• 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 22 [HbA2 (2% Hb) is 22]

• Early Embryo has 22 (like and)

• Later Embryo 22 to 22 = Hb F (fetus)

• and have 141 A.A.’s, slightly different

• , , and 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)

» 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 O2 reversibly

• Conserve AA’s inclue F8 His and E7 His

• Polar/Polar and Nonpolar/Nonpolar subst.

In contrast to myoglobin hemoglobin has 4°structureIn contrast to myoglobin

hemoglobin has 4°structure

• 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

10sites of # total

sites occupied #Y

o2O2 PRESSURE (torr)

SATURATION

1

010 50

myoglobin

hemoglobin

•Shapes of the curves

•Mb has the shape of hyperbola

•Hb has sigmoidal shape502

2

2

2

2

2

2

2

PpO

pO

[K]][O

][OY

[Mb]][MbO

][MbOY

][MbO

][Mb][OK

n

50

2n

50n

2

n2

P

pO

Y-1

Y

)P()(pO

)(pOY

MbO2 Mb + O2K

KHb + nO2Hb(O2)n

» 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

502 P logn - pO logn Y-1

Ylog

Log (pO2)

Y-1

Ylog

Mbn = 1.0

Hbn =2.8

– 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

» CO2 effect on Hb binding

glucose 5000glucose- 6- P 83fructose- 6- P 14fructose- 1,6- P 31dihydroxyacetone- P 138glyceraldehyde- 3- P 191,3 bisphosphoglycerate 12,3 bisphosphoglycerate 4000 (BPG)3 phosphoglycerate 1182 phosphoglycerate 30phosphoenolpyruvate 23pyruvate 51lactate 2900

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 O2 binding

»BPG is an organic phosphate

2,3 bisphospho-glycerate (BPG)

C

C

-O O

CH2

O

P

O-

O P O-

O-

O

O-O

H

o2O2 PRESSURE (torr)

SATURATION

1

010 50

No BPG

With BPG

BPG Lowers the binding affinity of Hb for O2

•[BPG] = 0, Hb P50 = 1 torr•[BPG] = 4000M, Hb P50 = 26 torr•Without BPG, Hb couldn’t unload O2 in cells

BPG acts by stabilizing deoxyHb

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 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

o2O2 PRESSURE (torr)

SATURATION

1

010 50

HbA

HbF

O2 flows frommom to baby !

– Structural Basis for Cooperativity

» Interactions between subunits

• A dissociated Hb subunit binds O2 like Mb

• A 4 tetramer binds O2 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 O2 binds, — 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”)

» 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 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.

N N

NNN

Fe2+

OO

Fe2+

N

N N

NN

– 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

O2O2 O2 O2 O2 O2 O2 O2

O2

O2

O2

O2 O2

O2

» 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.

O2O2O2

O2 O2 O2

O2

O2 O2O2

O2

O2

O2

O2 O2O2 O2 O2 O2

O2 O2

o2O2 PRESSURE (torr)

SATURATION

1

010 50

myoglobin

hemoglobin

Sickle-cell anemiaSickle-cell anemia

• A Glu normally resides at position 6 of each- subunit. In HbS this amino is mutated to Val

Glu 6

Glu 6

• the Val for Glu mutation makes deoxy-HbS insoluble -findout why!

• the Val for Glu mutation makes deoxy-HbS insoluble

Sickle-cell anemiaSickle-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 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

Hemoglobin : a portrait of a soluble protein with 4° stucture

A SUMMARY

Hemoglobin : a portrait of a soluble protein with 4° stucture

A SUMMARY