1. GLOBULAR HEMOPROTEINS

2. Hemeproteins are a group of specialized proteins that contain heme as a tightly bound prosthetic group.
Heme is a complex of protoporphyrin IX and ferrous iron (Fe2+) .
The iron is held in the center of the heme molecule by bonds to the four nitrogens of the porphyrin ring.
The heme Fe2+ can form two additional bonds, one on each side of the planar porphyrin ring.
In myoglobin and hemoglobin, one of these positions is coordinated to the side chain of a histidine residue of the globin molecule, whereas the other position is available to bind oxygen

3. A. Hemeprotein (cytochrome c(
B. Structure of heme

4. Myoglobin Structure and function:
Myoglobin, a hemeprotein present in heart and skeletal muscle, functions both as a reservoir for oxygen, and as an oxygen carrier that increases the rate of transport of oxygen within the muscle cell.
Myoglobin consists of a single polypeptide chain that is structurally similar to the individual subunit polypeptide chains of the hemoglobin molecule.
Myoglobin is a compact molecule, with approximately 80 % of its polypeptide chain folded into 8 stretches of α-helix. These α-helical regions, labeled A to H

5. The interior of the myoglobin molecule is composed almost entirely of nonpolar amino acids.
In contrast, polar amino acids are located almost exclusively on the surface of the molecule.
The heme group of myoglobin is located in a crevice in the molecule between helix E and helix F, which is lined with nonpolar amino acids. Notable exceptions are two histidine residues.

6. Model of myoglobin showing helices A to H.
Schematic diagram of the oxygen-binding site of myoglobin

7. One, the proximal histidine (F8), binds directly to the iron of heme.
The second, or distal histidine (E7), does not directly interact with the heme group, but helps stabilize the binding of oxygen to the ferrous iron.
The protein, or globin, portion of myoglobin prevents the oxidation of iron of heme.

8. Hemoglobin Structure and function:
Hemoglobin is found exclusively in red blood cells, where its main function is to transport oxygen from the lungs to the capillaries of the tissues.
Hemoglobin A, the major hemoglobin in adults, is composed of four polypeptide chains - two α chains and two β chains - held together by noncovalent interactions.
Each subunit has stretches of α-helical structure, and a heme-binding pocket similar to that described for myoglobin.

9. However, the tetrameric hemoglobin molecule is structurally and functionally more complex than myoglobin.
For example, hemoglobin can transport H+ and CO2 from the tissues to the lungs, and can carry four molecules of O2 from the lungs to the cells of the body.
Furthermore, the oxygen-binding properties of hemoglobin are regulated by interaction with allosteric effectors .

10. 3–6 % Normal adult human hemoglobins.
[Note: The α-chains in these hemo-globins are identical]
HbA1c could be used as a monitor for the control of the blood
glucose level during the last 2 months for diabetic patients

11. Schematic diagram showing structural changes resulting from oxygenation and deoxygenation of hemoglobin.

12. Quaternary structure of hemoglobin:
The hemoglobin tetramer can be envisioned as being composed of two identical dimers, (αβ)1 and (αβ)2, in which the numbers refer to dimers one and two.
The two polypeptide chains within each dimer are held tightly together, primarily by hydrophobic interactions
In contrast, the two dimers are able to move with respect to each other, being held together primarily by polar bonds.
The weaker interactions between these mobile dimers result in the two dimers occupying different relative positions in deoxyhemoglobin as compared with oxyhemoglobin

13. T form: The deoxy form of hemoglobin is called the “T,” or taut (tense) form.
In the T form, the two αβ dimers interact through a network of ionic bonds that constrain the movement of the polypeptide chains. The T form is the low-oxygen-affinity form of hemoglobin.
R form: The binding of oxygen to hemoglobin causes the rupture of some of the ionic bonds between the αβ dimers.
This leads to a structure called the “R,” or relaxed form, in which the polypeptide chains have more freedom of movement . The R form is the high- oxygen-affinity form of hemoglobin.

14. Binding of oxygen to myoglobin and hemoglobin
Myoglobin can bind only one molecule of oxygen (O2), because it contains only one heme group.
In contrast, hemoglobin can bind four oxygen molecules—one at each of its four heme groups.
The degree of saturation (Y) of these oxygen-binding sites on all myoglobin or hemoglobin molecules can vary between zero (all sites are empty) and 100% (all sites are full

15. Oxygen dissociation curves for myoglobin and hemoglobin

16. Oxygen dissociation curve:
A plot of Y measured at different partial pressures of oxygen (pO2) is called the oxygen dissociation curve.
The curves for myoglobin and hemoglobin show important differences.
This graph illustrates that myoglobin has a higher oxygen affinity at all pO2 values than does hemoglobin

17. Allosteric effects:
The ability of hemoglobin to reversibly bind oxygen is affected by the pO2 (through heme-heme interactions , the pH of the environment, the pCO2, the availability of 2,3-bisphosphoglycerate and CO.
These are collectively called allosteric (“other site”) effectors, because their interaction at one site on the hemoglobin molecule affects the binding of oxygen to heme groups at other locations on the molecule.
[Note: The binding of oxygen to myoglobin is not influenced by allosteric effectors.]

18. Effect of O2 binding (Heme-heme interactions):
The sigmoidal oxygen-binding curve reflects specific structural changes that are initiated at one heme group and transmitted to other heme groups in the hemoglobin tetramer.
The net effect is that the affinity of hemoglobin for the last oxygen bound is approximately 300 times greater than its affinity for the first oxygen bound.
O2 favors the R- form of Hb.
Deoxygenation favors the T- form of Hb.

19. Effect of pH and CO2 (Bohr effect):
The release of oxygen from hemoglobin is enhanced when the pH is lowered ( H+) or when the hemoglobin is in the presence of an increased partial pressure of CO2.
Both result in a decreased oxygen affinity of hemoglobin and both, stabilize the T state.
In the tissues, CO2 is converted by carbonic anhydrase (CA) to carbonic acid:
CO2 + H2O H2CO3
which spontaneously loses a proton, becoming bicarbonate
(the major blood buffer):
H2CO HCO3¯ + H+
The H+ produced by this pair of reactions   pH
H+ increases the ionic bonds on Hb T-form  O2 release to the tissues.
Lactic acid produced during muscular exercise   pH

20. Effect of 2,3-bisphosphoglycerate on oxygen affinity:
2,3- Bisphosphoglycerate (2,3-BPG) is an important regulator of the binding of oxygen to hemoglobin.
It is the most abundant organic phosphate in the red blood cell, where its concentration is approximately that of hemoglobin.
2,3-BPG is synthesized from an intermediate of the glycolysis.
Binding of 2,3-BPG to deoxyhemoglobin:
2,3-BPG decreases the oxygen affinity of hemoglobin by binding to deoxyhemoglobin but not to oxyhemoglobin.
This preferential binding stabilizes the T conformation of deoxyhemoglobin.
Response of 2,3-BPG levels to chronic hypoxia or anemia:
The concentration of 2,3-BPG in the red blood cell increases in response to chronic hypoxia or anemia.
Elevated 2,3-BPG levels lower the oxygen affinity of hemoglobin, permitting greater unloading of oxygen in the capillaries of the tissues.

21. Binding of CO:
Carbon monoxide (CO) binds tightly (but reversibly) to the hemoglobin iron, forming carboxyhemoglobin.
When carbon monoxide binds to one or more of the four heme sites, hemoglobin shifts to the relaxed conformation (R-form), causing the remaining heme sites to bind oxygen with high affinity.
As a result, the affected hemoglobin is unable to release oxygen to the tissues.
[Note: The affinity of hemoglobin for CO is 220 times greater than for oxygen.

22. Factors favoring the T-form of Hb. are:
Deoxygenation
Low pH (  H+)
CO2
Lactic acid
2,3 bisphosphoglycerate
Factors favoring the R-form of Hb. are: O2 CO

23. Hemoglobinopathies:
Hemoglobinopathies have traditionally been defined as a family of genetic disorders caused by production of a structurally abnormal hemoglobin molecule, synthesis of insufficient quantities of normal hemoglobin, or, rarely, both.
Sickle cell anemia (Hb S), hemoglobin C disease (Hb C), and the thalassemia syndromes are representative hemoglobinopathies that can have severe clinical consequences.
The first two conditions result from production of hemoglobin with an altered amino acid sequence (qualitative hemoglobinopathy), whereas the thalassemias are caused by decreased production of normal hemoglobin (quantitative hemoglobinopathy).

24. Sickle cell disease (hemoglobin S disease) (SCD)
Sickle cell disease (also called sickle cell anemia) is a genetic disorder of the blood caused by a single nucleotide alteration (a point mutation) in the β-globin gene.
Sickle cell disease is an autosomal recessive disorder.
It occurs in individuals who have inherited two mutant genes (one from each parent) that code for synthesis of the β chains of the globin molecules.
A molecule of Hb S contains two normal α-globin chains and two mutant β-globin chains (βS), in which glutamate at position six has been replaced with valine .

25. Therefore, during electrophoresis at alkaline pH, Hb S migrates more slowly toward the anode (positive electrode) than does Hb A .
Electrophoresis of hemoglobin obtained from lysed red blood cells is routinely used in the diagnosis of sickle cell trait and sickle cell disease.
HbS is slower in electrophoretic motility than HbA at pH 8.6
The lifetime of an erythrocyte in SCD is less than 20 days compared to 120 days in normal RBC, hence causing anemia.
HbS is less soluble than HbA and precipitates specially when in T-form giving the RBCs the sickle shape.