B.Sc III and M.Sc Chemistry

1. Prof. G.M.Dongare
Dept. of Chemistry,
Shri Shivaji College Of Arts, Commerce and
Science, Akola Maharashtra
Bioinorganic Chemistry
Essential and Non-essential Elements
B.Sc III VI Semester
Academic year 2021-22

2. 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.

7. 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.

8. 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.

9. 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.

10. 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).

11. 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 C-terminal 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.

12. 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.

13. 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.

14. 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. 5-12). 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.

15. 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.

16. 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
high-affinity 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).

17. 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.

18. 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.

19. 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.

20. 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.

21. 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.

22. 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).

23. 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.

24. 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.

25. 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.

26. 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.