1. Myoglobin and Hemoglobin
Protein Function
Myoglobin and Hemoglobin

2. O2 Binding and Allosteric Properties of Hemoglobin
Hemoglobin binds and transports H+, O2 and CO2 in an allosteric manner
Allosteric interaction – of, relating to, undergoing, or being a change in the shape and activity of a protein (as an enzyme) that results from combination with another substance at a point other than the chemically active site
a regulatory mechanism where a small molecule(effector) binds and alters an enzymes activity

3. Protein Function Physiological roles of: Myoglobin Hemoglobin
O2 does not easily diffuse in muscle and O2 is toxic to biological systems, so living systems have developed a way around this.
Physiological roles of:
Myoglobin
Transports O2 in rapidly respiring muscle
Monomer - single unit
Store of O2 in muscle high affinity for O2
Diving animals have large concentration of myoglobin to keep O2 supplied to muscles
Hemoglobin
Found in red blood cells
Carries O2 from lungs to tissues and removes CO2 and H+ from blood to lungs
Lower affinity for O2 than myoglobin
Tetrameter - two sets of similar units (22)

4. x-ray crystallography of myoglobin
Made up of 8  helix A to H (proline near end)
very small due to the folding
hydrophobic residues oriented towards the interior of the protein
only polar AAs inside are 2 histidines
Red indicates Heme group

5. Structure of heme prosthetic group
Protoporphyrin ring w/ iron = heme
Four Pyrrole groups [A to D] linked by methane bridges
Fe+2 coordinated by prophyrin N atoms and a N from Histidine (blue)
This is known as His F8 (8th residue of the F  helix
Iron is out of plane due to His 8 bond
A molecule of O2 acts as 6th ligand

6. Structure of heme prosthetic group
Two hydrophobic sidechains on O2 binding site of heme elp hold it in place
Valine E11 and phenylalanine CD1
oxygenation changes state of Fe
Purple to red color of blood, Fe+3 – brown
Oxidation of Fe+2 destroys biological activity of myoglobin
Physical barrier of protein is to maintain oxidation state of Fe+2

7. free vs. bound heme - role of apoprotein
Apoprotein - the protein moiety of a molecule or complex
restricts heme dimers
keeps iron reduced
stabilizes transition state (O2 binding)
CO, NO and H2S binding - poison of O2 binding bind with greater affinity than O2
His E 7 decreases affinity of ligands (CO and O2 ) for Fe+2

8. Myoglobin and Hemoglobin
Hemoglobin is structurally related to myoglobin
very different primary sequence about an 18% homology in the primary sequence
2 alpha subunits and 2 beta subunits
in adults there are very small amount of alpha 2delta 2 hemoglobin
- significance of conserved amino acids between myoglobin and hemoglobin
these are the important aas which keep hemes in contact with the protein
Stabilizes helical arraignment
Interacts with heme/iron

9. There are two general structural states - the deoxy or T form and the oxy or R form.
One type of interactions shift is the polar bonds between the alpha 1 and the beta 2 subunits.

10. 3D structure of hemoglobin and myoglobin
a1- b 1 units have 35 interactions
a 1- b 2 units have 19 interaction sites
similar units have few polar contacts
the two aand two b subunitsface each other through aqueous channels

11. Binding of oxygen dramatically alters the interactions and brings about a twisting of the two halves (alpha beta pairs)
Much of the quaternary changes takes place in the salt bonds between the C terminals of all four chains

12. Y = fraction of globin bound to O2
Myoglobin O2 affinity
myoglobin -oxygen dissociation
MbO2 <-> Mb + O2
[Mb] [O ] 2
K =
[MbO ] 2
[MbO ] 2
Y =
[MbO
] + [O ] 2 2 [O ] 2 2
Y = [O
] + K 2 pO 2
Y = pO
+ P 2 50
Y = fraction of globin bound to O2
K = dissociation constant

13. Myoglobin vs. Hemoglobin O2 affinity
Note the hyperbolic vs sigmoidal nature of the two curves!
Sigmoidal curves indicate cooperativity

14. Hemoglobin -sigmoidal dissociation
Hb(O )
=Hb +n O X
= number of Hb subunits 2 n 2 n Y
Y = fraction of globin bound to O2
log = n
log pO - n
log P 2 50
1 - Y
n = Hill constant - determined graphically by the - hill plot
n is the slope at midpoint of binding of log (Y/1-Y) vs log of pO2
if n = 1 then non cooperativity
if n < 1 then negative cooperativity
if n >1 then positive cooperativity
The experimentally determined slope does not reflect the number of binding sites however. It reflects, instead, the degree of cooperativity.

15. So how does this relate the biological effect of O2 affinity of Hb?
look at pO2 of alveoli vs. metabolically active tissue
What if oxygen dissociation were hyperbolic rather than sigmoidal?

16. Alteration in oxygen binding by:
Alteration in oxygen binding by: - H+, CO2 2,3 bisphosphoglycerate (BPG)
% O 2
Sat pO
effect of pH
effect of BPG
Bohr Effect (CO2 & pH effect on O2 binding)
Simple rule of thumb-
High H+ conc and higher CO2 levels decrease O2 affinity for Hb
High O2 concentration decrease H+ and CO2 affinity for Hb
Hb acts as a H+ buffer in respiring tissue

17. Cooperative Binding of O2 to HemoglobinNo
cooperativity
pH = 7.4
pH = 7.2
% Saturation
pH = 7.6
O2 affinity of hemoglobin increases with increasing pH.
This makes sense
In the muscle, rapidly respiring, acid will be building up – lactic acid.
During those times we know that the muscles need oxygen – so the affinity of hemoglobin goes down so more O2 can be delivered to the tissues. 20 40 60 80
100
120
pO2 (torr)

18. BPG and CO2 Effects on O2 Binding
Stripped Hb
Hb + BPG
Hb + BPG + CO2
% Saturation
Hb + CO2
BPG and CO2 binding shifts the oxygen affinity to the right. Lowers the affinity so more oxygen can be delivered to the tissues.
BPG – long term adaptation
Altitude adaptation – BPG levels increase as one goes to a higher altitude for a period of time.
Bisphosphoglycerate binds to deoxyhemoglobin, therefore decreasing hemoglobin’s oxygen affinity by keeping it in the deoxy form.
Very important molecule 20 40 60 80
100
120
pO2 (torr)

19. Functional Structure of Hemoglobin - allosteric regulations
Allosteric interaction - the binding of one ligand at one site in a protein that affects the binding of other ligands at other sites in the protein. This can be affect on binding can be cooperative (pos or neg). Allosterism is typically seen when sigmoidal binding / activity curves.

20. Simple definition of the concept of cooperativity
Simple definition of the concept of cooperativity. In its simplest meaning, cooperativity is a measure of communication between binding sites. Positive cooperativity describes a relationship in which the binding to one site facilitates the binding to the second, third, ect. No cooperativity puts all sites on equal footing and indicates no site to site influence. Negative cooperativity implies binding to one site hinders binding to subsequent sites.

21. So why is this important
So why is this important. Look at the Hill plot and the plot of the of binding vs pO2 (figs 7-8 and 7-7 respectively). Think of when hemoglobin should be mostly saturated and when it would be best if it had a low saturation / affinity and thus "give up" its oxygen. Use the table below to help your thoughts.
Oxygen pressure in various fluids
Region or fluid
Inspired Air
Aveolar Air
Arterial Blood
Capilary
Insterstitual Fluid
Cell Cytosol
pO2 (Torr)
158
100 90 40 30 10

22. Look at the pO2 pressure and think of where Hb will be loaded with oxygen and where it will deliver oxygen

23. How is this cooperation actually forced on hemoglobin?
The structures in one subunit effects the others
Structural differences of bound and unbound Hb
Two structures - T and R form depending on the oxygenation of Hb
T is the deoxy form, R is oxy form
allosteric modifiers shift or stabilize one particular conformation over the other
Monomer interactions, most chain interaction occurs between  and  chains
through mostly hydrophobic residues
 and interactions are few and polar
 and  contact act as a switch

24. oxy and deoxy quaternary structures are different
change takes place between 1 - 2 and 2 - 1
amino acids between 1 and 2 help to stabilize each forms

25. Oxygen binding shifts quaternary structure at long distances
binding of O2 ligand at 6th coordinate position pulls Fe into heme
moves proximal histadine (F8) and the alpha helix it is attached to.
shift in the helix is transmitted throughout of molecule

26. The T form finds the terminals in several important H bonds and salt bridges.
In the T form the C terminus of each subunit are "locked" into position through several hydrogen and ionic bonds.
Shifts into the R state break these and allow an increased movement throughout the molecule.
Note that binding of one or more oxygen can have a dramatic affect on the other subunits that have not yet bound an O2.

27. How Do Myoglobin (Hemoglobin) Bind to Oxygen
Move down
upon O2
binding
His E7

28. How Do Myoglobin (Hemoglobin) Bind to Oxygen
Move down
upon O2
binding
His E7

29. T and R State of Hemoglobin - Review
Below are the two major conformations of hemoglobin as predicted by the models for allosteric activation.
Oxygen will bind to hemoglobin in either state; however, it has a signficantly higher affinity for hemoglobin in the R state.
In the absence of oxygen, hemoglobin is more stable in the T state, and is therefore the predominant form of deoxyhemoglobin. R stands for relaxed, while T stands for tense, since this is stabilized by a greater number of ion pairs.
Upon a conformational change from the T state to the R state, ion pairs are broken mainly between the a1b2 subunits.

30. Structural Changes when converting R to T Hb

31. Bottom Line:
CO2, BPG and H+ all help to stabilize Hb in R or T form.

32. Bohr Effect Continued
The Bohr effect is the reversible shift in Hb affinity for O2 with changes in pH.
H+ Transport (effect) - O2 binding to Hb releases H+ due to conformational changes in Hb
- deoxyform (T form) brings Asp 94 close to His 146 (fig 7-11 (b))
- the proximity of an acidic amino acid increases the pK of histidine (pKa is now above the pH) and results in H+ “binding” to deoxyHb - in other words the His becomes protonated where it normally would be ionized
increasing pH stimulates Hb to bind to O2
Bottom line - when O2 binds Hb, H+ is released from several amino acid's functional groups. When O2 is released, the amino acids become protonized and then "picks" up a H+.
So when the H+ is high (acidic conditions) the H+ is driven onto the terminal amino acids driving it into the T conformation

33. CO2 + H2O <-> HCO3- + H+
CO2 effects - In the red blood cells the picture is even more complicated. CO2 is removed by converting CO2 to bicarbonate
CO2 + H2O <-> HCO3- + H+

34. bicarbonate (HCO3-) formed by the enzyme carbonic anhydrase
H+ produced by this enzyme is removed by the Hb as described above
This allows more CO2 to be removed in the form of bicarbonate

35. CO2 binding aids in reducing O2 affinity by changing conformation by the production of more H+ (R to T change)

36. In the lungs the O2 binds Hb and forces the R conformation

37. The carbamide increases the T formation - deoxy form.
This all aids the function of Hb. In active tissues respiration, (glycolysis) results in lactic acid formation. These tissues need more O2. Without the H+ effect Hb would hold on to more of the O2. The H+ induces Hb to dump 10% more of it's O2.
CO2 reversibly binds to N term (carbamate) to remove remaining CO2
R - NH2 + CO2 <-> R - NH - COO- + H+
R is the Hb N term amide
The carbamide increases the T formation - deoxy form.
The reverse occurs in the lungs. This results in 1/2 of CO2 removal from tissues.

39. 2,3 bisphosphoglycerate (BPG) Effects
Purified Hb has a different O2 affinity than it does in blood
26 fold decrease change in affinity is due to 2,-3 diphosphoglycerate BPG

40. diphosphoglycerate BPG
present in human red blood cells at approximately 5 mmol/L.
It binds with greater affinity to deoxygenated hemoglobin
In bonding to partially deoxygenated hemoglobin it allosterically upregulates the release of the remaining oxygen molecules bound to the hemoglobin, thus enhancing the ability of RBCs to release oxygen near tissues that need it most

41. 2,3 bisphosphoglycerate (BPG)
Purified Hb has a different O2 affinity than it does in blood
26 fold decrease change in affinity is due to 2,-3 diphosphoglycerate BPG
(BPG replaced by nucleotides IHP and ATP in fish and birds)
- 1 BPG per Hb - binds in central cavity of Hb
- binds preferentially to deoxy Hb
- hydrophobic bonds with Lys and salt bridge with His
- O2 binding changes conformation and “kicks out” BPG
change in altitude increases concentration of BPG
Fetal F Hb has replaced His 143 with Ser - What might the consequences be?

42. Cooperative interactions between subunits
Both models do not fully account for the effects of allosteric effectors
sequential model (D Koshland)
binding of one O2 induces T-R conformation change
1st change is most difficult due to influence by 3 other subunits
binding of next three subunits happens sequentially, with higher affinity (easier T-R changes)
kinetics increase to the fully oxy Hb state as more O2 is bound

43. Concerted model (J Monod)
All R or all T no in between as in the Koshland model
concerted model means as more O2 binds, the R conformation is favored until all units are in the R conformation regardless of the total units bound to O2
Affinities do NOT change until conformation changes
1 O2 - all T; 2 O2 - nearly even equilibrium; 3 O2 mostly R; 4 O2 - mostly R form
energy from O2 binding causes the change in equilibrium
this model best fits O2 dissociation curve but with limits.

44. Sickle-Cell Anemia, a Molecular Disease
One of the first “molecular” diseases found - sickle cell anemia
sickle cell - blood cell is elongated , mis-shaped (sickle)
occurs at low O2 concentration
caused by hemoglobin aggregates
inflammation in capillaries and pain
red blood cells break down - anemia
between 10% of American blacks and 25% of African blacks are heterozygous for sickle cell anemia
homozygous usually do not survive into adult hood
heterozygous individuals usually have no problem except when in severe oxygen deprivation

46. Single amino acid (point mutation) HbS vs. HbA changes structure
sickle cell b chains have a valine in place of glutamate
leads to more Hb S (sickle cell) has 2 more + charges than normal hemoglobin
Glu -Val occurs on exterior of protein - does not change O2 dissociation/allosteric properties of protein

47. Deoxy HbS precipitates
oxyHb phenylalanine b85 and leucine b88 interior
phe and leu shift to exterior
create a sticky patch with valine (hydrophobic bonding)
nucleation (cluster of aggregate) occurs logarithmically
homozygous times faster than heterozygous
that means mixed genes can re-oxygenate faster than polymerization can occur

49. how can such a disease occur?
highest concentration of gene mutation occurs where there is high incidence of malaria
heterozygous individuals survive this disease better than those without
malaria causing parasite lives in red blood cells during part of its life cycle
partial sickling must interrupt life cycle of malaria parasite

50. Any Questions?