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Hemoglobin Structure

Hemoglobin Structure Hemoglobin is tetrameric O 2 transport protein found in vertebrate erythrocytes (red blood cells) Hb has changing X 2 Y 2 composition over life Always has 2 pairs of polypeptide chains Hb A (adult) is a 2 b 2 [HbA 2 (2% Hb) is a 2 d 2 ]

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Hemoglobin Structure

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  1. Hemoglobin Structure • Hemoglobin is tetrameric O2 transport protein found in vertebrate erythrocytes (red blood cells) • Hb has changing X2Y2 composition over life • Always has 2 pairs of polypeptide chains • Hb A (adult) is a2b2 [HbA2 (2% Hb) is a2d2] • Early Embryo has z2e2 (like a and b) • Later Embryo z2e2 to a2g2 = Hb F (fetus) • a and z have 141 A.A.’s, slightly different • b, g, and d have 145 A.A.’s • Different oxygen affinities allow passing of O2 from mother to fetus (more later) • X-Ray Crystal Structure • 23 year project of Max Perutz (1959) • 4 subunits packed in tetrahedral array • One heme/subunit, near surface (25Å apart) • a contacts both b; no a—a or b—b contact • Hb subunits are similar to Mb • Only 18% of AA’s conserved; same shape • “Globin Fold” common to all vertebrates • Places Heme in correct environment to bind O2 reversibly • Conserve AA’s inclue F8 His and E7 His • Polar/Polar and Nonpolar/Nonpolar subst.

  2. In contrast to myoglobin hemoglobin has 4°structure

  3. Allosteric Interactions of Hb O2 Binding • Allosteric Interactions = those between spatially separated parts of a protein • O2 binding is cooperative • O2 binding is affected by H+, CO2 binding and vice versa • The organic phosphate BPG regulates O2 binding • Cooperative O2 Binding of Hb • Saturation • Mb vs. Hb Y (Oxygen Dissociation Curves) • YMb > YHb at any pO2 (partial pressure O2) • P50 = pO2 at which Y = 50% • Mb P50 = 1 torr (1 atm = 760 torr) • Hb P50 = 26 torr

  4. Shapes of the curves • Mb has the shape of hyperbola • Hb has sigmoidal shape myoglobin 1 SATURATION hemoglobin 0 10 50 o2O2 PRESSURE (torr)

  5. Mb n = 1.0 Hb n =2.8 • Hill Plots Tell Us About Cooperativity • n = Hill Coefficient indicates cooperativity • Mb: n = 1.0 = independent O2 binding • Hb: n = 2.8 = O2 cooperative binding • Binding the first O2 makes it easier to bind the next, and so on • Dissociating the first O2 makes it easier to dissociate the next one • Why is Cooperativity good in Hb? • Y changes very rapidly with pO2 • Lung pO2 = 100torr, Muscle pO2 = 20 n = 1 then Ylung = 0.79, Ymuscle = 0.43 (0.36 delivered) n = 2.8 then Ylung = 0.98, Ymuscle = 0.32 (0.66 delivered) Hb is 1.8 times as efficient as Mb Hb P50 lies between lungs and muscle Log (pO2)

  6. H+ and CO2 effects on Hb O2 Binding • Bohr Effect: Increased [H+] decreases binding • Mb O2 binding is not affected by [H+] • Contracting muscle generates H+ and CO2 • This helps Hb release O2 • Deoxy-Hb binds H+ stronger than oxy-Hb • The effect is mutual: high [O2] causes H+ to dissociate from Hb

  7. CO2 effect on Hb binding

  8. Organic Phosphate Regulation of Hb O2 binding • BPG is an organic phosphate Concentrations of glycolytic intermediates in erthyrocytes mM glucose 5000 glucose- 6- P 83 fructose- 6- P 14 fructose- 1,6- P 31 dihydroxyacetone- P 138 glyceraldehyde- 3- P 19 1,3 bisphosphoglycerate 1 2,3 bisphosphoglycerate 4000 (BPG) 3 phosphoglycerate 118 2 phosphoglycerate 30 phosphoenolpyruvate 23 pyruvate 51 lactate 2900 From S. Minakami and H. Yoshikawa. Biochem.Biophys.Res.Comm. 18(1965):345. 2,3 bisphospho-glycerate (BPG)

  9. BPG Lowers the binding affinity of Hb for O2 • [BPG] = 0, Hb P50 = 1 torr • [BPG] = 4000mM, Hb P50 = 26 torr • Without BPG, Hb couldn’t unload O2 in cells No BPG 1 SATURATION With BPG 0 10 50 o2O2 PRESSURE (torr)

  10. BPG acts by stabilizing deoxyHb BPG binds by electrostatic interactions to the highly electropositive region (red) in a crevice between the 4 subunits BPG binding site

  11. 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 oxyHb whose changed conformation prevents BPG binding because the binding cavity becomes too small • Fetal Hb has a lower affinity for 2,3-BPG and therefore has a higher affinity 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 His 1 HbF SATURATION HbA O2 flows from mom to baby ! 0 10 50 o2O2 PRESSURE (torr)

  12. 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

  13. OxyHb and DeoxyHb have very different quaternary structures • OxyHb is more compact (bFe—bFe changes from 40 to 33Å) • When O2 binds, a—b contacts change as H-bonds are adjusted • Electrostatic bonds (Salt Links) also change: OxyHb the CO2- termini can freely rotate, DeoxyHb CO2- termini salt linked • DeoxyHb has T-form (“taut”) • OxyHb has R-form (“relaxed”)

  14. Changes at the Heme initiate structure switch • DeoxyHb has Fe 0.3Å 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 the cavity gets too small. This favors the R-form as well.

  15. Models for Allosteric Interactions • Sequential Model • Only T and R forms possible for each unit • T to R transition of each subunit is induced by O2 binding, but this does not change the form of other subunits • Conformational changes enhance O2 binding at the next subunit, but O2 must bind each subunit before it switches to R

  16. Concerted Model • Whole protein changes from T to R form upon initial O2 binding • O2 has higher affinity for the unbound R subunits • This explains cooperativity • Actual: mix of the two models. Hb is predominantly T until ~2 O2 molecules are bound, then it goes all R. myoglobin 1 SATURATION hemoglobin 0 10 50 o2O2 PRESSURE (torr)

  17. Sickle-cell anemia • A Glu normally resides at position 6 of each b- subunit. In HbS this amino is mutated to Val Glu 6 a b b a Glu 6 • the Val for Glu mutation makes deoxy-HbS insoluble -findout why!

  18. Sickle-cell anemia In deoxy-HbS, b-subunit residues Phe 85 and Leu 88 reside at the surface and bond with Val 6 on another b-subunit. This leads to the formation of long filamentous strands of deoxy-HbS and to the sickling deformation of the erthyrocytes • the Val for Glu mutation makes deoxy-HbS insoluble In oxy-HbS, b-subunit residues Phe 85 and Leu 88 do not reside at the cell surface, so oxy-HbS does not aggregate. Thus, its oxygen binding capacity and allosteric properties are largely retained.

  19. Hemoglobin : a portrait of a soluble protein with 4° stuctureA SUMMARY • the heme prosthetic group is tightly bound in the protein and is essential for function • steric relationships within Hb ensure that the heme group has appropriate reactivity • hemoglobin has quaternary structure which gives it unique O2 binding properties - allosterism and cooperativity of binding • 2,3-bisphosphoglycerate is a regulatory molecule that stabilizes deoxy-Hb and is essential for the allosterism and cooperativity of binding in Hb • there is considerable interplay between the oxygen binding affinity of Hb and [H+], [CO2] and [2,3-BPG] • the interplay between various sites in Hb is mediated through changes in quaternary structure • Sickle-cell anemia is an example of a genetically transmitted disease which highlights the effect of one amino acid substitution on protein structure and function

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