Hemoglobin: A Paradigm for Cooperativity and Allosteric Regulation

1. Hemoglobin:A Paradigm for Cooperativity and Allosteric Regulation

2. Why do we breathe? http://www.uni.edu/schneidj/webquests/spring04/tvbroadcast/circulatorysystem.html

3. Cellular Requirement for O2

4. Oxygen Carriers Diffusion Limited solubility of O2 in Blood and Cell Water

5. Myoglobin and Hemoglobin • Myoglobin (Mb) • Increases O2 solubility in tissues (muscle) • Facilitates O2 diffusion • Stores O2 in tissues • Hemoglobin (Hb) • Transports O2 from lungs to peripheral tissues (erythrocytes)

6. Oxygen Transport

7. MyoglobinSmall Intracellular Protein in Vertebrate Muscle

8. Function(s) of Myoglobin Facilitate O2 Diffusion in Muscle O2 Storage (aquatic mammals)

9. Structure of Sperm Whale Myoglobin Figure 7-1

10. The Heme Prosthetic Group Figure 7-2

11. Properties of Heme Prosthetic Group in Myoglobin • Tightly bound • Synthesized separately from myoglobin • Fe2+ Coordination • Nitrogens of heme (4) • His (F8): proximal histidine • His (E7): distal histidine • Ligands: O2, CO, and NO

12. Ligands Small molecules that bind to proteins by non-covalent interactions (e.g. O2 to myoglobin)

13. Ligand Binding • usually transient and reversible interaction with others molecule (= ligands) such as metals, hormones • often involves “molecular breathing” of the protein, i.e. ability to undergo small conformational changes • often induces molecular rearrangements in the protein • ligand binding sites are • - highly conserved • - complementary in size, shape, and charge

14. Heme • prosthetic (permanent, non-proteinaceous) • groupof Mb and Hb • incorporated into Hb and Mb during folding • responsible for reversible O2 binding • responsible for red color of blood and muscles

15. Heme – Structure central Fe2+ 4 methyl groups 2 vinyl groups (buriedin protein) 2 propionate groups(exposed)

16. Heme – Iron Coordination • Fe2+ has 6 coordination sites • 4 with N of pyrrole rings, • 2 perpendicular to ring • Mb/Hb: 5th coordination site is occupied with proximal His • 6th coordination site: • O2 oxyhemoglobin • none deoxyhemoglobin • CO carboxyhemoglobin

17. Heme – Binding of CO vs. O2 • free heme binds C0 105 times better than O2 • kinked binding topology in Mb/Hbfavors O2 (100-fold) • TOTAL: CO binding ~ 230 fold stronger than O2 binding (Carbon monoxide poisoning)

18. Function(s) of Myoglobin Facilitate O2 Diffusion in Muscle O2 Storage (aquatic mammals)

19. Myoglobin (Mb) • primarily found in muscle (highly abundant in marine mammals such as whales) • single polypeptide (153 aa) with one bound heme • very simple oxygen binder: binds oxygen at high pO2, releases it at low pO2 • Mb + O2 MbO2 • typical globin fold

20. The Globin Fold 8 helices (A-H) and loops in between MCDB310 – Chapter 5: Protein Function

21. Myoglobin – Oxygen Binding Curve

22. Binding/Association Constant Ka Quantitatively describes the affinity of a protein P for its ligand L P + L PL the higher the binding affinity, the higher Ka

23. Dissociation Constant Kd P + L PL the higher the binding affinity, the smaller Kd Example: Ka = 106 M-1Kd = 10-6 M

24. Degree of Saturation, Fraction of binding sites that are occupied by ligand at any given ligand concentration 0    1

25. Degree of Saturation, Using  If [L] = Kd  = 0.5 Kd is the ligand concentration at which 50% of the binding sites are occupied

26. Ligand Binding Curve

27. Some Examples with KD = 1 µM Question: What fraction of the protein has ligand bound when the [L] is 1 µM or 10 µM? [L] = 1 µM: [L] = 10 µM:

28. Some Examples for Dissociation Constants

29. Myoglobin – Oxygen Binding Curve Revisited When ligand is a gas, partial pressures = concentrations

30. Myoglobin – Oxygen Binding Curve Revisited • Saturation of Mb depends on • the binding constant of Mb for O2 (KD = p50 = 2.8 torr) • the concentration of O2 (pO2) • Question: What is the fractional saturation of Mb? • pO2 = 1 torr: • pO2 = 10 torr:

31. Myoglobin – An Oxygen Storage! pO2 in lung ~ 13 kPa pO2 in tissue ~ 4 kPa 10 kPa = 76 torr

32. Hemoglobin(22)

33. Hemoglobin (Hb) • present in erythrocytes (makes blood look red, 34% of weight is Hb) • Different Hb subtypes: • Hb A (adult): two  (141 aa) and two  (146 aa) subunits that are arranged as a pair of identical  subunits (2  subunits) • Hb F (fetal): two  and two  chains

34. Hemoglobin – 3D Structure a2 b1 a1 b2

35. Each subunit has 1 heme, which binds 1 O2 O2 Heme Lehninger, Figure 7-5, 7-6

36. Hemoglobin • Erythrocytes: • 1 ml blood: 5 x 109 erythrocytes • 1 erythrocyte: 3 x 108 Hb molecules • Hb is a good marker for number of red blood cells • Homology: • 50% of AA are identical between  and  subunits • 20% of AA are identical between / and Mb

37. Function of Hemoglobin O2 binding in lungs O2 release in tissues

38. Oxygen Transport

39. Oxygen binds to hemoglobin and myoglobin differently Myoglobin Hemoglobin

40. Oxygen binding to hemoglobin pO2 = partial pressure of oxygen Θ = fraction of binding sites that are occupied

41. p50 is the pO2 where half the binding sites are occupied p50

42. Hb has evolved to transport O2 pO2 In Tissues pO2 In Lungs 38% p50

43. Hb gains cooperativity by switching between 2 states T state (Low Affinity) R state (high affinity) Lehninger Figure 7-10

44. The Concerted Model All or nothing mechanism T R Lehninger, Figure 7-14

45. The Concerted Model All or nothing mechanism T R Lehninger, Figure 7-14

46. The Sequential Model

47. Hb follows a little of both T R Lehninger, Figure 7-14

48. Movements of the Heme and the F Helix During the T —> R Transition Figure 7-8

49. Local structural changes around the Heme are communicated to the rest of Hb By Janet Iwasa, https://iwasa.hms.harvard.edu/project_pages/hemoglobin/hemoglobin.html

50. Changes in the 1–2 Interface during the T —> R Transition in Hemoglobin Figure 7-9