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Slide 1
Module 7 – Protein structure

Dr Lakshmi Wijeyewickrema

Department of Biochemistry and Genetics Slide 2

Intended Learning Outcomes
(ILOs)
After this lesson students will be able to:
• Understanding the influence of protein structure on function.
• Describe the properties of heme in haemoglobin function.
• Describe what the quaternary structure of haemoglobin is.
• Interpret what the oxygen saturation curves of haemoglobin
are.

Slide 3
 Quaternary structure

• Structure of proteins made up of more than one

subunit
• Two subunits = dimer
• three subunits = trimer
• many subunits = oligomer
• Haemoglobin is a tetramer (4 subunits)
• Immunoglobulins are also tetramers

Slide 4
 Haemoglobin
• The main function of a red blood cell (RBC)
• Transfer of O2 from lungs to tissue
• Transfer of CO2 from tissues to lungs
• To accomplish this function RBC’s have hemoglobin (Hb)
• Each RBC has about 270 million molecules of Hb

Slide 5
 Structure of Haemoglobin
Primary structure- 4 polypeptide chains.
Secondary structure – two a chains and two b chains
Tertiary structure – each chain is folded into a precise shape.
Quaternary structure – 4 chains are linked together to form an
almost spherical molecule. Each chain has a heme group.

Slide 6
 Structure of Haemoglobin
• Haemoglobin is an oligomeric protein made up of 2 αβ dimers,
a total of 4 polypeptide chains: α1β1α2 β2.

• Total Mr of haemoglobin is 64,500.

• The α (141 aa) and β (146 aa) subunits have < 50 % identity.

• Each Hb subunit consists of 7 (α) or 8 (β) alpha helices and

several bends and loops folded into a single globin domain.

• Each subunit has a heme-binding pocket.

Slide 7
 Structure of Haemoglobin
Tertiary folding gives rise to at least 3 functionally important
characteristics of the haemoglobin molecule
• 1- Polar or charged side chains tend to be directed to the
outside surface of the subunit and, conversely, non-polar
structures tend to be directed inwards. The effect of this is to
make the surface of the molecule hydrophilic and the interior
hydrophobic.
• 2- An open-toped cleft in the surface of the subunit known as
heme pocket is created. This hydrophobic cleft protects the
ferrous ion from oxidation.
• 3- The amino acids, which form the inter-subunit bonds
responsible for maintaining the quaternary structure, and thus
the function.

Slide 8
 Structure of Heme

• Heme contains: conjugated

system of double bonds →
red colour
• 4 nitrogen (N) atoms
• At the core of the molecule
is porphyrin ring which
holds an iron atom.
• An iron containing porphyrin
is termed a heme.
• This iron atom is the site of
oxygen binding.

Slide 9
 Structure of Heme
The heme group is responsible for the O2-binding capacity of
hemoglobin.

• The heme group consists of the planar aromatic protoporphyrin

made up of four pyrrole rings linked by methane bridges.
• A Fe atom in its ferrous state (Fe+2) is at the center of
protoporphyrin.
• Fe+2 has 6 coordination bonds, four bonded to the 4 pyrrole N
atoms. The nucleophilic N prevent oxidation of Fe+2.
• The two additional binding sites are one on either side of the
heme plane.
• One of these is occupied by the imidazole group of His.
• The second site can be reversibly occupied by O2, which is
hydrogen-bonded to another His.
Slide 10
 Different forms of Haemoglobin

• When haemoglobin is bound to O2, it is called oxyhemoglobin.

This is the relaxed (R ) state.

• The form with a vacant O2 binding site is called

deoxyhemoglobin and corresponds to the tense (T) state.

• If iron is in the oxidized state as Fe+3, it is unable to bind O2

and this form is called methemoglobin

• CO and NO have higher affinity for heme Fe+2 than O2 and can
displace O2 from Hb, accounting for their toxicity.

Slide 11
 T and R states of Haemoglobin

• Haemoglobin exists in two major conformational states:

Relaxed (R ) and Tense (T)

• R state has a higher affinity for O2.

• In the absence of O2, T state is more stable; when O2 binds,

R state is more stable, so haemoglobin undergoes a
conformational change to the R state.

• The structural change involves readjustment of interactions

between subunits.

Slide 12
Department of Biochemistry and Genetics Slide 13
Department of Biochemistry and Genetics Slide 14
L5-10, p165
 Changes Induced by O2 Binding
• O2 binding rearranges electrons within Fe+2 making it more
compact so that it fits snugly within the plane of porphyrin.

• Since Fe is bound to histidine of the globin domain, when Fe

moves, the entire subunit undergoes a conformational change.

• This causes haemoglobin to transition from the tense (T) state

to the relaxed (R) state.

• The α1β1 and α2β2 dimers rearrange and rotate approximately

15 degrees with respect to each other

• Inter-subunit interactions influence O2 binding to all 4 subunits

resulting in cooperativity.
Slide 16
Department of Biochemistry and Genetics Slide 17
 O2 Binding kinetics
• Four subunits, so four O2-binding sites
• O2 binding is cooperative meaning that each subsequent O2
binds with a higher affinity than the previous one
• Similarly, when one O2 is dissociated, the other three will
dissociate at a sequentially faster rate.
• Due to positive cooperativity, a single molecule is very rarely
partially oxygenated.
• There is always a combination of oxygenated and
deoxygenated haemoglobin molecules. The percentage of
haemoglobin molecules that remain oxygenated is represented
by its oxygen saturation.
• O2-binding curves show haemoglobin saturation as a function of
the partial pressure for O2. Slide 18
Oxygen Saturation Curve

Department of Biochemistry and Genetics Slide 19

 Oxygen Saturation Curve
• Saturation is maximum at very high

• O2 pressure in the lungs (pO2 = ~ 13 kPa).

• As haemoglobin moves to peripheral organs and the O2

pressure drops (pO2 = ~4 kPa), saturation also drops allowing
O2 to be supplied to the tissues.

• Due to co-operative binding of O2 to haemoglobin, its oxygen

saturation curve is sigmoid.

• Such a curve ensures that at lower pO2, small differences in O2

pressure result in big changes in O2 saturation of haemoglobin.
This facilitates dissociation of O2 in peripheral tissues.
Slide 20
 Summary
• Quaternary structure describes the arrangement of sub-units
in a protein consisting of more than one polypeptide chain,
where the sub-units may be identical or different.
• Haemoglobin’s primary function is to bind oxygen that
diffuses into the bloodstream from the lungs and then
transport it to outlying tissues where it is released primarily
for aerobic respiration.
• The cooperative loading or unloading of oxygen from
haemoglobin, depending on the environmental concentration
of oxygen,. effectively enhances the oxygen uptake and
delivery capacity of haemoglobin.
• Cooperative ligand binding is the remarkable product of the
evolutionary moulding of haemoglobin’s structure such that it
can adopt more than one functional shape or conformation.
Slide 21
Resources

• Lehninger Principles of Biochemistry Seventh

Edition (2017), W. H. Freeman and Company •
Chapter 5.1

Slide 22