Part II => PROTEINS and ENZYMES
§2.4 PROTEIN FUNCTION
§2.4a Oxygen Transport
Section 2.4a:
Oxygen Transport
Synopsis 2.4a
- Myoglobin (Mb) and hemoglobin (Hb) are two of the best characterized
members of the heme-based family of oxygen-binding proteins
- While Hb carries oxygen (oxygen carrier) in the bloodstream to make it
available to all tissues, Mb largely serves to store oxygen (oxygen
reservoir/facilitator) so that it can be quickly made available to the
muscle tissue during high oxygen demand (eg strenuous exercise)
- Mb exhibits a hyperbolic oxygen-binding curve (a non-cooperative
response)—such behavior is ideally suited to its role as an oxygen
reservoir!
- By virtue of its ability to undergo a conformational change, Hb displays
a sigmoidal oxygen-binding curve (a cooperative response)—such
behavior is ideally suited to its role as an oxygen carrier!
- The ability of allosteric factors such as pH (Bohr effect) and 2,3bisphosphoglycerate (BPG) to modulate oxygen binding to Hb is of
immense physiological significance
Oxygen Transporters: Myoglobin (Mb) and Hemoglobin (Hb)
PDBID 1MBO
PDBID 1GZX
Myoglobin
(monomer)
myo  “of muscle”
heme  “of blood”
globin  “globe(sphere)-like”
Hemoglobin
(α2β2tetramer)
- Oxygen is essential for most living organisms as it is required for the breakdown of food to release energy in
a phenomenon referred to as “respiration”
- In addition to microbes, more complex organisms such as sponges and jellyfish also rely on direct diffusion
of oxygen from the environment without the need for organs such as lungs/heart/blood—no brains either!!
- However—in higher-order organisms such as vertebrates, the direct diffusion of oxygen to specific tissues is
not sufficient to maintain life
- Vertebrates rely on Hb (the mainstay of red blood cells) to efficiently transport oxygen from lungs to other
tissues in the body
- On the other hand, Mb (predominantly located in the muscle tissue) binds and relays (facilitates) oxygen
from the capillaries to muscle cells—additionally, Mb also stores oxygen so that it can be made available in
drastic times such as during exercise and diving (particularly in aquatic animals)
- Both Mb and Hb harbor a prosthetic group (a type of cofactor) called “heme”—which is buried in a deep
hydrophobic cleft within each globin and serves as the oxygen-carrier
Evolutionary Relationship Between Mb and Hb:
Sequence Alignment
Mb
HbA
HbB
MG-LSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASEDLKKHGATVLTALG
MV-LSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHF-DLS--H---GSAQVKGHGKKVADALT
MVHLTPEEKSAVTALWGKV--NVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFS
Mb
HbA
HbB
GILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKHPGDFGADAQGAMNKALELFRKDMASNYKE
NAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYRDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH-
- The α (HbA) and β (HbB) chains of Hb are
evolutionarily related to each other and to Mb
polypeptide chain
- Out of nearly 150 residues, only 28 are IDENTICAL—
the three polypeptide chains of Mb and Hb only
share a meager 18% amino acid identity!
- Yet, all three polypeptide chains of Mb and Hb
share a remarkably similar 3D structure—with each
polypeptide comprised of eight helices (designated
A-H) wound around each other into a globular fold
with an equally remarkable shared function
- The only difference being that while Mb adopts a
monomeric conformation, Hb is a tetramer (α2β2)
C
D
COOH
H 2N
Myoglobin and Hemoglobin: Heme Cofactor
- In a manner akin to chlorophyll (the light-absorbing pigment
critical for photosynthesis in plants), heme is a porphyrin
derivative containing four pyrrole groups (designated A-D)
linked together via methine bridges
- At the heart of heme lies the iron divalent metal ion (Fe2+)—
hereinafter referred to as Fe(II)
- The porphyrin ring of heme is stabilized via numerous van
der Waals contacts with specific sidechain moieties located
in the globin
ε7
(7th residue
of helix E)
Pyrrole
Oxygen
Distal Face
- Fe(II) is stabilized via coordination by five ligands: one N
atom from each of the four pyrrole rings and an additional
N atom courtesy of a highly conserved histidine (φ8) residue
located on the proximal face of heme
- Oxygen (O2) binds as a sixth ligand to Fe(II) in a reversible
manner—bound under high concentrations of oxygen
(arterial blood) and released in regions with low oxygen
(venous blood)
- Additionally, an highly conserved histidine (ε7) located on
the distal face of heme “sandwiches” the oxygen molecule
via hydrogen bonding—this interaction caps the oxygen
binding “crevice” so as to keep larger molecules out
Proximal Face
φ8
(8th residue
of helix F)
Myoglobin
Myoglobin: Binding of oxygen
ε7 and φ8 histidine residues in human Mb and Hb
Globin
Length
ε7
φ8
Mb
154*
H65*
H94*
Hbα
142*
H59*
H88*
Hbβ
147*
H64*
H93*
Myoglobin in complex
with heme and oxygen
*Including the N-terminal MET residue cleaved
via post-translational modification (PTM)
- φ8 histidine serves as one of the five
N ligands of heme (note the planar
conformation of the porphyrin ring)
ε7
O2
Heme
- ε7 histidine hydrogen bonds to oxygen
- In addition to oxygen (O2), heme also binds to
toxic gases such as carbon monoxide (CO), nitric
oxide (NO) and hydrogen sulfide (H2S) but with
much higher affinity—thereby accounting for
their toxicity
- But, there is no obvious route for the entry of O2
(as well as other toxic gases) to coordinate with
heme! So what gives?! Enter allostery.
φ8
PDBID 1A6M
Myoglobin: Allosteric binding of oxygen
Note the subtle planarization
of the porphyrin ring of
heme upon oxygen binding
- Static images do little justice to the remarkable
flexibility that the proteins harbor—various
regions of protein are under constant
fluctuations at the very minimum
- Such protein motions (or dynamics) are
central to protein function in a manner
akin to human body (flexible) versus a
robot (rigid)!
- Indeed, many proteins undergo a varying
degree of conformational change upon
binding to their ligands (small molecules
or other protein partners)
ε7
O2
Heme
Fe(II)
φ8
- Such conformational change (involving changes in
either structure and/or dynamics) in response to
ligand binding has come to be known as “allostery”
- Allostery plays a key role in the ability of Mb to bind
O2—albeit in a highly subtle manner—ie the binding
of O2 induces a conformational change in Mb
Animation generated via
morphing of deoxyMb (PDBID
1A6N) and oxyMb (PDBID 1A6M)
Myoglobin: Hyperbolic binding of oxygen
- The reversible binding of oxygen (O2) to myoglobin (Mb) can be described as:
Mb + O2 <==> MbO2
When [Mb] = [MbO2]:
=> K = [O2]
ie K is the [ligand] @ which
the protein is half-saturated!
- The equilibrium dissociation constant (K) is given by:
K = [Mb].[O2] / [MbO2]
[1]
⇒ [MbO2] = [Mb].[O2] / K
[2]
where [Mb] and [MbO2] are respectively the concentrations of
free and oxygen-bound myoglobin @ equilibrium
-
Thus, the fractional saturation (Y) of myoglobin with oxygen is:
Y = [MbO2] / {[Mb]+[MbO2]}
[3]
-
Combining Eqs [2] and [3] and factoring out the [Mb]/K term yields:
Y = {[Mb] .[O2]/K} / {[Mb] + {[Mb].[O2]/K}} [4]
⇒ Y = {{[Mb]/K} [O2]} / {{[Mb]/K} {K+[O2]}}
[5]
⇒ Y = [O2] / {K+[O2]}
[6]
-
Since oxygen is a gas, its concentration can be expressed by its partial pressure to be pO2—now,
rewriting Eq [6] in terms of the partial pressure of oxygen gives:
Y = pO2 / (K + pO2)
[7]
-
Eq [7] is the equation of a hyperbola—a plot of pO2 versus Y will almost linearly increase at lower
concentrations of oxygen and eventually plateau out at saturating oxygen!
-
Such hyperbolic behavior is characteristic of many biological phenomena such as drug-receptor
interactions and substrate binding to enzymes (Michaelis-Menten kinetics)
Myoglobin: Hyperbolic response curve
1 Torr = 1/760 atm = 133 Pa
pO2 (21% air) = 160 Torr
KO2 = 3 Torr = 50*KCO
CO binds to Mb by 50-fold
stronger than O2!
T in “Torr” must be capitalized
as a unit—named after
Torricelli, the inventor of
barometer
Y
Saturation of Mb in response
to oxygen concentration
Evangelista Torricelli
(1608-1647)
pO2 / Torr
- As shown earlier, the degree of saturation of Mb in response to oxygen concentration is given by:
Y = pO2 / (K + pO2)
- But, K is the concentration of oxygen (pO2) at which Mb is half-saturated—thus, from the hyperbolic
curve shown:
K = 3 Torr @ 50% Mb saturation—why is CO so toxic?!
- The fact that the physiological range of pO2 in the blood is 30-100 Torr is telling—lower the K, higher the
ligand binding affinity!!!!
- This implies that Mb is almost always saturated under physiological conditions, thereby accounting for its
ability to not only serve as an efficient oxygen reservoir but also being able to relay oxygen from a region
of high (arterial blood) to a region of low concentration (venous blood)
Hemoglobin
Hemoglobin: Binding of oxygen
ε7 and φ8 histidine residues in human Mb and Hb
Globin
Length
ε7
φ8
Mb
154*
H65*
H94*
Hbα
142*
H59*
H88*
Hbβ
147*
H64*
H93*
Hemoglobin in complex
with heme and oxygen
(Only α monomer shown)
*Including the N-terminal MET residue cleaved
via post-translational modification (PTM)
ε7
O2
- φ8 histidine serves as one of the five N
ligands of heme (note the planar
conformation of the porphyrin ring)
Heme
Fe(II)
φ8
- ε7 histidine hydrogen bonds to oxygen
- As noted earlier for Mb, there is no obvious
route for the entry of O2 to coordinate with
heme! So what does hemoglobin do?
PDBID 1HHO
Hemoglobin Allostery:
Deoxygenated Conformation (deoxyHb)
deoxyHb
(Only α monomer shown)
Valence Electrons
Electrons in the outer shells
directly involved in bonding
between adjacent atoms
ε7
In the deoxyHb conformation:
- Porphyrin ring of heme is non-planar
- Fe(II) atom is pulled out of the porphyrin
ring toward φ8 due to repulsion between
the valence electrons in heme and φ8
- Under such electronic and conformational
properties of heme and Fe(II), deoxyHb
predominantly absorbs visible light at
wavelengths between 500-600nm—ie
venous blood appears dark red!
Heme
Fe(II)
φ8
PDBID 2HHB
In the oxyHb conformation:
Hemoglobin Allostery:
Oxygenated Conformation (oxyHb)
- Binding of O2 as a sixth ligand to Fe(II) overcomes the repulsion
between the valence electrons in heme and φ8 so as to allow
Fe(II) to move into the center of the porphyrin ring, thereby
resulting in the planarization of the porphyrin ring
oxyHb
(Only α monomer shown)
- The change in the electronic and conformational properties
of Fe(II) and heme upon planarization of its porphyrin ring
enables oxyHb to predominantly absorb visible light at all
wavelengths but above 600nm—ie arterial blood appears
bright red!
- The planarization of porphyrin ring also draws φ8
upwards and closer to heme—such movement of φ8
subsequently results in the conformational shift of
numerous other residues so as to adopt the oxyHb state
- The resulting intra-subunit conformational change from
deoxyHb to oxyHb state (triggered by O2 binding) ultimately
culminates in the likewise conformational re-arrangement of
the other three subunits within the α2β2 tetramer to oxyHb
(without the intervention of O2!)—thereby enabling them to
bind O2 much more easily than the first monomer
- The conformational change in α2β2 tetramer in response to
oxygen thus enhances its ability to rapidly load additional
molecules of O2—a phenomenon referred to as “cooperative
binding”—a form of allosteric regulation
ε7
O2
Heme
Fe(II)
φ8
PDBID 1HHO
Hemoglobin Allostery: Intra-Subunit (α)
deoxyHb <=> oxyHb
(Only α monomer shown)
Unlike Mb, Hb undergoes a
substantial intra-subunit
conformational change
upon oxygen binding
ε7
O2
Heme
Fe(II)
φ8
Note the planarization of the
porphyrin ring of heme upon
oxygen binding
Animation generated via
morphing of deoxyHb (PDBID
2HHB) and oxyHb (PDBID 1HHO)
Hemoglobin Allostery: Inter-Subunit (α2β2)
- O2 binding to heme within α2β2 tetramer does not
occur simultaneously!
- Binding of O2 to one of the four heme groups
triggers a conformational transition of its
deoxyHb component to oxyHb state
deoxyHb <=> oxyHb
(α2β2 tetramer shown)
β2
α1
- Such intra-subunit conformational transition
causes the other three neighboring subunits
to quickly adopt the oxyHb conformation,
thereby facilitating O2 binding
Central
Cavity
- Thus, the binding of first O2 molecule
is slow at first but the subsequent O2
molecules are loaded rapidly by virtue
of the ability of α2β2 tetramer to undergo
a change in quaternary structure—note
the narrowing of the central cavity due to
the rotation of each αβ protomer by about
15° relative to each other
O2
- In thermodynamic terms, the α2β2 subunits
of Hb bind O2 in a cooperative manner
- How can we thermodynamically rationalize such
binding cooperativity?
- But, first, the physics of hemoglobin color
(spectroscopy)!
Fe(II)
Heme
α2
Animation generated via
morphing of deoxyHb (PDBID
2HHB) and oxyHb (PDBID 1HHO)
β1
Hemoglobin Color: Absorption Spectra
Visible Spectrum
oxyHb
oxyHb
deoxyHb
Bright Red
oxyHb:
λabsorb < 600nm
λreflect > 600nm
Red
deoxyHb
Blue
deoxyHb:
500nm < λabsorb < 600nm
λreflect < 500nm
λreflect > 600nm
Blue + Red = Dark Red/Purple!
Red
Dark Red
Hemoglobin Color: Why Does Venous Blood Appear Blue?!
I ∝ 1/λ4
Blue  1/(400)4 = 4*10-11
Red  1/(700)4 = 4*10-12
Why is the sky blue?
Because of the reflection of light by the sea! True or false?
- White light is made up of visible spectrum with wavelength roughly stretching from about 350-750nm
- Three primary components of white light are blue (350-500nm), green (500-600nm) and red (600-750nm)
- Particles in the atmosphere scatter white light from the sun (according to Rayleigh scattering—particles
much smaller than the wavelength!) on its way to us in a wavelength-dependent manner—the intensity
(I) of scattered light is inversely proportional to the wavelength (λ) raised to the 4th (quartic)
exponent/power—simply put, I ∝ 1/λ4
- Thus, blue light (shorter λ) is scattered by an order of magnitude greater than red light (longer λ) such
that the blue light is radiated in all directions making the sky appear blue from the earth’s surface
- For the same reason, the blue light component of deoxyHb is much more scattered as it travels through
the skin than the red component, making venous blood appear BLUE through the skin—but, inside the
veins, it is DARK RED!
O2 Binding to Hemoglobin: Hill Equation
- The reversible and cooperative binding of oxygen (O2) to hemoglobin (Hb) was first
described by Hill in 1910 via a single step:
Hb + nO2 <==> Hb(O2)n
where n is the number of O2 molecules bound—but, more commonly, referred to as
the Hill coefficient (or degree of cooperativity)
- The above model assumes simultaneous (rather than sequential) binding of n molecules
of O2 to Hb—thermodynamically valid but not how Hb binds O2 in kinetic terms!
- The overall equilibrium dissociation constant (K) is given by:
Kn = {[Hb].[O2]n} / [Hb(O2)n]
⇒
[Hb(O2)n] = {[Hb].[O2]n} / Kn
where [Hb] and [Hb(O2)n] are respectively the concentrations of
free and oxygen-bound hemoglobin
[1]
[2]
Archibald Hill
(1886-1977)
- Thus, the fractional saturation (Y) of hemoglobin with oxygen is:
Y = [Hb(O2)n] / {[Hb]+[Hb(O2)n]}
[3]
- Combining Eqs [2] and [3] and factoring out the [Hb]/Kn term yields:
Y = {[Hb].[O2]n/Kn} / {[Hb] + {[Hb].[O2]n/Kn}}
⇒
Y = {{[Hb]/Kn} [O2]n} / {{[Hb]/Kn} {Kn + [O2]n}}
⇒
Y = [O2]n / {Kn + [O2]n}
[4]
[5]
[6]
- Since oxygen is a gas, its concentration can be expressed by its partial pressure to be pO2—now, rewriting Eq [6] in
terms of the partial pressure of oxygen gives:
[7]
Y = (pO2)n / {Kn + (pO2)n}
- Eq [7] is the Hill Equation describing the binding of oxygen to hemoglobin (n > 1) in a cooperative manner (sigmoidal
response curve)—a plot of pO2 versus Y progresses slowly at the beginning and then rapidly accelerates (due to
cooperative binding) before plateauing out at saturating oxygen so as to generate an S-shaped curve!
O2 Binding to Hemoglobin: Sigmoidal response curve
1 Torr = 1/760 atm = 133 Pa
pO2 (21% air) = 160 Torr
Hypothetical
hyperbolic curve
for HB (n=1)
Y
KO2(Hb) = 30 Torr = 200*KCO
KO2(Mb) = 3 Torr = 50*KCO
CO binds to Hb by 200-fold
stronger than O2!
pO2 / Torr
- As shown earlier, the degree of saturation of Hb in response to oxygen concentration is given by:
Y = (pO2)n / {Kn + (pO2)n}
- But, K is the concentration of oxygen (pO2) at which Hb is half-saturated—thus, from the sigmoidal curve shown:
K = 30 Torr @ 50% Hb saturation
- The partial pressures of oxygen in the VENOUS and ARTERIAL blood are respectively 30 Torr and 100 Torr!
- Sigmoidal binding response enables Hb to deliver more O2 to the tissues than would be achieved via hyperbolic
binding (indicated by dashed curve)—a greater fraction of Hb becomes unloaded below the venous pressure of
oxygen (30 Torr) and, conversely, a greater fraction of Hb becomes loaded above this threshold
- Such a remarkable virtue of Hb is made possible by its ability to bind oxygen in a cooperative manner—in other
words, the α2β2 subunits bind oxygen with differential affinities—how can we tell?!
O2 Binding to Hemoglobin: Linearization of Hill Equation
- The Hill equation describing the cooperative binding of
oxygen to hemoglobin is as follows:
Y = (pO2)n / {Kn + (pO2)n}
[1]
Hill Plot
- Taking the reciprocal of both sides of Eq [1] gives:
1/Y = {Kn + (pO2)n } / (pO2)n
=>
1/Y = {Kn / (pO2)n)} + 1
=> {(1/Y) – 1} = {Kn / (pO2)n)}
=> {(1-Y)/Y} = {Kn / (pO2)n)}
[2]
- Now, inverting both sides of Eq [2] and taking logs, we have:
=>
{Y/(1-Y)} = {(pO2)n / Kn}
=> log{Y/(1-Y)} = log{(pO2)n / Kn}
=> log{Y/(1-Y)} = log(pO2)n – log(Kn)
=> log{Y/(1-Y)} = nlog(pO2) – nlogK
[3]
cf:
y = mx
+b
n
-nlogK
log(pO2)
- Thus, Eq [3] is the equation of a straight line with a slope of n
and y-intercept equal to -nlogK!
What does n mean?!
n > 1 => Positive cooperativity
n = 1 =>
No cooperativity
n < 1 => Negative cooperativity
- A plot of log(pO2) versus log{Y/(1-Y)} would yield a straight
line (or lines!) from which one can extrapolate the Hill
coefficient (n) and the dissociation constant (K)—this is the
so-called Hill Plot
What would you expect n to be
for Mb and Hb?
O2 Binding to Hemoglobin: Hill Plot
Myoglobin—Hill plot is essentially linear—indicative of
non-cooperative binding as expected (n=1 and K=3 Torr) :
-nlogK = -0.5
@ log(pO2)=0
=> logK = 0.5
=>
K = 10(0.5) = 3 Torr
A
B
C
Hemoglobin—Hill plot exhibits three distinct phases (A-C):
(A) pO2 < 10 Torr => log(pO2) < 1
All four subunits compete with each other for binding to
O2 in a non-cooperative manner (n=1 and K=100 Torr):
-nlogK = -2
@ log(pO2)=0
=> logK = 2
=>
K = 102 = 100 Torr (binding affinity of the first subunit!)
(C) pO2 > 55 Torr => log(pO2) > 1.75
Since at least three of the four subunits are already occupied, O2 binds to the
fourth subunit independently in a non-cooperative manner (n=1 and K=1 Torr):
-nlogK = 0
@ log(pO2)=0
=> logK = 0
=>
K = 100 = 1 Torr (binding affinity of the fourth subunit!)
log(pO2)
(B) 10 < pO2 < 55 Torr => 1 < log(pO2) < 1.75
In between the two extremes (regions A and C), the binding of O2 occurs in a highly cooperative manner
as evidenced by an accelerated slope (n=3 and K=30 Torr):
-nlogK = -4.5 @ log(pO2)=0
=> logK = (4.5)/3 => logK = 1.5
=>
K = 10(1.5) = 30 Torr (the average binding affinity of all four subunits)
Hemoglobin as a Model for Allosteric Regulation
Allosteric (“other site”)—
change occurs at a site other
than where the ligand binds!
allo  “other”
stereo  “solid/site”
T (-O2) <<<<=> R (-O2)
deoxyHb
oxyHb
- Allosteric regulation is a hallmark of modular and multi-subunit proteins —
ligand binding induces conformational changes within the protein so as to
facilitate subsequent binding
- According to the contemporary Equilibrium Shift model, such proteins exist in
an equilibrium between two conformations designated T (taut) and R (relaxed)
- In the context of Hb, the T and R states would respectively be the deoxyHb and
oxyHb conformations—In other words T is the unbound (free) state and R is
the state that assumes the conformation of oxyHb but in the absence of O2!
- In the absence of ligand or substrate (S), the equilibrium lies well over to the
left in the direction of T—the ligand-free state
- Upon the introduction of the ligand S, it only binds to the R state and merely
serves to shift the equilibrium in its direction—when R becomes usurped upon
binding S, more of T will undergo conformational shift to R due to cooperative
interactions
O2 Binding to Hb Is Coupled to Proton Release
- In the T (deoxyHb) state, there exists an intricate network of ion pairs and hydrogen bonding between
charged residues such as the sidechain groups of Asp94 and His146—the stabilization of imidazole
proton on His146 effectively makes it less likely to be released, thereby increasing its pK to around 8
- Upon O2 binding, the T (deoxyHb) state undergoes equilibrium shift to R (oxyHb) and such
conformational switch results in the disruption of the network of ion pairs and hydrogen bonding,
including the Asp94-His146 pair—this exposes the imidazole proton to solution, thereby decreasing
its pK value to around 6
- Consequently, under physiological conditions (pH 7.4), oxygen binding to Hb is coupled to proton
release from residues such as His146—one proton released for every two O2 molecules bound!
- Thus, a decrease in pH would favor the protonation of Hb (and hence favor deoxyHb over oxyHb),
thereby mitigating its affinity for O2—this phenomenon is called the the Bohr effect
Bohr Effect: Role of pH on O2 binding to Hb
Bohr Effect:
Lowering the pH lowers the
affinity of Hb for oxygen!
Y
KO2(pH7.4) = 30 Torr
KO2(pH7.2) = 35 Torr
@ Tissue pO2 (20 Torr):
Hb-saturation(pH7.4) = 32%
Hb-saturation(pH7.2) = 22%
pO2 / Torr
- In the tissues, the pH is lower (~7.2) due to metabolic
production of CO2 (catalyzed by carbonic anhydrase):
CO2 + H2O <==> HCO3- + H+
Christian Bohr
(1855-1911)
Effect of H+ on Hb:
H+
T (deoxyHb)
R (oxyHb)
H+
- Thus, the lower pH shifts the equilibrium in favor of the T (deoxyHb) state, thereby
facilitating the release of O2 from Hb where it is needed most—this is further added by
lower pO2 in the tissues (20 Torr)—neat!
- pH thus acts as a negative allosteric effector of Hb—and the delivery of O2 to tissues is
boosted by about 10% thanks to the Bohr effect
Bohr Effect: Role in CO2 Transport to Lungs
- In respiring muscles (pO2 = 20 Torr), the metabolic production of CO2 generates
bicarbonate and Bohr protons—which facilitate Hb to unload O2—and bicarbonate is
carried in the blood back to the lungs
- In the lungs (pO2 = 100 Torr), binding of O2 to Hb is favored—such that the released Bohr
protons combine with bicarbonate to regenerate CO2, which is subsequently exhaled
- The bound O2 is transported back to the muscles, where it can either directly diffuse into
the cells, or in the case of rapidly respiring muscles, O2 is first taken up by Mb before
being made available
BPG Effect: Role in O2 Release to Tissues
Crystal structure of deoxyHb
(α2β2) bound to BPG (red)
(-BPG)
Y
(+BPG)
KO2(+BPG) = 30 Torr
KO2(-BPG) = 10 Torr
@ Tissue pO2 (20 Torr):
Hb-saturation(+BPG) = 30%
Hb-saturation(-BPG) = 80%
pO2 / Torr
- Human blood contains high concentrations of 2,3-bisphosphoglycerate (BPG)—an indispensable
allosteric effector in that Hb would not be able to release O2 to our tissues without it!
- In a manner akin to pH, BPG also facilitates the release of O2 from Hb by virtue of its ability to bind to
the T (deoxyHb) state but not R (oxyHb)—thereby shifting the equilibrium in favor of deoxyHb
- BPG specifically docks into the rather wide central cavity of α2β2 tetramer in the T state via an
extensive network of ion pairing and hydrogen bonding
- Since the central cavity narrows upon T -> R transition (see the animation on Slide 15), the binding of
BPG to oxyHb is hindered on steric grounds
- By virtue of its ability to act as a negative allosteric effector of Hb, BPG boosts the delivery of O2 to
tissues by about 50%!
Hemoglobin Variants in Health and Disease
Variant
Topology
Mutation
Biochemical and Clinical Consequences
HbA
α2β2
Wild type
Most common variant in human population with > 95% occurrence
(K=30Torr).
HbC
α2β2
β -> E7K
Prevalent among Africans. May cause hemolytic anemia but also
confers resistance to malaria.
HbD
α2β2
β -> E122Q Prevalent among Euroasians. Protects against sickle cell anemia.
HbE
α2β2
β -> E27K Prevalent among Asians. May cause β-thalassemia
HbF
α2γ2
β2 -> γ2
Fetal hemoglobin (K=20Torr). Binds O2 with greater affinity than HbA
due to its diminished interaction with BPG.
HbH
β4
α2 -> β2
Causes α-thalassemia (production of α chains is impaired). Much
lower binding affinity for O2 than HbA.
HbO
α2β2
HbS
α2β2
β -> E122K Prevalent among Arabs and Africans. May cause moderate anemia.
β -> E7V
Prevalent among Africans. The E7V mutation induces polymerization of
deoxyHb giving erythrocytes the appearance of a “sickle”—the cause
of sickle cell anemia. Protects against malaria.
Exercise 2.4a
-
Describe the O2-binding behavior of myoglobin in terms of pO2 and K.
How is K defined?
-
Explain the structural basis for cooperative oxygen binding to hemoglobin
-
Sketch a binding curve (% bound ligand versus ligand concentration) for
cooperative and noncooperative binding
-
Describe how myoglobin and hemoglobin function in delivering O2 from
the lungs to respiring tissues
-
What is the physiological relevance of the Bohr effect and BPG?