1. The heme group.

2. The visible absorption spectra of oxygenated and deoxygenated hemoglobins.

3. Oxygen dissociation curves of Mb and of Hb in whole blood.

4. Hill plots for Mb and purified (“stripped”) Hb.

5. A picket-fence Fe(II)–porphyrin complex with bound O2 (prevents auto-oxidation via dimerization)

6. Effect of pH on the O2-dissociation curve of Hb: the Bohr effect.
Hb(O2)nHx + O2  Hb(O2)n+1 + xH+
x ≈ 0.6

7. DeoxyHb binds more CO2 as carbamate
CO2 + H2O  H+ + HCO3-
catalyzed by carbonic anhydrase in erythrocytes
Carbamate Formation (N-termini)
R-NH2 + CO2  R-NH-COO- + H+
DeoxyHb binds more CO2 as carbamate
than does oxyHb

8. The effect of
2,3-BPG on
Hb oxygen
affinity
Comparison of the O2-dissociation curves of “stripped” Hb and whole blood in 0.01M NaCl at pH 7.0.

9. The effects of 2,3-BPG and CO2, both separately and combined, on hemoglobin’s O2-dissociation curve compared with that of whole blood (red curve).

10. The effect of high-altitude exposure on the p50 and the BPG concentration of blood in sea level–adapted individuals.

11. The O2-dissociation curves of blood adapted to sea level (black curve) and to high altitude (red curve).

12. Structure of sperm whale myoglobin (Mb)
Contains 8
helices: A-H
Contains some
310 helices
Subunits of Hb
are similar to Mb
Structure of sperm whale myoglobin (Mb)

13. The Amino Acid Sequences of the a and b Chains of Human Hemoglobin and of Human Myoglobin

14. The Amino Acid Sequences of the a and b Chains of Human Hemoglobin and of Human Myoglobin

15. Stereo drawings of the heme complex in oxyMb.
Heme located in
a hydrophobic pocket
formed mainly by
helices E and F
Fe(II) is 0.22 Å out
of the heme plane in
oxyMb on the proximal
His side; O2 in bent
geometry
Fe(II) is 0.55 Å out
of plane in deoxyMb
Structures of oxyMb and
deoxyMb are superimposable
Stereo drawings of the heme complex in oxyMb.

16. The X-ray structure of deoxyHb as viewed down its exact 2-fold axes.
Contains two
ab protomers
Tertiary structures
of a and b subunits
are similar to each
other and to Mb
There is extensive
interactions between
unlike subunits
(a1-b1 and a2-b2); hydrophobic
in character
Contacts between like
subunits few and polar
The X-ray structure of deoxyHb as viewed down its exact 2-fold axes.

17. The X-ray structure of oxyHb as viewed down its exact 2-fold axes.
Extensive quaternary
structural changes occur
to Hb upon oxgenation
Changes occur at the
a1-b2 and a2-b1 interfaces
The X-ray structure of oxyHb as viewed down its exact 2-fold axes.

18. respect to the a2-b2 dimer; two-fold symmetry is maintained
Oxygenation rotates
the a1-b1 dimer by 15o with
respect to the a2-b2 dimer;
two-fold symmetry
is maintained
4o forms:
deoxyHb = T state (tense)
oxyHb = R state (relaxed)
The major structural differences between the quaternary conformations of (a) deoxyHb and (b) oxyHb

19. (based on X-ray analyses)
Explaining
cooperativity:
Perutz mechanism
(based on X-ray analyses)
Note out-of-plane
Fe(II) in deoxyHb;
ion moves in-plane in
oxyHb, and pulls
on the proximal
His; F helix is moved
The heme group and its environment in the unliganded a chain of human Hb.

20. Triggering mechanism for the T ® R transition in Hb (T = blue; R = pink)

21. The a1C–b2FG interface of Hb in (a) the T state and (b) the R state.
No stable intermediate
states are allowed:
a binary switch
The a1C–b2FG interface of Hb in (a) the T state and (b) the R state.

22. The hemoglobin a1b2 interface as viewed perpendicularly to Fig. 10-13.
black: deoxyHb
blue: oxyHb
The hemoglobin a1b2 interface as viewed perpendicularly to Fig

23. Salt bridges
must break in T to
R transition
Val-1 on a2:
Bohr effect
Networks of salt bridges and hydrogen bonds in deoxyHb. (a) Last two residues of the a chains.

24. His-146 on b2:
Bohr effect
Networks of salt bridges and hydrogen bonds in deoxyHb. (b) Last two residues of the b chains.

25. Free energy and saturation curves for O2 binding to hemoglobin
Relative free energies of the
T and R states vary with
fractional saturation
Overall binding curve for Hb
is a composite of the hyperbolic
binding curves for pure T and R
Free energy and saturation curves for O2 binding to hemoglobin

26. Hb with carbamoylated a subunits (N-terminal amino
Reaction of cyanate with the unprotonated (nucleophilic) forms of primary amino groups.
Hb with carbamoylated a subunits (N-terminal amino
groups) lacks 20-30% of the Bohr effect.

27. Binding of BPG to deoxyHb: selective stabilization of the T form
BPG binding pocket is lined
with positive charge
(Lys, His, N-termini):
complementary to BPG’s
negative charge
BPG preferentially
binds to deoxyHb: central
cavity is smaller in oxyHb
Binding of BPG to deoxyHb: selective stabilization of the T form

28. Abnormal Hemoglobins:
Hemoglobinopathies -
860 variant Hbs in humans
Mutations stabilizing the Fe(III) oxidation state of heme. (a) Alterations in the heme pocket of the a subunit on changing from deoxyHbA to Hb Boston.

29. Mutations stabilizing the Fe(III) oxidation state of heme
Mutations stabilizing the Fe(III) oxidation state of heme. (b) The structure of the heme pocket of the b subunit in Hb Milwaukee.

30. Sickle-Cell
Anemia: HbS
Single-site
mutation:
Valine replaces
Glu A3(6)b
Electron micrograph of deoxyHbS fibers spilling out of a ruptured erythrocyte.

31. 220-Å in diameter fibers of deoxyHbS: an electron micrograph of a negatively stained fiber

32. 220-Å in diameter fibers of deoxyHbS: a model, viewed in cross section, of the HbS fiber.

33. Structure of the deoxyHbS fiber: arrangement of the deoxyHbS molecules in the fiber.

34. Intermolecular association
Val 6 involving b2;
Val 6 of b1 - pocket
Structure of the deoxyHbS fiber: a schematic diagram indicating the intermolecular contacts in the crystal structure of deoxyHbS.

35. Molecular
Basis for
Fibril Formation
In HbS
Structure of the deoxyHbS fiber: the mutant Val 6b2 fits neatly into a hydrophobic pocket formed mainly by Phe 85 and Leu 88 of an adjacent b1 subunit.

36. Note delay, td
1/td = k(ct/cs)n: concentration dependence of the delay time
Time course of deoxyHbS gelation: the extent of gelation as monitored calorimetrically (yellow) and optically (purple).

37. concentration dependence
Implies a 30th power
concentration dependence
Time course of deoxyHbS gelation: a log–log plot showing the concentration dependence of 1/td for the gelation of deoxyHbS at 30°C.

38. Double nucleation mechanism for deoxyHbS gelation

39. Allosteric regulation:
two general models
Monod, Wyman, Changeux:
symmetry model
conformational change alters
affinity for ligand: molecular
symmetry conserved
The species and reactions permitted under the symmetry model of allosterism

40. Models of ligand binding

41. The sequential model of allosterism
Koshland, Nemethy, Filmer
Binding to T-state induces conformational changes in
unliganded subunits (intermediate affinity between T and R)

42. Sequential binding of ligand in the sequential model of allosterism
Ligand affinity varies with number of bound ligands;
intermediate conformations
Sequential binding of ligand in the sequential model of allosterism

43. The sequential and the symmetry models of allosterism can provide equally good fits to the measured O2-dissociation curve of Hb.

44. More complex
model of Hb
allosterism
Free energy penalties for binding O2 to various ligation states of Hb tetramers relative to O2-binding to noncooperative Hb ab dimers.

45. END

46. Adair Constants for Hemoglobin A at pH 7.40.