Hemoglobin Structure –Hemoglobin is tetrameric O 2 transport protein found in vertebrate...
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Transcript of Hemoglobin Structure –Hemoglobin is tetrameric O 2 transport protein found in vertebrate...
• 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