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Chap. 5B Protein Function
Topics
• Reversible Binding of a Protein to a Ligand:
Oxygen-binding Proteins
• The heme group
• Myoglobin
• Hemoglobin
• Sickle-cell Anemia
Fig. 5-19. Normal (a) and sickle-cell
anemia (b) erythrocytes.
Erythrocytes, Hb, and Oxygen Transport
Nearly all of the O2 carried by whole blood in animals is bound
and transported by hemoglobin (Hb) in erythrocytes (red blood
cells). Erythrocytes lack a nucleus and organelles such as
mitochondria and have a half-life in the bloodstream of 120 days.
Their main function is to carry Hb, which is dissolved in the
cytosol at a very high concentration of ~34% by weight.
Erythrocyte Hb is about 96% saturated with O2 in the lungs. In
venous blood, erythrocyte Hb is 64% saturated. Thus about a
third of the O2 carried by erythrocytes is released to peripheral
tissues during one pass through the circulation. The binding of O2
to Hb occurs with less affinity than to Mb. Thus, Hb is more
suited for delivery of O2 to tissues. Furthermore, ligands such as
H+ and CO2, which are in relatively higher concentrations in the
peripheral circulation, improve the release of O2 from Hb and its
delivery to peripheral tissues.
Hemoglobin Structure (I)
Hb (Mr 64,500) is a tetrameric protein that is roughly spherical
and has a diameter of about 5.5 nm. Each subunit contains a
bound heme group that has the same structure as the heme of
myoglobin. There are two types of chains in adult Hb, two 
chains (141 residues each), and two ß chains (146 residues each).
Although fewer than half of the amino acid residues in the two
chains are identical, the structures of the  and ß chains are
nearly superimposable. Furthermore their structures are very
similar to that of myoglobin
(Fig. 5-6), which has a more
dissimilar amino acid sequence.
Note that the helix naming
conventions are the same for
the Mb and Hb chains, except
that the  subunit lacks the
short D helix. Like Mb, the
heme-binding pocket is made
up of the E and F helices in
the  and ß chains of Hb.
Hemoglobin Structure (II)
Despite the fact that the amino acid
sequences of the three polypeptides are
identical at only 27 positions (Fig. 5-7),
the structures of the myoglobin and Hb
chains are nearly identical. This
illustrates how similar amino acids can
form similar secondary and tertiary
structures.
Hemoglobin Structure (III)
Strong interactions involving more than 30 residues occur within the
1ß1 and 2ß2 protomers of the Hb tetramer (Fig. 5-8). Fewer
interactions involving 19 amino acids connect the 1-ß2 and 2-ß1
contact interfaces. Hydrophobic interactions predominate at all
interfaces, but there are also hydrogen bonds and a few ion pairs
that provide connections between subunits. When O2 binds to Hb,
contacts within the protomers change little. However, there are
large changes between the 1-ß2 and 2-ß1 interfaces, with several
ion pairs being broken.
Structural Changes in Hb on O2 Binding (I)
X-ray diffraction studies revealed that Hb transitions between
two major conformational states depending on whether oxygen is
present or not. These are the R state (relaxed) and T state
(tense). Although oxygen binds to Hb in either structural state,
O2 binding stabilizes and favors the R state. In the absence of
O2, the T state is more stable and is the predominant
conformation. The two states are also referred to as oxy- and
deoxyhemoglobin, respectively.
The terms tense and relaxed
were originally coined because
the T state is stabilized by a
greater number of ion pairs at
the 1-ß2 and 2-ß1 interfaces
than are present in the R
state. Some of these T state
interactions are shown in Fig.
5-9 a, and more are shown in
Fig. 5-9 b (next slide).
Structural Changes in Hb on O2 Binding (II)
One important contact occurring between the 1-ß2 and 2-ß1
interfaces in the T state is the His HC3 to Lys C5 -carboxyl
group/-amino group interaction (Fig. 5-9 a&b). His HC3 is the Cterminal residue in the ß subunits. His HC3 of the ß subunits also
forms an interaction between its side-chain and Asp FG1 within
the ß subunits. As shown in the next slide, the His HC3 to Lys C5
-carboxyl group/-amino group interaction is broken on binding of
O2.
Structural Changes in Hb on O2 Binding (III)
The binding of O2 to a Hb subunit in the T state triggers a change
in conformation to the R state. When the entire protein undergoes
this transition, the structures of the individual subunits change
little. However, the aß protomers slide past each other and rotate,
narrowing the pocket between the ß subunits (Fig. 5-10). A number
of contacts that stabilize the T state are broken and new ones are
formed. The His HC3 to Lys C5 contact is one of the ones that is
broken in the T  R transition. As shown in Fig. 5-10, the His
HC3 residues at the C-termini of the ß subunits rotate in the R
state towards the center of the molecule where they are no longer
involved in ionic interactions to the  subunits. The size of the
pocket between the ß chains also narrows as as a result of the T 
R transition.
Structural Changes in Hb on O2 Binding (IV)
The structural changes that occur at the 1-ß2 and 2-ß1
interfaces on O2 binding are ultimately triggered by movement of
the proximal histidines, His F8, of the four subunits when O2
binds to the heme groups (Fig. 5-11). In the T state, the
porphyrin is slightly puckered, causing the heme iron to protrude
on the side where the proximal histidine is located. The binding of
O2 causes the heme to assume a more planar conformation,
shifting the position of the proximal His and the attached F helix
in the R state. These movements lead to changes in the ion pairs
at the 1-ß2 and 2-ß1 interfaces.
The Hb O2 Binding Curve
Hb must bind O2 efficiently in the lungs, where pO2 is about 13.3
kPa, and release O2 in the tissues, where pO2 is about 4 kPa.
Myoglobin, or any protein that binds O2 with a hyperbolic binding
curve, would be ill-suited to this function. For example, a protein
such as myoglobin that binds O2 with high affinity would bind it
efficiently in the lungs, but would not release much of it in the
tissues. On the other hand if the protein bound O2 with sufficiently
low affinity to release it in the tissues, it would not pick up much
O2 in the lungs. Hb solves the problem by undergoing a structural
transition from a low-affinity T state to a high-affinity R state as
more O2 molecules are bound.
As a result, Hb has a hybrid S-shaped,
or sigmoid, binding curve for O2 (Fig. 512). A sigmoid curve can be viewed as a
hybrid curve reflecting the transition
between low- and high-affinity structural
states in Hb on O2 binding. Sigmoidal
ligand binding curves are indicative of
cooperative binding of a ligand to a
protein. In cooperative binding, the
binding of one ligand to a protein alters
its binding affinity for subsequent
ligands.
Cooperative Ligand Binding to Allosteric
Proteins (I)
Proteins such as Hb, in which structural transitions occur due to
ligand binding and affect ligand-binding affinity, are called
allosteric (Greek for “other shape”) proteins. The ligands
themselves are broadly called structural modulators. Modulators
for allosteric proteins can be either inhibitors or activators.
When the normal ligand and modulator are identical as they are
with Hb (i.e., O2), the modulator is termed homotropic. If the
modulator is a molecule other than the normal ligand, the
interaction is heterotropic. Some proteins can have two or more
modulators, and therefore can have both homotropic and
heterotropic interactions. As a result of allosteric transitions in
the packing of Hb subunits, Hb binds O2 cooperatively.
Cooperative binding confers a much more sensitive response to
ligand concentration. It is important to the function of many
multisubunit proteins. The principle of allostery extends readily
to regulatory enzymes, as covered in Chap. 6.
Cooperative Ligand Binding to Allosteric
Proteins (II)
Cooperative conformational changes
generally depend on variations in
the structural stability of
different parts of a protein. The
binding sites of an allosteric
protein typically consist of stable
segments in proximity to relatively
unstable segments, with the later
capable of frequent changes in
conformation or intrinsic disorder
(Fig. 5-13). When a ligand binds,
the moving parts of the protein’s
binding site may be stabilized in a
particular conformation, affecting
the conformation of adjacent
polypeptide subunits. The
conformational changes that occur
as the ligand binds can convert the
protein from a low- to a highaffinity state, which is a form of
induced fit. The degree of
cooperativity in ligand binding to
an allosteric protein can be
calculated using the Hill equation
(not covered).
Models for Cooperative O2 Binding to Hb (I)
Although much is known about the structures of the T and R states
of Hb, the mechanism by which this structural transition occurs on
sequential ligand binding still is unsolved. Two principle models for
cooperative binding of O2 to Hb (and ligand binding to any allosteric
protein) are widely used to explain structural transitions. In the
Monod, Wyman, and Changeux (MWC, or concerted) model, all
subunits of Hb undergo the transition from one conformation to the
other simultaneously (Fig. 5-15a). No tetramer has individual
subunits in different conformations, and the two conformations are
in equilibrium. The ligand can bind to either conformation, but binds
each with different affinity. Successive binding of ligand molecules
to the low-affinity conformation (which is more stable in the
absence of ligand) makes the transition to the high-affinity
conformation more likely.
Models for Cooperative O2 Binding to Hb (II)
In the second model, known as the sequential model proposed by
Koshland (Fig. 5-15b), ligand binding can induce a change of
conformation in an individual subunit. A conformational change in
one subunit makes a similar change in a adjacent subunit, as well
as the binding of a second ligand molecule, more likely. There are
more potential intermediate states in the sequential model than in
the concerted model. The two models are not mutually exclusive:
the concerted model may be viewed as the all-or-none limiting
case of the sequential model.
Transport of H+ and CO2 by Hb (I)
Hb binds to and transports about 40%
of the total H+ and 15% to 20% of the
CO2 formed in peripheral tissues to the
lungs and kidneys. The remainder of
the H+ is absorbed by the plasma’s
bicarbonate buffer system. The
remainder of the CO2 is transported as
dissolved HCO3- and CO2. [Note that
the solubility of CO2 in the blood is
increased by the carbonic anhydrase
reaction (CO2 + H2O  H+ + HCO3-)
which occurs in erythrocytes.] The
binding of H+ and CO2 to Hb decreases
the affinity of
Hb for O2, favoring the release of O2 to the tissues where the
concentrations of these components are relatively high. Conversely,
in the capillaries of the lung, as CO2 is excreted and the blood pH
consequently rises, the affinity of Hb for O2 increases and the
protein binds more O2 for transport to the peripheral tissues. The
effect of pH and CO2 concentration on the binding and release of
O2 by Hb is known as the Bohr effect. The effect of pH on Hb O2
binding curves is shown in Fig. 5-16.
Transport of H+ and CO2 by Hb (II)
H+ binds to several amino acid side-chains in Hb. His HC3 at the
C-termini of the ß chains makes a major contribution to H+ binding.
When His HC3 is protonated, it forms a salt bridge to Asp FG1
that helps stabilize deoxyhemoglobin in the T state. The ion pair
stabilizes the protonated form of His HC3, giving this residue an
abnormally high pKa in the T state. The proton is released from His
HC3 in the lungs (pH 7.6) when the binding of O2 to Hb drives the
conformation to the R state. Carbon dioxide binds as a carbamate
group to the -amino groups at the N-terminal ends of each globin
chain, forming carbaminohemoglobin (see below). This reaction
produces H+, contributing to the Bohr effect. The bound
carbamates also form additional salt bridges that help to stabilize
the T state and promote the release of O2. When Hb reaches the
lungs, the high O2 concentration promotes binding of O2 and the
release of bound CO2.
BPG Regulation of O2 Binding to Hb (I)
2,3-bisphosphoglycerate (BPG), is a negative heterotropic
modulator of O2 binding to Hb. BPG is important in physiological
adaptation to the lower pO2 values that are present at high
elevations. As discussed below, BPG binds to and favors the
formation of deoxyhemoglobin (the T state). This facilitates O2
delivery to tissues. Synthesis of BPB in erythrocytes increases
when an individual moves to higher elevation.
BPG Regulation of O2 Binding to Hb (II)
As shown in Fig. 5-17, the BPG
concentration in normal human blood is
about 5 mM at sea level and about 8 mM
at high elevations (e.g., 4,500 m). Thus,
at sea level, Hb is nearly saturated with
O2 in the lungs, but is just over 60%
saturated in the tissues, so the amount
of O2 released in the tissues is about
38% of the maximum carried in the
blood. At high elevations, O2 delivery
declines about one-fourth to 30% of
maximum in the absence of increased BPG
production. When BPG synthesis increases
after time spent at high elevation, the
affinity of Hb for O2 decreases, so
approximately 37% of what can be
carried is again delivered to the tissues.
Note that Hb binds very tightly to O2 in
the absence of BPG (curve at left).
BPG Regulation of O2 Binding to Hb (III)
One molecule of BPG binds to Hb in the cavity between the ß
subunits that is present in the T state (Fig. 5-18a). This cavity is
lined with positively charged amino acid R groups that interact with
the negatively charged groups of BPG. The binding site is absent in
the R state, precluding BPG binding (Fig. 5-18b). BPG lowers Hb’s
affinity for O2 by stabilizing the T state. Thus, BPG favors the
release of O2 from Hb and increases its delivery to peripheral
tissues. Interestingly, BPG does not bind to fetal Hb which has a
subunit composition of 22. (Note that the  subunit is expressed
instead of the ß subunit during the last two trimesters of fetal
life.) The  subunit lacks the basic residues that are needed for
BPG binding to Hb. For this reason, fetal Hb has a higher affinity
for O2 than maternal Hb, and transfer of O2 from the maternal to
the fetal circulation in the placenta is facilitated.
Carbon Monoxide Poisoning
Carbon monoxide (CO) is a colorless, odorless gas that is
responsible for more than half of the annual poisoning deaths
worldwide. CO has a 250-fold greater affinity for Hb than O2,
and exposure to CO reduces the oxygen-carrying capacity of the
blood. Symptoms of CO poisoning depend on the percent of total
Hb that is bound to the gas. At 10% bound, symptoms are rarely
observed. At 20% to 30%, the individual will experience a severe
headache accompanied by nausea, dizziness, confusion, and visual
disturbances. At CO Hb levels of 30% to 50%, neurological
symptoms become more severe, and at levels near 50%, the
individual loses consciousness and can fall into a coma. Respiratory
failure may follow. Death normally occurs rapidly when CO Hb
levels rise above 60%. CO is a component of tobacco smoke, and
chain-smokers can have CO Hb levels of 15%. Thus, smoking
predisposes individuals to the effects of CO poisoning. In addition,
the fetus of a pregnant woman is highly susceptible because fetal
Hb has a higher affinity for CO than maternal Hb. As CO binds to
one or two subunits of a Hb tetramer, the affinity of the
remaining subunits for O2 is increased substantially. Thus a Hb
tetramer with two bound CO molecules can efficiently bind O2 in
the lungs; however it releases O2 very inefficiently in the
peripheral tissues. Poisoning is further exacerbated by the binding
of CO to cytochromes of the mitochondrial electron transport
chain.
Sickle-cell Anemia (I)
Sickle-cell anemia is a hereditary human disease that is caused by
the expression of an altered form of Hb known as Hb S in
erythrocytes. The disorder is inherited as an autosomal recessive
trait and requires that the individual have two copies of the Hb S
allele. Individuals with the disease produce variably shaped
erythrocytes (Fig. 5-19) that are prone to lysis and can clog the
microvasculature. Abnormal erythrocytes only form when Hb S
undergoes deoxygenation in capillaries. The sickled erythrocytes
are fragile and rupture easily.
This results in a significant
anemia wherein the Hb content
of the blood is only about 50%
of normal. In addition,
individuals experience painful
episodes due to the clogging of
capillaries. These episodes often
are brought on by physical
exertion. Ultimately, impaired
organ function caused by poor
oxygen delivery can cause death,
typically in childhood.
Sickle-cell Anemia (II)
The abnormal structure of sickle-cell
erythrocytes is caused by subtle differences
in the conformation of sickle-cell Hb (Hb S)
(Fig. 5-20). Namely, the substitution of Val
for Glu at position 6 of the ß chains creates
a sticky hydrophobic contact point at this
site which is located on the outer surface of
the Hb S molecules. These sticky spots
cause deoxyhemoglobin S molecules to
associate abnormally with each other,
forming long fibrous aggregates (Fig. 5-20
b). These fibers distort the shape of
erythrocytes that contain them. Individuals
who inherit only one Hb S allele, and are
thus heterozygous, experience a milder
medical condition known as sickle-cell trait.
Only about 1% of their erythrocytes become
sickled on deoxygenation. These individuals
may live completely normal lives if they avoid
vigorous exercise and other stresses on the
circulatory system. The Hb S allele is most
prevalent in people of African descent.
Heterozygotes who express a single copy of
the Hb S allele have some protection against
malaria. For this reason, the allele is
prevalent in this population group.