Chemistry and function of Hemoglobin

Dioxygen-carrier protein, Heme group, Protoporphyrin IX, globin, Tetrameric structure, T and R states, Functions, Deoxy-hemoglobin and oxy-hemoglobin, Oxygen Dissociation Curve, Cooperativity in oxygen binding, steric conformational changes, Spin states of iron, Pauling model, Weiss model, Enzymes involved, Allosteric regulation, Bohr effect, Effect of BPG, CO poisoning, CN poisoning, Anemia, Hemoglobinopathy

1. Chemistry and Functions of Hemoglobin Dr . Santarupa Thakurta Assistant Professor in Chemistry Prabhu Jagatbandhu College West Bengal, India Course : Undergraduate Chemistry Honours Topic : Bio-inorganic Chemistry

2. Arterial blood (red, oxygen-rich blood) flows from the heart to each part of the body to provide oxygen and nutrients. The venous blood (blue, oxygen-poor blood) returns from the body to the heart. The blood then travels through the lungs to exchange carbon dioxide for new oxygen. The heart is a pump which moves the blood. The arteries and veins are the pipes through which the blood flows.  The lungs provide a place to exchange carbon dioxide for oxygen . Circulatory system

3. Dioxygen -carrier proteins Most organisms require molecular oxygen for their survival. Simple diffusion does not supply sufficient dioxygen for respiration. Specialized molecules for the transport and storage oxygen are necessary for higher organisms. These functions are carried out by dioxygen -carrier protein. The dioxygen -binding site in the protein i.e., " active site," is a complex either of copper or of iron. A good oxygen carrier should bind O 2 at high pressure, should not oxidize cellular components and give up oxygen on demand. Heme proteins are involved in oxygen transport and storage.

4. Hemoglobin is the iron-containing protein molecule in red blood cells of verrebrates that carries oxygen from the lungs to the body's tissues and returns carbon dioxide from the tissues back to the lungs. Myoglobin is an iron- and oxygen-binding protein found in the muscle tissue of vertebrates in general and in almost all mammals. Hemocyanin are proteins that transport oxygen throughout the bodies of some invertebrate animals. These metalloproteins contain two copper atoms that reversibly bind a single oxygen molecule. They are second only to hemoglobin in frequency of use as an oxygen transport molecule. Hemerythrin is used to transport O 2 in a variety of marine invertebrates. Deoxyhemerythrin contains two Fe 2+ ions per subunit and is colorless , whereas oxyhemerythrin contains two Fe 3+ ions and is bright reddish violet.

5. Importance of hemoglobin O 2 is a nonpolar molecule, and therefore does not dissolve well in the aqueous environment of the blood . Only about 1.5% of the oxygen transported by the blood is dissolved directly in the blood plasma. In fact, most of the oxygen is carried via a more sophisticated mechanism that utilizes the metal complex heme. This mechanism is capable of transporting the large amount of oxygen required by the body and allowing it to leave the blood when it reaches the tissues that demand the most oxygen. Hemoglobin increases O 2 solubility in blood by about a hundredfold. This means that without hemoglobin , in order to provide sufficient oxygen to the tissues, blood would have to make a complete circuit through the body in less than a second, instead of the minute that it actually takes . That would take a mighty powerful heart!

6. What Is Hemoglobin? Hemoglobin(Hb) is a major Hemoprotein of Human body ( Hemo - comes from the Greek haîma , meaning “blood) Hemoglobin Chemically is: Conjugated Protein In Hemoglobin Heme is a Prosthetic group Globin is a Protein part (Hemoglobin = Heme + Globin)

7. Hemoglobin(Hb) is Red color pigment Location Of Hemoglobin- Inside Red blood cells/Erythrocytes of blood . Hemoglobin In RBCs Occupies: 33% of the RBC volume (1/3) 90-95% of the dry weight of RBC is by Hb . Normal concentration of Hemoglobin in the Human Blood: Adult Males- 13.5–17.5 gm / dL Adult Females- 12.5–16.5 gm / dL

8. Terminologies of Hemoglobin Hemoprotein – Heme is a prosthetic group Chromoprotein - Red in color Metalloprotein - Metal Iron (Fe) present Respiratory Protein - Connected to Respiration process and Respiratory Chain ( Electron Transport Chain) Oxygen Binding Protein - Binds with molecular Oxygen and transports it . Allosteric protein - Binding of oxygen to one of the subunits is affected by its interactions with the other subunits.

9. Iron containing pigment heme is attached to protein globin Heme consists of a porphyrin ring chelated to an iron atom. Heme is iron porphyrin complex called Iron protoporphyrin IX Globin is a protein One of the first proteins to be studied by X-ray crystallography, and earned Max Perutz the Nobel Prize in Chemistry in 1962. Structure of Hemoglobin

10. Structure o f p orphyrin One of the most important classes of chelating agents in nature is the   porphyrins . The porphyrin ring consists of four pyrrole rings linked by methene bridges . The rigidity of the system derives from the delocalization of  electrons of the conjugated macrocyclic system. A porphyrin molecule can coordinate to a metal using its four nitrogen atoms as electron-pair donors, thus prophyrins are tetradentate ligands.  The porphyrin ring can donate two protons and become -2 dianion . So it can coordinate to dipositive metal ions, to form metalloporphyrin complexes. The size of the cavity in the center of the porphyrin ring is ideal for accommodating metal ions of 1 st transition series Various substituents are attached to the perimeter

11. Protoporphyrin IX The porphyrin in heme, with its particular arrangement of four methyl, two propionate, and two vinyl substituents, is known as protoporphyrin IX.

12. The Roman numeral "IX" indicates that these chains occur in the circular order MV-MV-MP-PM around the outer cycle. Methyl (-CH 3 ) Vinyl (-CH=CH 2 ) Propionic acid (-CH 2 -CH 2 -COOH) Protoporphyrin is a cyclic tetrapyrrole that consists of porphyrin bearing four methyl (M) substituents at positions 3, 8, 13 and 17, two vinyl substituents (V) at positions 7 and 12 and two 2-carboxyethyl/propionic acid (P) substituents at positions 2 and 18. 

13. Heme group Important role in binding of dioxygen In vertebrates, two heme -iron containing proteins are hemoglobin and myoglobin. Hemoglobin picks up O 2 from lungs and transports it to tissues where it is stored by myoglobin. These proteins are non-redox proteins, i.e. there is no change in the oxidation state of metal during the transport or storage of O 2 . Myoglobin has a molecular weight of 17000 and is a monomer Hemoglobin has a molecular weight of 64500 and is a tetramer.

14. Distal and Proximal regions In common terminology, the side of the heme plane at which exchangeable ligands are bound is known as the distal region , while that below the heme plane is known as the proximal region . The histidine on helix F is one of two that are present in all species. Such ‘highly conserved’ amino acids are a strong indication that evolution has determined that they are essential for function. The other conserved histidine is located on helix E . proximal (near) histidine : His F8 - the eighth residue of the helix F distal ( distant) histidine : His E7 - the seventh residue of helix E

15. Iron in Heme Iron remains in Ferrous form (Fe ++ ) Reduced state whether or not the heme is oxygenated The heme consists of a ferrous ion held in the center of a porphyrin and coordinated by the four nitrogen atoms of the porphyrin ring. The Fe is also covalently anchored to Hb at the heme proximal pocket by an N atom of imidazole of a histidine residue located on the F helix (His F8). The Fe(II) centre is in a square-based pyramidal environment when in its ‘rest state’, also referred to as the deoxy -form. This setup allows the Fe to bind O 2 or other gasses at the distal pocket of the heme by a covalent bond to fulfill the octahedral coordination of six ligands.

16. Oxygen binding to Heme group Fe ++ of Heme is linked to Proximal Histidine (F8) When O 2 binds to the heme group, it enters trans to the His F8 residue End-on bent (angular) coordination of O 2 to heme with an Fe–O–O angle of about 130° The imidazole of a histidine residue at the distal pocket (His E7) stabilizes the bound O 2 through hydrogen-bond interaction. Thus to attain stability, oxygen is bound to both Heme and Globin .

17. Crystal structures

18. Structure of Globin Adult Hemoglobin Hb has 4 p olypeptide chains held together by noncovalent interactions  So, the quaternary structure is a tetramer consisting of two α-subunits (α 1  and α 2 :141 amino acids ) and two β-subunits (β 1  and β 2 : 146 amino acids) that are structurally similar and about the same size

19. Heme -binding p ocket Heme Pocket is a crevice/ hollow hydrophobic area Formed in the interior of Globin subunits Heme group is tucked between E and F helices of Globin subunit. The α-subunits and β-subunits are formed of 7 and 8 alpha helices , respectively named A–H that are joined by non-helical segments (referred to as corners ). Each subunit has a binding pocket for heme formed by the E and F helices.

20. The hemoglobin tetramer are visualized as composed two identical dimers, (αβ)1 and (αβ)2 . Two polypeptide chains within each dimer are held tightly together primarily   by h ydrophobic interactions, which prevent their movement relative to each other . The two αβ dimers are symmetrically related by a 2- fold rotation. Two dimers are linked to each other primarily by polar bonds ( network of ionic bonds and hydrogen bonds ) The weak er interaction allows movement at the interface of these two more freely . Tetrameric structure of Hb α 2 β 2 tetramer (a dimer of αβ protomers ).

21. Oxygenation rotates one αβ dimer ∼ 15° with respect to the other αβ dimer These movements are the result of a change in the quaternary structure. The quaternary structure exhibited by deoxy form is called the T state (T = tense) and that of the oxy form is R state (R = relaxed). There is a large central water cavity in the T state and a narrower cavity in the R state. Two conformations of the quaternary structure : T and R states

22. The function of hemoglobin as an oxygen-carrier in the blood is fundamentally linked to the equilibrium between the two main states of its quaternary structure, the unliganded " deoxy " or "T state" versus the liganded "oxy" or "R state".

23. T Form of Hb R Form Of Hb Deoxy Hb is in T form binds with CO 2 ,H + and 2,3BPG Oxy Hb is in R form binds only with Oxygen T form has 8 salt bridges linked in between the dimer subunits Salt bridges are broken in between the dimer subunits during oxygenation of Hb. More constrained form Less constrained f orm T form has low affinity for Oxygen R form has higher affinity for Oxygen T form of Hb predominates in low pO2 R form of Hb predominates at high pO2

24. Hemoglobin has important role in Respiration mechanism- Hb Majorly Transports- Oxygen (97% - 100 %) Hb Minorly Transports – Carbon dioxide (15% - 25 %) Oxygen transported by Hb and reached to every cell is used up in Mitochondrial ETC (Respiratory Chain/Cellular respiration) To generate ATP (Oxidative Phosphorylation) Functions of Hemoglobin The primary function of Hb is to transport oxygen (O 2 ) from the lung to tissues

25. Hemoglobin Plays Role as Buffer - (Hb/Hb-H + ) in the Erythrocytes r esists change in pH Imidazole group of amino acid Histidine of Hb molecule – p articipates in buffering mechanism of Hb.

26. During oxygenation, one Hb molecule with 4 Heme can bind to four O 2 molecules Oxygen links to Ferrous form of Iron, of Heme non enzymatically, loosely and reversibly . The relationship between these forms can be written as follows: deoxy -hemoglobin + O 2  oxy-hemoglobin Because oxygen binding is reversible, the two forms of hemoglobin are said to be in equilibrium with one another. Under certain conditions, the deoxy form is favored, and under other conditions the oxy form is favored. In the lungs, where O 2 pressure is highest , hemoglobin is completely loaded with O 2 , giving four O 2 molecules per Hb . In the tissues, however, where the oxygen pressure is much lower, hemoglobin releases O 2 Features of oxygenation of Hb

27. If free Fe(II)-heme in aqueous solution is exposed to dioxygen , it is immediately converted to Fe(III) form known as hematin through an intermediate - oxo dimer ( Fe III -O- Fe III ) Function of protein part is to provide crevice which keeps heme from getting oxidized. In the absence of globins , Fe 2 + will get converted to Fe 3+ All the heme in living systems would be precipitated as hematin . Heme buried in hydrophobic environment of protein so that O 2 binding does not result in oxidation. Thus, the globins of Mb and Hb function to prevent the auto-oxidation of oxyheme During Oxygenation Ferrous of Heme is not oxidized to Ferric

28. Oxygenation and deoxygenation of hemoglobin Hemoglobin gets o xygenated At l ungs At increased pO 2 concentration (100-120 mm Hg) At decreased pCO 2 Hemoglobin gets deoxygenated At t issues With Increased pCO 2 Decreased pO 2 levels (40 mm Hg ) Hemoglobin is virtually 100% saturated in the lungs , pressure of oxygen 100 mm Hg Hemoglobin is only about 50% saturated in working muscles , the pressure of oxygen decreases to about 25 mm Hg.

29. Oxygen Dissociation Curve ( ODC ) The oxygen dissociation curve ( ODC ), represents O 2  saturation of Hb at varying partial pressures of O 2 Plot of sO 2 , % vs. pO 2 , mm Hg Hemoglobin’s sigmoidal O 2 -dissociation curve is of great physiological importance Sigmoidal (“S-shaped”) curve can be described only in terms of a cooperative interaction between the four hemes . binding and releasing O 2   occur in a cooperative manner Pulse Oximeter Is An Instrument That Measures The Percentage Hb Fully Saturated With Oxygen In Arterial Blood

30. Cooperativity in oxygen binding Although each of the four units in hemoglobin contains a heme group, the four groups do not operate independently of each other: the binding (and release) of O 2 is a cooperative process . The secret to hemoglobin’s success as an oxygen delivery molecule is the fact that it has four subunits that “talk” to each other. In other words, the binding of one O 2 molecule affects the binding of others Hemoglobin has a relatively low attraction for oxygen, B ut when one molecule of oxygen binds to a heme group, the structure changes to the oxygenated form, which has a greater attraction for oxygen. Therefore, the second molecule of O 2 binds more easily, and the third and fourth even more easily. The oxygen affinity of oxy-hemoglobin is many times greater than that of deoxy -hemoglobin.

31. Cooperative binding and ODC Steep part of the sigmoidal curve occurs at about the oxygen pressure found within the tissues, allowing hemoglobin to deliver a significant amount of oxygen over a fairly narrow range of pressures. That is, it binds oxygen at the relatively high partial pressures in the lungs (the red region in Figure) and releases oxygen at the lower partial pressures in the peripheral tissues (the blue region in Figure).

32. Features of cooperative binding of oxygen E nhances the efficiency of haemoglobin as an oxygen transporter. Without positive cooperativity, an 81-fold increase of the pO 2 would be required to raise the oxygen saturation from 10% to 90%. However, about 5- fold increase is sufficient to do the same. This means that haemoglobin can rapidly bind oxygen in lungs (where pO 2 is high) and then readily liberate it in the tissue capillaries (where pO 2 is low).

33. Hill coefficient is an indicator of cooperative binding Extent to which the binding of first oxygen molecule to one subunit of Hb increases the subsequent oxygen binding to other subunits. Max.4, practically 2.5

34. Historically, Hb function has been explained in terms of equilibrium between two classical states: the tense (T) state which exhibits low affinity for O 2 , and the relaxed (R) state which exhibits high affinity for O 2 , This provides a structural basis for cooperative effects that facilitate the efficient uptake and release of O 2  in vivo. Oxygen binding to Hemoglobin triggers a conformational change from T to R . Mechanism of cooperativity

35. Deoxy form, T state Oxy form, R state

36. The interdimer interface (α 1 β 1 –α 2 β 2 ) of T state structure is characterized by more salt-bridge/hydrogen-bond interactions than R state structure. So, the unliganded ( deoxy ) form is called the "T" (for "tense") state because it contains extra stabilizing interactions between the subunits. In the high-affinity R-state conformation the interactions which oppose oxygen binding and stabilize the tetramer are somewhat weaker or "relaxed". The lower affinity of the T form is responsible for a slow start of the sigmoidal curve, The higher affinity of the R form causes rapid rise in the curve. The structural differences between the quaternary conformations

37. In the deoxygenated state, the iron ion is domed out of the heme plane because of its attraction to a histidine side chain (His F8 ) O 2 binding causes the iron ion to move back toward the center of the plane of the porphyrin ring such that it becomes planar with the rest of the heme group This causes a pull of the histidine residue with it towards the porphyrin ring , His F8 is part of Helix F, which is attached to the rest of the hemoglobin molecule, and when it moves, other parts of the protein also move with it This interaction induces a strain in the protein helix and the strain is transmitted to the remaining three monomers in the tetramer, where it induces a similar conformational change in the other heme sites The result is a switch to the oxy form, with its enhanced ability to bind oxygen Binding of subsequent molecules of O 2 to the other subunits is therefore easier Cooperative binding is achieved through steric conformational changes of the hemoglobin protein complex

38. Oxygenation of Hemoglobin causes considerable structural conformational change in Globin subunits. The movement of Fe(II) into the heme plane triggers the T to R conformational shift

39. Effect of Spin states of iron In oxygen free heme, Fe(II) is in high spin state (d 6 : t 2g 4 e g 2 ) The radius of Fe 2+ is too large to fit in the plane of four porphyrin nitrogen atoms Fe(II) lies 40 pm out of the plane of the N,N’,N’’,N’’’-donor set of the porphyrin group and is drawn towards the proximal His residue When O 2 enters the sixth coordination site, it causes a spin pairing of the iron centre ( d 6 : t 2g 6 ) The radius of low-spin Fe(II ) is 17 pm shorter than high spin Fe(II) Now the low-spin Fe(II) moves into the plane of the porphyrin ring and pulls the His residue with it

40. Iron's oxidation state in oxyhemoglobin Oxyhemoglobin (Hb-O 2 ), by experimental measurement, is diamagnetic (no net unpaired electrons ) According to old Pauling model, there is a low spin Fe(II) ion that is bound to singlet O 2  in oxy- Hb , as we already discussed Fe II : S = 0, O 2 : S = 0 Fe(II) in high spin Fe(II) in low spin

41. Current model According to Weiss model, coordination of O 2 to the low-spin Fe(II) centre is accompanied by electron transfer, oxidizing high-spin Fe(II) to low-spin Fe(III ) and reducing O 2 to [O 2 ] .  . there is low-spin Fe(III ) ( d 5 : t 2g 5 ) ion bound to superoxide radical anion [O 2 ] .  . Fe III S = ½ , O 2  S = ½ Though both are paramagnetic, a strong antiferromagnetic coupling between them ensues diamagnetic behavior. This model is supported by the O-O stretching frequency at 1105 cm -1  in resonance raman spectrum that is consistent with the fact that O 2  is in superoxide form . The order of the O—O bond is about 1.5. This considers to be more accurate and modern explanation .

42. low-spin Fe(III) superoxide radical anion

43. Enzymes involved The Fe(II) of Hb and Mb can be oxidized to Fe(III) to form methemoglobin ( metHb ) and metmyoglobin ( metMb ). Type of Hb /Mb that has  hematin  or ferriheme as a prosthetic group instead of ferrous heme. MetHb and metMb do not bind O 2 ; their Fe(III) is already octahedrally coordinated with OH- in the sixth liganding position . The brown color of dried blood and old meat is that of metHb and metMb . Globin chain provides some protection by providing a hydrophobic environment. Still about 3% Hb is oxidized to methemoglobin ( MetHb ) daily. Erythrocytes contain the enzyme methemoglobin reductase , which converts the small amount of metHb that spontaneously forms back to the Fe(II) form.

44. hematin

45. Hemoglobin binding ligands include allosteric ligands and competitive inhibitors Binding for ligands other than oxygen

46. Regulation of ligand binding to proteins through allosteric interactions Greek : allos , other stereos, solid or space. These cooperative interactions occur when the binding of one ligand at a specific site is influenced by the binding of another ligand , known as an effector or modulator, at a different (allosteric ) site on the protein . If the ligands are identical, this is known as a homotropic effect, whereas if they are different , it is described as a heterotropic effect. These effects are termed positive or negative depending on whether the effector increases or decreases the protein’s ligand-binding affinity. Hemoglobin, as we have seen, exhibits both homotropic and heterotropic effects. Allosteric regulation

47. Factors affecting ODC ( Allosteric Effectors) Hemoglobin is an allosteric protein .  The binding of O 2 to Hb results in a positive homotropic effect since it increases hemoglobin’s O 2 affinity. In contrast, BPG, CO 2 , H + , and Cl - are negative heterotropic effectors of O 2 binding to Hb because they decrease its affinity for O 2 (negative ) and are chemically different from O 2 ( heterotropic ). Factors causing Right Shift of ODC CADET C – CO 2 A – Acid (H + ) D– 2,3-BPG /2,3 DPG E – Exercise T – Temperature

48. Oxygen D issociation C urve (ODC) The equilibrium between the T and R states is affected by endogenous heterotropic ligands. They modulate Hb-O 2 affinity, either by stabilizing the R state Hb (left-shift the ODC ) or stabilizing the T state Hb (right-shift the ODC )

49. Bohr effect The release of oxygen from hemoglobin is enhanced when the pH is lowered or when the hemoglobin is in the presence of an increased pCO 2 . Both result in a decreased oxygen affinity of hemoglobin and, therefore, a shift to the right in the oxygen-dissociation curve, and both, then, stabilize the T ( deoxy ) form. This change in oxygen binding is called the Bohr effect. Conversely , raising the pH or lowering the concentration of CO 2 results in a greater affinity for oxygen, a shift to the left in the oxygen-dissociation curve, and stabilization of the R (oxy) form. Christian Bohr, father of Niels Bohr discovered this effect.

50. Chemical basis of the Bohr effect The Bohr effect reflects the fact that the deoxy form of hemoglobin has a greater affinity for protons than does oxyhemoglobin . This effect is caused by ionizable groups such as specific histidine side chains that have a higher pK a in deoxyhemoglobin than in oxyhemoglobin . Therefore , an increase in the concentration of protons (resulting in a decrease in pH) causes these groups to become protonated (charged) favoring salt bridge formation. These bonds preferentially stabilize the deoxy form of hemoglobin, producing a decrease in oxygen affinity. Note : Hemoglobin, then, is an important blood buffer . The Bohr effect can be represented schematically as :

51. Carbon Dioxide Transport and the Bohr Effect In addition to being an O 2 carrier, Hb plays an important role in the transport of CO 2 by the blood . CO 2 modulates O 2 binding directly and by combining reversibly with the N-terminal amino groups of blood proteins to form carbamates : The conformation of deoxygenated Hb ( deoxyHb ) is significantly different from that of oxygenated Hb ( oxyHb ). Consequently , deoxyHb binds more CO 2 as carbamate than does oxyHb . CO 2 is therefore a modulator of hemoglobin’s O 2 affinity : A high CO 2 concentration, as occurs in the capillaries, stimulates Hb to release its bound O 2 . The carbon dioxide is bound to amino groups of the globin proteins to form carbaminohemoglobin ; this mechanism is thought to account for about 10% of carbon dioxide transport in mammals.

52. Bohr effect

53. Effect of BPG on O 2 Binding Purified (stripped) hemoglobin has a much greater O 2 affinity than does hemoglobin in whole blood. This observation led to speculate that blood contains some other substance that complexes with Hb so as to reduce its O 2 affinity. This substance is D-2,3-bisphosphoglycerate (BPG), previously known as 2,3-diphosphoglycerate (DPG ). 2,3-BPG is synthesized from an intermediate of the glycolytic pathway

54. Binding of 2.3-BPG to d eoxyhemoglobin BPG binds in the central cavity of deoxyhemoglobin , but not oxyhemoglobin . A single molecule of BPG binds to the positively cavity formed by both β subunits of deoxyhemoglobin . This pocket contains several positively charged amino acids that form ionic bonds with the negatively charged phosphate groups of 2,3-BPG . This preferential binding stabilizes the T conformation. In the R state, the central cavity is too narrow to contain BPG . After oxygenation , BPG is expelled from the cavity.

55. High-altitude adaptation is a complex physiological process that involves an increase in the amount of hemoglobin per erythrocyte and in the number of erythrocytes. It normally requires several weeks to complete . Yet , as is clear to anyone who has quickly climbed to high altitude, even a 1-day stay there results in a noticeable degree of adaptation. This effect results from a rapid increase BPG concentration in the blood . The consequent decrease in O 2 -binding affinity increases the amount of O 2 that hemoglobin unloads in the tissues  under conditions of lower oxygen tension . Similar increases in BPG concentration occur in individuals suffering from disorders that limit the oxygenation of the blood (hypoxia ), such as various anemias and cardiopulmonary insufficiency. Role of BPG in high altitude acclimatization

56. Competitive ligands Certain small molecules, such as CO,NO,CN, and H 2 S, coordinate to the sixth liganding position of the Fe(II) in Hb and Mb with much greater affinity than does O 2 . This, together with their similar binding to the hemes of cytochromes, accounts for the highly toxic properties of these substances

57. CO poisoning In binding to a heme group, O 2 acts as a  -acceptor ligand. It is not surprising, therefore, that other  -acceptor ligands can take the place of O 2 in haemoglobin or myoglobin, and this is the basis of the toxicity of CO. Because the π* orbitals in CO are empty and those in NO are singly occupied, these ligands interact more strongly with Fe 2+ than does O 2 , in which the π* orbitals of the neutral ligand are doubly occupied . Carbon monoxide (CO) binds tightly (but reversibly) to the hemoglobin iron, forming carboxyhemoglobin . Carbon monoxide has 210 times greater affinity for hemoglobin than oxygen. A small environmental concentration will thus cause toxic levels of carboxyhemoglobin .

58. After the carbon monoxide has selectively bound to haemoglobin the oxygen-hemoglobin dissociation curve of the remaining oxyhemoglobin shifts to the left, reducing oxygen release The affinity of carbon monoxide for myoglobin is even greater than for hemoglobin. Binding to cardiac myoglobin causes myocardial depression, hypotension and arrhythmias. Cardiac decompensation results in further tissue hypoxia and is ultimately the cause of death.

59. carboxyhemoglobin

60. Cyanide poisoning Cyanide is isoelectronic with CO. But, CN- poisoning is not caused by CN − blocking the O2-binding sites in haemoglobin . Cyanide, although a  -acceptor ligand, favours higher oxidation state metal centres Cyanide binds to Fe(III) in cytochrome oxidase and stabilizes Fe(III) so that it can be no longer be readily reduced and take part in electron shuttle. Cyanide does not bind to hemoglobin, it binds only to Fe(III) hemoglobin ( methemoglobin , metHb ). Cyanide poisoning is thus not the result of lack of the Hb function.

61. Treatment Treatment of cyanide poisoning consists of diverting the cyanide into the production of cyanmetHb . First, some of the normal hemoglobin is converted to methemoglobin by intravenous infusion of a solution of NaNO 2 or by inhalation of amyl nitrite. Once metHb is formed, CN − can replace OH − at position 6 of the iron, since it has a higher affinity for Fe 3+ than OH − . CyanmetHb is no more toxic than metHb , and cells containing it can be eliminated by normal body processes.

62. Diseases related to hemoglobin : Anemia Anemia is a blood disorder in which there is a deficiency of actual or available hemoglobin in blood. There is a reduced oxygen capacity leading to oxygen deficiency . A Hb level below 8 mg/ dL results severe anemia . Some common causes are listed below. Defect in heme synthesis : Nutritional deficiency of iron, copper, pyridoxal phosphate, folic acid, vitamin B 12 or vitamin C. Mal-absorption where the body is not able to properly absorb or use the nutrients in the diet. Enzyme deficiencies: deficiency of glucose-6-phosphate dehydrogenase Hemoglobinopathies : sickle-cell anemia ( HbS ), hemoglobin C disease ( HbC ), Thalassemia Infections : Malarial parasites Internal hemorrhage that cause blood loss, like gastric ulcer, colorectal cancer Anemia is associated with the symptoms of apathy (dull and inactive), sluggish metabolic activities, retarded growth, loss of appetite, weakness, shortness of breath, fast or irregular heartbeat, headache, dizziness or lightheadedness, cold hands or feet, pale or yellow skin and chest pain.

63. Hemoglobinopathy Hemoglobinopathy - a group of blood disorders and diseases that affect red blood cells . It is a kind of genetic defect that results in abnormal structure of one of the globin chains of the hemoglobin molecule . Sickle cell anemia is caused by a defective form of hemoglobin ( HbS ) due to a single amino acid change in the β-chain of hemoglobin (glutamic acid replaced by valine ) Thalassemias usually result in underproduction of normal globin proteins, often through mutations in regulatory genes.