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  2  2 [HbA 2 (2% Hb) is  2  2 ] Early Embryo has  2  2 (like  and  ) Later Embryo  2  2 to  2  2 = Hb F (fetus)  and  have 141 A.A.’s, slightly different , , and  have 145 A.A.’s »Different oxygen affinities allow passing of O 2 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) »  contacts both  ; no  —  or  —  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 O 2 reversibly Conserve AA’s inclue F8 His and E7 His Polar/Polar and Nonpolar/Nonpolar subst.

In contrast to myoglobin hemoglobin has 4°structure

Allosteric Interactions of Hb O 2 Binding –Allosteric Interactions = those between spatially separated parts of a protein »O 2 binding is cooperative »O 2 binding is affected by H +, CO 2 binding and vice versa »The organic phosphate BPG regulates O 2 binding –Cooperative O 2 Binding of Hb »Saturation »Mb vs. Hb Y (Oxygen Dissociation Curves) Y Mb > Y Hb at any pO 2 (partial pressure O 2 ) P 50 = pO 2 at which Y = 50% Mb P 50 = 1 torr (1 atm = 760 torr) Hb P 50 = 26 torr

o2O 2 PRESSURE (torr) SATURATION myoglobin hemoglobin Shapes of the curves Mb has the shape of hyperbola Hb has sigmoidal shape

»Hill Plots Tell Us About Cooperativity n = Hill Coefficient indicates cooperativity Mb: n = 1.0 = independent O 2 binding Hb: n = 2.8 = O 2 cooperative binding –Binding the first O 2 makes it easier to bind the next, and so on –Dissociating the first O 2 makes it easier to dissociate the next one »Why is Cooperativity good in Hb? Y changes very rapidly with pO 2 Lung pO 2 = 100torr, Muscle pO 2 = 20 n = 1 then Y lung = 0.79, Y muscle = 0.43 (0.36 delivered) n = 2.8 then Y lung = 0.98, Y muscle = 0.32 (0.66 delivered ) Hb is 1.8 times as efficient as Mb Hb P50 lies between lungs and muscle Log (pO 2 ) Mb n = 1.0 Hb n =2.8

–H + and CO 2 effects on Hb O 2 Binding »Bohr Effect: Increased [H + ] decreases binding Mb O 2 binding is not affected by [H + ] Contracting muscle generates H + and CO 2 This helps Hb release O 2 Deoxy-Hb binds H + stronger than oxy-Hb »The effect is mutual: high [O 2 ] causes H+ to dissociate from Hb

»CO 2 effect on Hb binding

glucose5000 glucose- 6- P83 fructose- 6- P14 fructose- 1,6- P31 dihydroxyacetone- P138 glyceraldehyde- 3- P19 1,3 bisphosphoglycerate1 2,3 bisphosphoglycerate 4000 (BPG) 3 phosphoglycerate118 2 phosphoglycerate30 phosphoenolpyruvate23 pyruvate51 lactate2900 MM From S. Minakami and H. Yoshikawa. Biochem.Biophys.Res.Comm. 18(1965):345. Concentrations of glycolytic intermediates in erthyrocytes –Organic Phosphate Regulation of Hb O 2 binding »BPG is an organic phosphate 2,3 bisphospho-glycerate (BPG)

o2O 2 PRESSURE (torr) SATURATION No BPG With BPG  BPG Lowers the binding affinity of Hb for O 2 [BPG] = 0, Hb P50 = 1 torr [BPG] = 4000  M, Hb P 50 = 26 torr Without BPG, Hb couldn’t unload O 2 in cells

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

»BPG ensures that O 2 can be unloaded at the peripheral tissues by decreasing the affinity of Hb for O 2 about 26 fold increasing O 2, 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 O 2 BPG regulates O 2 binding between Hb types This allows transfer of O 2 from mother to child This explains the need for multiple Hb types If [BPG] = 0, HbA > HbF for O 2 binding HbF has neutral Serine in place of HbA His o2O 2 PRESSURE (torr) SATURATION HbA HbF O 2 flows from mom to baby !

–Structural Basis for Cooperativity »Interactions between subunits A dissociated Hb subunit binds O 2 like Mb A  4 tetramer binds O 2 like Mb Cooperativity must involve subunit interactions

»OxyHb and DeoxyHb have very different quaternary structures OxyHb is more compact (  Fe —  Fe changes from 40 to 33Å) When O 2 binds,  —  contacts change as H-bonds are adjusted Electrostatic bonds (Salt Links) also change: OxyHb the CO 2 - termini can freely rotate, DeoxyHb CO 2 - termini salt linked DeoxyHb has T-form (“taut”) OxyHb has R-form (“relaxed”)

»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  interface –T-form becomes R-form –R-form has greater O 2 affinity –Cooperativity set in motion BPG stabilizes deoxyHb T-form by creating more contacts O 2 binding to Hb causes dissociation of BPG because the cavity gets too small. This favors the R-form as well.

–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 O 2 binding, but this does not change the form of other subunits Conformational changes enhance O 2 binding at the next subunit, but O 2 must bind each subunit before it switches to R

»Concerted Model Whole protein changes from T to R form upon initial O 2 binding O 2 has higher affinity for the unbound R subunits This explains cooperativity »Actual: mix of the two models. Hb is predominantly T until ~2 O 2 molecules are bound, then it goes all R. o2O 2 PRESSURE (torr) SATURATION myoglobin hemoglobin

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

the Val for Glu mutation makes deoxy- HbS insoluble Sickle-cell anemia In deoxy-HbS,  -subunit residues Phe 85 and Leu 88 reside at the surface and bond with Val 6 on another  -subunit. This leads to the formation of long filamentous strands of deoxy-HbS and to the sickling deformation of the erthyrocytes In oxy-HbS,  -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.

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 O 2 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 + ], [CO 2 ] 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 Hemoglobin : a portrait of a soluble protein with 4° stucture A SUMMARY