture 15: Regulation of Proteins 2: Allosteric Control of Hemoglobin Hemoglobin and Myoglobin Allosteric Transition in Hemoglobin Physiological Role of Hemoglobin

2. Myoglobin and Hemoglobin Monomer Heterotetramer (  2  2 ) Myoglobin Hemoglobin Similar tertiary structure, different quaternary structure. The quaternary structure of Hemoglobin confers allosteric properties.

3. Myoglobin: Biological role: oxygen storage protein (1 binding site) Binds oxygen in muscle cells, keeping it until needed. Hemoglobin: Biological role: oxygen transport protein. (4 binding sites.) Circulates in red blood cells. Binds oxygen when in lungs, and releases it when in oxygen-requiring tissues. Affinity for oxygen is modulated so as to bind tightly in lungs, but release easily when in tissues. The modulation relies on the allosteric properties of hemoglobin.

4. Binding of Oxygen by Hemoglobin (In solution, dissolved gases such as oxygen are described in terms of partial pressure, not concentration. ) In lungs, the partial pressure of oxygen is high, ~100 torr. In tissues, the partial pressure of oxygen is lower, ~20 torr. Hemoglobin must bind oxygen tightly enough that it can fill up its sites when in the lungs, but not so tightly that it cannot release oxygen when it reaches tissues where oxygen is needed. In other words, it would be most efficient if, over the range of oxygen levels between lungs and tissues, the molecule could go from completely saturated (4 sites occupied) to completely unloaded (zero sites occupied.) Hemoglobin comes close to achieving this through cooperativity.

5. Cooperative Binding The fractional saturation is the percentage of the total binding sites occupied. Hemoglobin has a sigmoidal binding response to oxygen- oxygen binding by hemoglobin is cooperative. (the binding of oxygen at one site influences binding at other sites) Hemoglobin: 100 torr: 98% saturated 20 torr: 32% saturated 66% change possible due to cooperativity (a non-cooperative transport protein could achieve 38% at best)

6. Basis of Hemoglobin Cooperativity The ability of the oxygen-binding sites to influence one another results from a conformational change in hemoglobin. The T state has a low affinity for oxygen, while the R state has a high affinity for oxygen. The abrupt change in oxygen affinity results from a switch between the low affinity state and the high affinity state.

7. Oxygen binding sites in Hemoglobin Oxygen is bound on an iron ion on prosthetic groups called heme. There are 4 hemes in hemoglobin between 24 and 40 Angstroms apart. Each iron has six coordination sites, one of which can be occupied by oxygen. Heme = Fe2+ ion and protoporphyrin N N N N Histidine Oxygen Fe

8. In the absence of oxygen, the iron ion lies 0.4 Angstroms outside the plane of the porphyrin ring.

9. When oxygen binds, the iron ion moves into the plane of the porphyrin ring, pulling the histidine upwards and closer to the porphyrin.

10. In turn, force on the histidine causes upward displacement of a helix which leads directly to the interface region of the tetramer. The changes at the heme due to oxygen binding are “communicated” through the protein to the interface, causing the rearrangement of the quaternary structure, which in turn affects the affinity for oxygen at the other sites. (Two distinct stable quaternary arrangements)

11. Free Energy All sites bound No sites bound Fractional Occupancy 0 1 R state T state In the absence of oxygen, the T state is more stable, and so a larger percentage of the tetramers are in the T state. But when oxygen levels are high, most of the sites are bound, and the R state predominates.

12. pO 2 Sites Occupied The sigmoidal shape of the binding curve results from the switch between the T and R states. Effect of the Allosteric Switch on Binding Affinity

13. All T All R Mixed T and R subunits Sequential Model Models for Cooperativity All R All T Concerted Model Most allosteric proteins fall somewhere in between the two extremes.

14. Allosteric Effectors of Hemoglobin The affinity of hemoglobin for oxygen can be controlled by external effectors, which affect the equilibrium between the T and R states. Favoring the T state assists hemoglobin in unloading its bound oxygen. 2,3-Bis-phosphoglycerate: BPG decreases the affinity of hemoglobin for oxygen, assisting it to unload the oxygen molecules where they are needed. The Bohr effect: Carbon dioxide and hydrogen ions also decrease the affinity of hemoglobin for oxygen, so that a larger fraction of its oxygen can be delivered in the tissues.

15. BPG Binding Stabilizes the T state ( - 5 charge) Purified hemoglobin has much higher affinity for oxygen than when in red blood cells. A substance in red blood cells, 2,3- bisphosphoglycerate (BPG), binds the T state of hemoglobin, effectively decreasing the affinity for oxygen. The binding of BPG is stabilized by ionic interactions.

16. Fetal hemoglobin is less sensitive to BPG During early development, the human fetus expresses different  and  hemoglobin genes. These are similar but not identical to the hemoglobin genes expressed in adults. The fetal  chain differs in that residue 143 (a histidine in adult hemoglobin) is a serine, which is uncharged. An ionic interaction at the BPG binding site is removed, decreasing the ability of BPG to bind. As a result, fetal hemoglobin has a higher affinity for oxygen, allowing transfer from the maternal adult hemoglobin.

17. Increased BPG Contributes to Altitude Adaptation The low levels of oxygen found at high altitude make it more difficult to transport sufficient oxygen from lungs to tissues. To overcome this difficult, a complicated adaptation process takes place in which more red blood cells and more hemoglobin are produced, which requires several weeks. (Mountain climbers often have a series of camps where they pause to allow time for this process.) There is also a faster physiological response, which takes place over the course of only a few hours. Red blood cells can increase their levels of BPG, decreasing the affinity of hemoglobin for oxygen, with the result that more oxygen can be unloaded in the tissues. 1 2 3

18. The Bohr Effect Carbon dioxide and H+ ions also are effectors of hemoglobin. In active muscle cells, oxygen is rapidly consumed and carbon dioxide and H+ ions are produced. Oxygen affinity decreases at lower pH. As hemoglobin moves into a tissue with lower pH, it can more easily unload oxygen. Increase in carbon dioxide concentrations have the same effect. In combination, the effect of pH and carbon dioxide allow nearly 90% of the oxygen bound in the lungs to be unloaded in tissues where it is required.

19. The Bohr Effect can be explained in terms of ionic interactions. Structural Basis of the Bohr Effect When protonated, His 146 on the  chain is positively charged, and can participate in an ionic interaction with Asp 94, which stabilizes the T state. At low pH, the T state is favored, decreasing the oxygen affinity. The amino termini of the hemoglobin chains lie at an interface between  and  subunits. Carbon dioxide can react with the positively charged amino termini to form negatively charged carbamate groups that form ionic interactions which also stabilize the T state.

20. Summary: Hemoglobin is an oxygen transport protein that carries oxygen from the lungs, where oxygen levels are high, to tissues where it is needed. Hemoglobin exhibits cooperative binding of oxygen which is the result of a conformational switch between a low-affinity state and a high-affinity state. The affinity of hemoglobin for oxygen can be regulated by allosteric effectors to improve its physiological performance. Key Concepts: Function of hemoglobin Conformational changes due to oxygen binding Cooperativity Role of BPG Role of Bohr Effect