• 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.
In 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 Y  # occupied sites  0  1
total # of sites
» 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
•Shapes of the curves
•Mb has the shape of hyperbola
MbO2
K
K
[Mb][O 2 ]
[MbO 2 ]
Y
Mb + O2
Y
[MbO 2 ]
[MbO 2 ]  [Mb]
[O 2 ]
pO 2

[O 2 ]  [K] pO 2  P50
•Hb has sigmoidal shape
K
Hb(O2)n
Hb + nO2
 pO 2 
(pO 2 )
Y

Y

 
n
n
(pO 2 )  (P50 )
1 - Y  P50 
n
n
myoglobin
1
SATURATION
hemoglobin
0
10
50
o2O2 PRESSURE (torr)
» Hill Plots Tell Us About Cooperativity
 Y 
log 
  n log pO2 - n log P50
 1- Y 
 Y 
log 

 1- Y 
Mb
n = 1.0
Hb
n =2.8
Log (pO2)
• 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
– 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
–Organic Phosphate Regulation of Hb O2 binding
»BPG is an organic phosphate
Concentrations of glycolytic intermediates in erthyrocytes
mM
5000
83
14
31
138
19
1
4000 (BPG)
118
30
23
51
2900
glucose
glucose- 6- P
fructose- 6- P
fructose- 1,6- P
dihydroxyacetone- P
glyceraldehyde- 3- P
1,3 bisphosphoglycerate
2,3 bisphosphoglycerate
3 phosphoglycerate
2 phosphoglycerate
phosphoenolpyruvate
pyruvate
lactate
From S. Minakami and H. Yoshikawa. Biochem.Biophys.Res.Comm. 18(1965):345.
-
O
O
C
H C O
CH2
O
P O
-
O-
O
-
O
P O
O
-
2,3 bisphospho-glycerate (BPG)
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)
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
1
SATURATION
HbF
HbA
O2 flows from
mom to baby !
0
10
50
o2O2 PRESSURE (torr)
– 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
» 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”)
» Changes at the Heme initiate structure switch
• DeoxyHb has Fe 0.3Å out of plane
N
N
Fe2+
N
N
N
• OxyHb has Fe in plane of porphyrin
O
O
N
2+ N
Fe
N
N
N
• 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.
– 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
O2
O2
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
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
» 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)
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!
Sickle-cell anemia
• the Val for Glu mutation makes deoxyHbS insoluble
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
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.
Hemoglobin : a portrait of a soluble
protein with 4° stucture
A 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