1. HemoglobinBy-Saurav K. Rawat(Rawat DA Greatt) 1

2. Hemoglobin andMyoglobinBecause of its red color, the red blood pigment has been ofinterest since antiquity.First protein to be crystallized - 1849.First protein to have its mass accurately measured.First protein to be studied by ultracentrifugation.First protein to associated with a physiologicalcondition.First protein to show that a point mutation can causeproblems.First proteins to have X-ray structures determined.Theories of cooperativity and control explainhemoglobin function 3. The visible absorptionspectra forhemoglobinThe red color arises from thedifferences between the energylevels of the d orbitals around theFerrous atom.There is an energy differencebetween them, which determinesthe size of the wavelength of themaximal absorbance band.Fe(II) = d6 electron configuration:Low spin state 4. The structure ofmyoglobin and hemoglobinAndrew Kendrew and Max Perutz solved the structure of thesemolecules in 1959 to 1968.The questions asked are basic. What chemistry is responsible foroxygen binding, cooperativity, BPG effects and what alterations inactivity does single mutations have on structure and function.Myoglobin: 44 x 44 x 25 single subunit 153 amino acid residues121 residues are in an a helix. Helices are named A, B, C, F. Theheme pocket is surrounded by E and F but not B, C, G, also H is nearthe heme.Amino acids are identified by the helix and position in the helix or bythe absolute numbering of the residue. 5. SSttrruuccttuurree ooff HHeemmoogglloobbiinn 6. MMyyoogglloobbiinnHHeemmeeHHeemmoogglloobbiinn2 a chains2 b chains 7. HemoglobinSpherical 64 x 55 x 50 two fold rotation ofsymmetry a and b subunits are similar and areplaced on the vertices of a tetrahedron. There is noD helix in the a chain of hemoglobin.Extensive interactions between unlikesubunits a2-b2 or a1-b1 interface has 35 residueswhile a1-b2 and a2-b1 have 19 residue contact.Oxygenation causes a considerablestructural conformational change 8. The Heme groupEach subunit of hemoglobin ormyoglobin contains a heme.Binds one molecule of oxygenHeterocyclic porphyrin derivativeSpecifically protoporphyrin IXThe iron must be in the Fe(II) form orreduced form. (ferrous oxidation) state.Loss of electrons oxidation LEOGain of electrons reduction GERLeo the lion says GER 9. 11GLOBIN chainHEME 10. HemoglobinBinding of O2 alters the structure 11. Function of the globinProtoporphyrin binds oxygen to thesixth ligand of Fe(II) out of theplane of the heme. The fifth ligandis a Histidine, F8 on the side acrossthe heme plane.His F8 binds to the proximal sideand the oxygen binds to the distalside.The heme alone interacts withoxygen such that the Fe(II) becomesoxidized to Fe(III) and no longer 12. HHeemmoogglloobbiinn aannddmmyyoogglloobbiinn HHeemmoogglloobbiinn oxygen transport protein of redblood cells. MMyyoogglloobbiinn oxygen storage protein of skeletalmuscles. As with the cytochrome example,both proteins use heme groups.It acts as the binding site formolecular oxygen. 13. Hemoglobin functiona2,b2 dimer which are structurally similar to myoglobinTransports oxygen from lungs to tissues.O2 diffusion alone is too poor for transport inlarger animals.Solubility of O2 is low in plasma i.e. 10-4 M.But bound to hemoglobin, [O2] = 0.01 M or thatof airTwo alternative O2 transporters are;Hemocyanin, a Cu containing protein.Hemoerythrin , a non-heme containingprotein. 14. Models for AllostericBehavior Monod, Wyman, Changeux (MWC)Model: allosteric proteins can exist in twostates: R (relaxed) and T (taut) In this model, all the subunits of anoligomer must be in the same state T state predominates in the absence ofsubstrate S S binds much tighter to R than to T 15. More about MWC Cooperativity is achieved because Sbinding increases the population of R,which increases the sites available to S Ligands such as S are positivehomotropic effectors Molecules that influence the binding ofsomething other than themselves areheterotropic effectors 16. Myoglobin facilitates rapidlyrespiring muscle tissueThe rate of O2 diffusion fromcapillaries to tissue is slowbecause of the solubility ofoxygen.Myoglobin increases the solubilityof oxygen.Myoglobin facilitates oxygendiffusion.Oxygen storage is also a functionbecause Myoglobin concentrationsare 10-fold greater in whales and 17. Fe O O FeA heme dimer isformed whichleads to theformation ofFe(III)By introducing steric hindrance on one side of the heme planeinteraction can be prevented and oxygen binding can occur.The globin acts to:a. Modulate oxygenbinding affinityb. Make reversibleoxygen bindingpossible 18. The globin surrounds the hemelike a hamburger is surrounded bya bun. Only the propionic acidside chains are exposed to thesolvent.Amino acid mutations in the hemepocket can cause autooxidation ofhemoglobin to formmethemoglobin. 19. When Fe(II) goes to Fe(III),oxidized, it producesmethemoglobin which is brownand coordinated with water inthe sixth position. Dried bloodand old meat have this browncolor.Butchers use ascorbic acid to reduce methemoglobin tomake the meat look fresh!!There is an enzyme methemoglobin reductase thatconverts methemoglobin to regular hemoglobin. 20. Hemoglobin as oxygencarrier In each hemoglobin molecule there are fourheme groups Heme = Fe2+ surrounded by phorphyringroup, four N act as ligands. As O2 carrier: O2 binds to Fe2+ as a ligand Reversible process CO and CN bind irreversible to Fe2+ 21. OOxxyyggeenn TTrraannssppoorrtt 22. N NegN N t2gFe2+NOON NFe2+N NNegt2g Changes at the Heme initiate structure switch DeoxyHb has Fe 0.3 out of plane OxyHb has Fe in plane of porphyrin Fe atom pulls the bound F8 His with it Shifts the whole F helix, EF corner Salt links are broken at ab interface T-form becomes R-form R-form has greater O2 affinity Cooperativity set in motion BPG stabilizes deoxyHb T-form by creating more contacts O2 binding to Hb causes dissociation of BPG because thecavity gets too small. This favors the R-form as well. 23. Quaternary structure of deoxy- andoxyhemoglobinT-state R-state 24. Structural Basis for Cooperativity Interactions between subunits A dissociated Hb subunit binds O2 like Mb A b4 tetramer binds O2 like Mb Cooperativity must involve subunit interactions 25. Functions ofHaemoglobin Oxygen delivery to the tissues Reaction of Hb & oxygen Oxygenation not oxidation One Hb can bind to four O2 molecules Less than .01 sec required foroxygenation"b chain move closer when oxygenated When oxygenated 2,3-DPG is pushed out"b chains are pulled apart when O2 isunloaded, permitting entry of 2,3-DPGresulting in lower affinity of O2 26. Oxy &deoxyhaemoglobin 27. Oxygen-haemoglobindissociation curve O2 carrying capacity of Hb atdifferent Po2 Sigmoid shape Binding of one molecule facilitate thesecond molecule bindingP 50 (partial pressure of O2 at whichHb is half saturated with O2)26.6mmHg 28. Hb-oxygen dissociationcurve 29. Hb-oxygen dissociationcurve The normal position of curvedepends on Concentration of 2,3-DPG H+ ion concentration (pH) CO2 in red blood cells Structure of Hb 30. Hb-oxygen dissociationcurve Right shift (easy oxygen delivery) High 2,3-DPG High H+ High CO2 HbS Left shift (give up oxygen lessreadily) Low 2,3-DPG HbF 31. Summary Normal structure including theproportion of globin chains arenecessary for the normal function ofhaemoglobin Reduced haemoglobin in the red bloodcells due to any abnormality of any ofits constituents result into a clinicalsituation called anaemia Metabolic & other abnormalities resultinto abnormal oxygen supply to the 32. OxyHb and DeoxyHb have very differentquaternary structures OxyHb is more compact (bFebchanges from 40 toFe 33) When Obinds, ab contacts change as H-bonds are2 adjusted Electrostatic bonds (Salt Links) also change: OxyHbthe CO- termini can freely rotate, DeoxyHb CO-22termini salt linked DeoxyHb has T-form (taut) OxyHb has R-form (relaxed) 33. Oxygenation rotates the a1b1 dimerin relation to a2b2 dimer about 15The conformation of the deoxy state iscalled the T stateThe conformation of the oxy state iscalled the R stateindividual subunits have a t or r if in the deoxy or oxy state.What causes the differences inthe conformation states?It is somehow associated withthe binding of oxygen, buthow? 34. The positive cooperativity of O2binding to Hb arises from theeffect of the ligand-bindingstate of one heme on theligand-binding affinity ofThe Fe iron ains oatbhoeur.t 0.6 outof the heme plane in the deoxystate. When oxygen binds itpulls the iron back into theheme plane. Since theproximal His F8 is attached tothe Fe this pulls the completeF helix like a lever on afulcrum. 35. HemoglobinA classic example of allostery Hemoglobin and myoglobin are oxygentransport and storage proteins Compare the oxygen binding curves forhemoglobin and myoglobin Myoglobin is monomeric; hemoglobinis tetrameric Mb: 153 aa, 17,200 MW Hb: two alphas of 141 residues, 2betas of 146 36. Hemoglobin FunctionHb must bind oxygen in lungsand release it in capillaries When a first oxygen binds to Fein heme of Hb, the heme Fe isdrawn into the plane of theporphyrin ring This initiates a series ofconformational changes thatare transmitted to adjacentsubunits 37. Hemoglobin FunctionHb must bind oxygen in lungsand release it in capillaries Adjacent subunits' affinity foroxygen increases This is called positivecooperativity 38. Myoglobin StructureMb is a monomeric heme protein Mb polypeptide "cradles" theheme group Fe in Mb is Fe2+ - ferrous iron - theform that binds oxygen Oxidation of Fe yields 3+ charge -ferric iron -metmyoglobin does notbind oxygen Oxygen binds as the sixth ligandto Fe See Figure 15.26 and discussion 39. The ConformationChangeThe secret of Mb and Hb! Oxygen binding changes the Mbconformation Without oxygen bound, Fe is out ofheme plane Oxygen binding pulls the Fe into theheme plane Fe pulls its His F8 ligand along with it The F helix moves when oxygen binds Total movement of Fe is 0.029 nm - 40. Binding of Oxygen by HbThe Physiological Significance Hb must be able to bind oxygen inthe lungs Hb must be able to release oxygenin capillaries If Hb behaved like Mb, very littleoxygen would be released incapillaries - see Figure 15.22! The sigmoid, cooperative oxygenbinding curve of Hb makes thispossible! 41. Oxygen Binding by HbA Quaternary Structure Change When deoxy-Hb crystals areexposed to oxygen, they shatter!Evidence of a structural change! One alpha-beta pair moves relativeto the other by 15 degrees uponoxygen binding This massive change is induced bymovement of Fe by 0.039 nm whenoxygen binds See Figure 15.32 42. Binding of the oxygen on one hemeis more difficult but its bindingcauses a shift in the a1-b2contacts and moves the distal HisE7 and Val E11 out of the oxygenspath to the Fe on the othersubunit. This process increasesthe affinity of the heme towardoxygen.The a1-b2 contacts have two stablepositions.These contacts, which are joinedby different but equivalent sets of 43. The energy in the formation of theFe-O2 bond formation drives the TR transition.Hemoglobins O2 -bindingCooperativity derives from the T R Conformational shift.The Fe of any subunit cannot move into its heme planewithout the reorientation of its proximal His so as toprevent this residue from bumping into the porphyrinring.The proximal His is so tightly packed by itssurrounding groups that it can not reorient unless thismovement is accompanied by the previously describedtranslation of the F helix across the heme plane.The F helix translation is only possible in concert withthe quaternary shift that steps the a1C-b2FG contactone turn along the a1C helix. 44. The inflexibility of the a1-b1 and the a2-b2 interfaces requiresthat this shift simultaneously occur at both the a1-b2 and a2-b1interfaces.No one subunit or dimer can change its conformation.The t state with reduced oxygenaffinity will be changed to the rstate without binding oxygenbecause the other subunits switchupon oxygen binding. Unbound rstate has a much higher affinity foroxygen, and this is the rational forcooperativity 45. a. Free energy changes withfractional saturationb. Sigmoidal binding curve as acomposite of the R state binding andthe T state binding. 46. Binding of oxygenrearranges the electronicdistribution and altersthe d orbital energy.This causes a differencein the absorptionspectra.Bluish for deoxy HbRedish for Oxy HbMeasuring theabsorption at 578 nmallows an easy method todetermine the percent ofOxygen bound tohemoglobin. 47. O2 binding to myoglobin2 2 Mb + O MbOKd = [Mb][O ]2[MbO ]2[O ]Kd [O ]Y [MbO ][Mb] [MbO ]2222=O2 ++=Written backwardswe can get thedissociationconstantFractional Saturation solve for [MbO2]and plug in 48. How do you measure theconcentration of oxygen?Use the partial pressure of O2 or O2tension = pO2= 2P= the partial50 Y pO 2 +O K pOd 2oxygen pressurewhen YO2 = 0.502Y pO 2 +O P pO50 2=What is theshape of thecurve if you plotYO2 vs. pO2What does the value of P50 tell youabout the O2 binding affinity? 49. P50 value for myoglobin is 2.8 torror1 torr = 1 mm Hg = 0.133 kPa760 torr = 1 atm of pressureMb gives up little O2 over normalphysiological range of oxygen concentrationsin the tissuei.e. 100 torr in arterial blood30 torr in venous bloodYO2 = 0.97 to YO2 = 0.91What is the P50 value for Hb?Should it be different than myoglobin? 50. The Hill EquationE = enzyme, S = ligand, n= smallnumberE + nS ESn This is for bindingof 1 or more ligandsOis considered a2 ligandn[ESn]K [E][S]= 1.Ys n[ESn]n([E] +[ESn])2. =Fractional Saturation =bound/total 51. As we did before, combine 1. + 2. = 3.n[E][S]KYs n( +)K[E] 1 [S]=nYs [S]3.or n+K [S]=Look similar to Mb + O2 except forthe n 52. Continuing as before:( )nK = P50( )( ) ( )n2Y pO 2 n+50n2= 4.O P pOn = Hill Constant, a non integral parameterrelatingDegree of Cooperativity among interactingligand-binding sites or subunitsThe bigger n the more cooperativity (positivevalue)If n = 1, non-cooperativen < 1, negative cooperativity 53. Hill PlotRearrange equation 4.Log Ys = - nLog[S] logK1-Ysy = mx + cn = slope and x intercept of-c/m 54. Things to rememberHb subunits independently compete for O2 forthe first oxygen molecule to bindWhen the YO2 is close to 1 i.e. 3 subunits areoccupied by O2 , O2 binding to the last site isindependent of the other sitesHowever by extrapolating slopes: the 4th O2binds to hemoglobin 100 fold greater than thefirst O2A DDG of 11.4 kJmol -1 in the binding affinityfor oxygenWhen one molecule binds, the rest bind andwhen one is released, the rest are released. 55. Contrast Mb O2 binding toHemoglobinYO2 = 0.95 at 100 torrbut0.55 at 30 torra DYO2 of 0.40Understand Fig 9-3Hb gives up O2 easier than Mb and thebinding is Cooperative!! 56. 72Oxygen and Carbon DioxideTransport in Blood 57. Basic Mechanism of the Gases TransportationTwo forms of the gases: physical dissolution andchemical combination.Most of oxygen and carbon dioxide in the blood istransported in chemical combinationOnly the gas in physical dissolution express PP anddiffuse to a place with low PP.Dynamic balance between the two forms:Physical dissolution P P Chemical combination73PP 58. 74I. Transport of Oxygen 59. 75Oxygen Transport Method Percentage Dissolved in Plasma 1.5 % Combined with Hemoglobin 98.5 %Bound to HgbDissolved 60. 76Oxyhemoglobin Formation An oxygen molecule reversibly attaches to theheme portion of hemoglobin. The heme unit contains iron ( +2 ) whichprovides the attractive force.O2 + Hb HbO2 61. In normal adults, most of the hemoglobincontains 2 and 2 chains.Each of the 4 iron atoms can bind reversibly onO2 molecule.The iron stays in the ferrous state, so that thereaction is an oxygenation, not an oxidation.77 62. When saturated with O2 (4 O2 in onehemoglobin molecule), it is always writtenHb4O8.The reaction is rapid, requiring less than 0.01second.The deoxygenation (reduction) of Hb4O8 is alsovery rapid.78 63. Oxygen Capacity: The maximum quantity ofoxygen that will combine chemically with thehemoglobin in a unit volume of blood;normally it amounts to 1.34 ml of O2 per gm of Hb or20 ml of O2 per 100 ml of blood.Oxygen Content: how much oxygen is in the bloodOxygen Saturation: A measure of how muchoxygen the blood is carrying as a percentage of themaximum it could carry79Basic Concepts: 64. The oxygen-hemoglobin dissociation curve:80The curverelatingpercentagesaturation ofthe O2-carrypower ofhemoglobin tothe PO2. 65. 81The oxygen-hemoglobin dissociation curveA. Flattened upper portionB. Steep middle portionC. Lower portion 66. 82Shifting the Curve 67. Factors that Shift the Oxygen-Hemoglobin DissociationCurve83 68. 841. pH and PCO2: Bohr effect 69. The Bohr EffectCompetition between oxygen and H+ Discovered by Christian Bohr Binding of protons diminishes oxygenbinding Binding of oxygen diminishes protonbinding Important physiological significance See Figure 15.34 70. Bohr Effect IICarbon dioxide diminishesoxygen binding Hydration of CO2 in tissues andextremities leads to protonproduction These protons are taken up byHb as oxygen dissociates The reverse occurs in the lungs 71. 892. Temperature 72. 3. 2,3-biphosphoglycerate, 2,3-BPGA byproduct of anaerobic glycolysis.Present in especially high concentration in red bloodcells because of their content of 2,3-BPG mutase.The affinity of hemoglobin for O2 diminishes as theconcentration of 2,3-BPG increase in the red bloodcells.90 73. 2,3-BisphosphoglycerateAn Allosteric Effector ofHemoglobin In the absence of 2,3-BPG,oxygen binding to Hb follows arectangular hyperbola! The sigmoid binding curve is onlyobserved in the presence of 2,3-BPG Since 2,3-BPG binds at a sitedistant from the Fe where oxygenbinds, it is called an allosteric 74. 2,3-BPG and HbThe "inside" story...... Where does 2,3-BPG bind? "Inside" in the central cavity What is special about 2,3-BPG? Negative charges interact with 2Lys, 4 His, 2 N-termini Fetal Hb - lower affinity for 2,3-BPG, higher affinity for oxygen, soit can get oxygen from mother 75. No BPGO2 PRESSURE (torr)SATURATION1010 50With BPGBPG Lowers the binding affinity of Hb for O2[BPG] = 0, Hb P50 = 1 torr[BPG] = 4000mM, Hb P50 = 26 torrWithout BPG, Hb couldnt unload O2 in cells 76. BPG acts BPG acts bbyy ssttaabbiilliizziinngg ddeeooxxyyHHbbBPG binds by electrostatic interactions tothe highly electropositive region (red) in acrevice between the 4 subunitsBPG binding site 77. BPG ensures that O2 can be unloaded at the peripheral tissues by decreasing the affinity of Hb for O2 about 26 fold increasing O2, on the other hand, promotes the formation of oxyHbwhose changed conformation prevents BPG binding because thebinding cavity becomes too small Fetal Hb has a lower affinity for 2,3-BPG and therefore has a higheraffinity for O2 BPG regulates O2 binding between Hb types This allows transfer of O2 from mother to child This explains the need for multiple Hb types If [BPG] = 0, HbA > HbF for O2 binding HbF has neutral Serine in place of HbA Hiso2O2 PRESSURE (torr)SATURATION1010 50HbAHbFO2 flows frommom to baby ! 78. Importance:The normal DPG in the blood Hypoxic condition that last longer thana few hoursDisadvantage:The excess DPG also makes it moredifficult for the hemoglobin tocombines with Oin the lungs.982 79. 4. Effect of Carbon Monoxide (CO)CO combines Hb at the same point as does O,2and can displace Ofrom hemoglobin.2 CO binds with about 250 times as much tenacityas O.2Therefore, a Ponly a little greater than 0.4CO mmHg can be lethal.In the presence of CO (low concentration), theaffinity of hemoglobin for Ois enhanced,992 80. Effect of CO & Anemia on Hb-O2 affinityNormal blood with Hb=15 gm/dl, anemia with Hb=7.5 gm/dl,and normal blood with 50% COHb (carboxyhemoglobin).100 81. 1015. Fetal HemoglobinAdvantageIncreased O2release to thefetal tissuesunder thehypoxiccondition. 82. 102II Carbon Dioxide TransportMethod Percentage Dissolved in Plasma 7 - 10 % Chemically Bound toHemoglobin in RBCs 20 - 30 % As Bicarbonate Ion inPlasma 60 -70 %Dissolvedbound to HbHCO3- 83. 103CarbaminohemoglobinFormation Carbon dioxide molecule reversibly attaches toan amino portion of hemoglobin.CO2 + Hb HbCO2 84. 104Carbonic Acid Formation The carbonic anhydrase stimulates waterto combine quickly with carbon dioxide.CO2 + H2 0 H2 CO3 85. 105Bicarbonate Ion Formation Carbonic acid breaks down to release ahydrogen ion and bicarbonate.H2 CO3 H+ + HCO-3 86. CO2 Transport and Cl- Movement106 87. Carbon Dioxide Dissociation Curve107Haldane effectFor any given PCO2,the blood will holdmore CO2 when thePO2 has beendiminished.Reflects thetendency for anincrease in PO2 todiminish the affinityof hemoglobin forCO2. 88. Mechanism of Haldane effectCombination of oxygen with hemoglobin in thelungs cause the hemoglobin to becomes astronger acid. Therefore:1) The more highly acidic hemoglobin has lesstendency to combine with CO2 to form CO2Hb2) The increased acidity of the hemoglobin alsocauses it to release an excess of hydrogen ions108 89. Interaction Between CO2 and O2 Transportation1. Bohr effect109 90. 1102. Haldane effect 91. SSiicckkllee--cceellll aanneemmiiaa A Glu normally resides at position 6 ofeach b- subunit. In HbS this amino ismutated to ValGlu 6Glu 6baba the Val for Glu mutation makes deoxy-HbS insoluble-findout why! 92. SSiicckkllee--cceellll AAnneemmiiaaThe Val for Glu mutation makes deoxy-HbS insolubleIn deoxy-HbS, b-subunit residues Phe 85 and Leu 88 reside at thesurface and bond with Val 6 on another b-subunit.This leads to the formation of long filamentous strands of deoxy-HbSand to the sickling deformation of the erthyrocytesIn oxy-HbS, b-subunit residues Phe 85 and Leu 88 do not reside at thecell surface, so oxy-HbS does not aggregate. Thus, its oxygen bindingcapacity and allosteric properties are largely retained. 93. Hemoglobin : a portrait of a soluble proteinwith 4 structure A SUMMARYHemoglobin : a portrait of a soluble proteinwith 4 structure A SUMMARY the heme prosthetic group is tightly bound in the protein and isessential for function steric relationships within Hb ensure that the heme group hasappropriate reactivity hemoglobin has quaternary structure which gives it unique O2 bindingproperties - allosterism and cooperativity of binding 2,3-bisphosphoglycerate is a regulatory molecule that stabilizesdeoxy-Hb and is essential for the allosterism and cooperativity ofbinding in Hb there is considerable interplay between the oxygen binding affinity ofHb and [H+], [CO2] and [2,3-BPG] the interplay between various sites in Hb is mediated through changesin quaternary structure Sickle-cell anemia is an example of a genetically transmitted diseasewhich highlights the effect of one amino acid substitution on proteinstructure and function 94. 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