1. Berg • Tymoczko • Stryer
Biochemistry
Sixth Edition
Chapter 7
Hemoglobin:
Portrait of a Protein in Action
Copyright © 2007 by W. H. Freeman and Company

5. Introduction to Heme Proteins
Biomedical importance
Structure of heme
Structure and function of Mb
Structure and function of Hb
O2 saturation curve of Mb and Hb differences
The Bohr effect
Effect of 2, 3 Bisphosphoglycerate
Hemoglobinopathies
Met Hb
HbS
 and  thalassemias

6. Biomedical Importance
Heme proteins function
O2 binding
O2 transport
Electron transport
Sickle cell disease:  subunit of Hb altered.
Cyanide and CO poisoning
2, 3 BPG (Bisphosphoglycerate) effect on Hb.

8. Heme Proteins Mb and Hb are “heme” proteins. Porphyrins are colored.
Iron porphyrin gives red color to blood.
Magnesium porphyrin gives green color to plants.
Fe2+ + protoporphyrin IX = HEME
Heme is a prosthetic group, or non-polypeptide unit necessary for the biological activity.
FerriMb-Iron is +3 oxidation state
OxyMb-Iron is +2 oxidation state and O2 is bound to 6th
DeoxyMb-Iron is +2 oxidation state

9. Heme Structure Four pyrolles linked  methene bridges
Four CH3, 2 vinyl, and 2 propionate side chains could be arranged in fifteen different ways.
Only one isomer, protoporphyrin IX, present.
Fe2+  Four nitrogen atoms
Fe2+ normally octahedrally coordinated.
6 ligands
4N of the porphyrin ring available
2 remaining
In cytochrome c  5th and 6th met and His
In Mb and Hb  5th coordinated to a His, 6th is for O2

10. Structure and Function of Myoglobin
Myoglobin, a heme protein in heart and skeletal muscles.
Myboglobin functions as
reservoir for O2.
carrier for O2.
Myoglobin is single polypeptide chain (MW 17,000), 153 amino acids
Surface is polar, interior is non-polar (characteristic pattern of globular proteins).
75% of polypeptide chain folded into eight stretches of -helical regions labeled A-H. Four segments terminated by proline.

11. Structure and Function Continued
Heme group of Mb in crevice in proteins (between E and F helices). Cavity lined with non-polar amino acids, except two His residues.
Proximal His binds Fe2+.
Distal His (E7) does not directly interact with heme group but helps stabilize ferrous form of iron.
Therefore, Mb creates special environment for heme that allows reversible binding of oxygen without simultaneous oxidation of Fe2+.
His F8 means 8th residue in F helix, it identifies it as a histidyl residue.
F8  proximal His
E7  distal His

12. More Structure and Function
CO binds 25,000 times stronger than O2 in isolated heme.
Because no distal His in isolated heme. In biological systems, distal His forces CO to bind at an angle, decreasing affinity of CO to heme (200 times). This protects us against CO poisoning.
Natural protection.

13. Electron-density Map of Myoglobin Near the Oxygen-binding Site

15. Oxygen Binding in Myoglobin

16. Preferred angles for bonding of oxygen and carbon monoxide to the iron atom of heme

19. Myoglobin

21. Mb Hb
Monomer Tetromer
Bind 1 O2 4 O2
Hyberbolic
 141 a.a.
 146
HbA (normal adult) = 22
HbF (fetal Hb) = 22
HbS (sickle cell Hb) = 2S2
HbA2 (a minor adult) = 22

22. Hb subunits closely resemble Mb in 3D.

24. Oxygen dissociation curves of Hb and Mb.

27. Benefit of Sigmoidal Curve
Why Mb unsuitable as O2 transport protein
PO2 in lung 100 mmHg
PO2 n venous blood 40 mmHg
It cannot serve as an effective vehicle for delivery of O2 from lungs to periphery
After exercise  PO2 of muscle 5 mmHg. Then Mb  releases its O2
Sigmoidal curve allows Hb to deliver more O2 to tissues in response to small S in PO2

28. Salt links between and within
subunits in deoxyhemoglobin

30. Change of quartenery structure upon oxygenation
Electron density map of oxyHb
Change of quartenery structure upon oxygenation
Model of oxyHb

31. Model of hemoglobin

32. Postage stamp analogy to sequential model

36. Switching of the contact region

37. Myoglobin and Hemoglobin

39. Bohr Effect

40. Chemical basis for the Bohr effect
Three amino acid residues form two
Salt bridges that stabilize the T state.
DeoxyHb protonation (low pH) helps this!

46. Binding of BPG to deoxyHb

49. Hb has allosteric properties
Allosterism (Allos: other, Steros: space): enzyme regulation that effector binds to one site on enzyme and increases or decreases the activity of another site.

50. Sickle Cell Anemia Genetically transmitted disease
4/1000 among blacks
Sickle cell anemia homozygous
Sickle cell trait is heterozygous
HbS: Sickle cell Hb; HbA: Normal Hb
The only difference is substitution of Val for Glu at position six of the beta chains!
Fetal DNA can be analyzed for sickle cell gene
Val  Glu results from T  A mutation
Detected by new techniques
Sickle cell gene has missing cleavage site for restriction endonuclease which produces 1.3 kb fragment instead 1.1 kb fragment on gel electrophoresis
DNA analysis performed early in pregnancy (8 weeks gestation)

51. Epidemics of sickle cell anemia in Africa

54. HbS

55. Why HbS aggregates? DeoxyHbS
New hydrophobic patch Val6 interacts Phe85 and Val88 of beta chain of neighboring molecule to initiate aggregation.
Why aggregates do not form when HbS oxygenated
In OxyHbS Phe85 and Val88 are largely buried inside Hb….

58. Ninhydrin staining is used for “fingerprinting”

59. Detect sickle cell gene by using restriction endonucleases