1. The Functional Diversity of Proteins: The Example of Hemoglobin

2. Two Oxygen-binding Proteins
Myoglobin Hemoglobin
Skeletal muscle Red blood cells (erythrocytes)
Oxygen storage Oxygen transport (lung to tissues)
Monomer Four subunits (two each of two different kinds)
Prosthetic group*: Prosthetic groups:
1 Heme-Fe Heme-Fe+2 (1 per subunit)
Binds oxygen Bind oxygen
* a nonpolypeptide moiety that forms a functional part of a protein

3. Myoglobin
153 amino acids

4. Myoglobin
153 amino acids

5. Naming Helices and Corners
“proximal”
His F8

6. Myoglobin Hemoglobin β-chain
Sequences: 26% identical 59% similar

7. Hemoglobin has:
2 α-chains (α1 and α2)
2 β-chains (β1 and β2)
α and β chains are 45% identical

8. α1 β2 β1 α2

9. α1 β2 β1 α2

10. α1 β2 β1 α2

11. Introduction to Hemoglobinopathies:
(Hereditary Disorders of Hemoglobin)
Structural variants
--altered amino acid sequence
>800 known
Thalassemias
--decreased abundance of one or more of the globin chains
Most common single-gene diseases

12. Sickle Cell Disease Red blood cells Oxygenated Deoxygenated
Normal: squeeze through blood capillaries single file
Sickle cells: block blood flow (local ischemia) (painful sickling crisis)
Sickle cells have weak membranes and break (hemolysis, anemia)

14. β-chain Glu 6: mutated to Val (Glu6Val) Called hemoglobin S (HbS)

15. Binding site for β-chain Val6
(normal hydrophobic patch; Ala, Phe, Leu)

17. Hemoglobin Polymerizes
and Precipitates

18. (heterozygous) “sickle cell trait” 2 bad β-chain genes
1 good β-chain gene
(heterozygous) “sickle cell trait”
2 bad β-chain genes
(homozygous) “sickle cell disease”
Prevalence of sickle cell trait (protects against malaria)
African-Americans: 8% trait; 1/600 disease

19. Heme Porphyrin + Fe+2 Planar.
One edge hydrophobic; other hydrophilic (proprionic acid groups, red).
Fe2+: ferrous. Two H2O molecules bind to Fe2+ above and below the plane.
(Fe3+: ferric. Oxidized, cannot carry O2; “methemoglobin”)

20. Heme Binding Site (Hydrophobic)

21. Heme Binding Site (Hydrophobic)

22. Heme Binding Site (Hydrophobic)
Heme carboxyl groups on the surface

23. Heme Binding Site (Hydrophobic)
β-chain Phe42 mutated to Ser
Hb Hammersmith:
Heme slips out of pocket
Hb unstable, precipitates

24. Oxygen Binding to Heme

25. Heme Stick Model

26. Heme from the Edge

27. Heme from the Edge

28. Heme from the Edge
F8 His
“proximal histidine”

29. Heme from the Edge
F8 His
“proximal histidine”

30. Heme from the Edge
F8 His
“proximal histidine”
E7 His
“distal histidine”

31. Heme from the Edge
Oxygen
F8 His
“proximal histidine”
E7 His
“distal histidine”

32. Binding of oxygen to hemoglobin results in
conformational changes in the protein structure
(“allosteric effect”)
Deoxyhemoglobin: T-conformation
(tissues) (“tense” or “taut”)
Oxyhemoglobin: R-conformation
(lung) (“relaxed”)
Oxygen affinity of R is 150 to 300-fold greater than that of T.

33. Positive Cooperativity Between Subunits

34. Mechanism of Cooperativity
Red: deoxyhemoglobin (T)
Black: oxyhemoglobin (R)

35. Effect on Adjacent Subunits

36. β –chain: Asp99Asn (FG corner) Hb Kempsey
A hemoglobinopathy caused by mutation of a residue at the α1 – β2 interface
β –chain: Asp99Asn (FG corner)
Hb Kempsey
Locks Hb in high oxygen affinity structure (R-conformation)

37. R-State (red)
T-State (blue)

38. Definitions:
Oxygen saturation: per cent (or fraction) of myoglobin or hemoglobin that has O2 bound.
pO2: the concentration of oxygen given as “partial pressure” in units of torr.
P50: the concentration of oxygen (pO2) when 50% of myoglobin or hemoglobin has oxygen bound.

39. Lung capillary pO2 = 90

40. More on Cooperativity
Cooperativity can be measured by the fold-change in O2 needed to go from 10% saturation to 90%.
For myoglobin: ≈ 0.11 to 9 torr (81-fold) (no cooperativity)
For hemoglobin: ≈ 11 to 52 torr (4.8-fold) (positive cooperativity)
(The smaller the range of fold-change, the more cooperativity)
Hill coefficient: = no cooperativity
>1 = positive cooperativity
<1 = negative cooperativity
The hemoglobin Hill coefficient is 2.8

41. Beyond Cooperativity:
Role of pH and BPG pH
Hb O Hb(O2) nH+
T R
[H+] ( pH)
tissues
lactic acid
carbonic acid (CO2)

42. BPG binds to the β1β2 interface of deoxy Hb (T) but not oxy Hb (R).
2,3-bisphosphoglycerate
-- made in RBC’s
-- 5 negative charges
hypoxia (anemia; high altitudes; lung diseases)
BPG binds to the β1β2 interface of deoxy Hb (T) but not oxy Hb (R).
BPG stabilizes the T conformation; forces equilibrium from R to T.

43. Enhance delivery of oxygen to tissues
Summary
[H+]
[BPG]
Hb Hb
(T) (R)
Enhance delivery of oxygen to tissues

44. Different Globin Genes
Hb F: high affinity for oxygen; enhances transfer from mother to fetus.

45. Thalassemias Imbalance of α- and β-globin subunits:
Reduced synthesis or stability of one of them.
Pathology: the excess subunit precipitates,
RBC’s destroyed (anemia).
Carrier (heterozygote) is resistant to malaria.
α-Thalassemia: mostly α-globin gene deletion.
Hemoglobin has 4 β-subunits, which can’t release O2.
β-Thalassemia: mostly single base pair mutations, leading to decreased or defective β-chain mRNA.