1
BI25M1
THE TERMINAL
RESPIRATORY SYSTEM
and
OXIDATIVE
PHOSPHORYLATION
LECTURE 1:
AIM:
To review
the way in which ‘high-energy’ electrons, stripped
from food molecules and part of the structure of
NADH + H+ and FADH2,
are passed through a series of redox carriers
(the terminal respiratory system), and eventually
combine with electronegative oxygen to form
water.
Lehninger:
Instant Notes:
Chapter 19
Section L2
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1 LINKS TO WHAT WENT BEFORE
(Citric Acid Cycle lectures)
An early form of catabolism involved
partial, anaerobic breakdown
(fermentation) of hexoses like glucose.
During the catabolism, some of the
potential energy of food molecules was
conserved in ATP synthesis
(substrate-level phosphorylation).
Such metabolism still occurs.
It is the only way in which ATP can be
generated from food molecules in the
absence of oxygen:
hence the importance of hexoses in life.
Evolution
of
an
oxygen-involving
‘apparatus’, however, allowed much more
potential energy of food molecules to be
‘released’ and ‘saved’,
and also widened the range of food
molecules to include amino-acids and,
particularly, fatty acids.
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The ‘apparatus’ involves electropositive H
atoms, containing ‘high-energy’ electrons,
being stripped from food molecules, two at
a time, as the molecules are completely
broken in the presence of O2.
The electrons are passed to the oxidised
form of a co-reactant, either NAD+ or FAD.
[The reduced co-reactant can then be used in
anabolism
Energy Transformations Lecture 3].
The reduced co-reactant can also pass on
the electrons, through a series of redox
carriers (the ‘terminal respiratory
system’; TRS), until, eventually, they
combine with O2 deliberately taken into
the cell, to form H2O.
The citric acid cycle, in which much of the
reduction of NAD+ and FAD occurs, is a
part of this process.
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In summary:
hexoses
fatty acids
(6 C)
glycolysis
(3 C)
(3 C)
 oxidation
glyceraldehyde 3-ph’ate
dehydrogenase
NADH + H+
ATP
1 NADH + H+
1 FADH2
(substrate-level
phosphorylation)
per pass through
the pathway
amino acids
pyruvate
(3 C)
pyruvate dehydrogenase
NADH + H+
acetyl CoA
(2 C)
citric acid cycle
3 NADH + 3H+
1 FADH2
1 GTP
per turn of the cycle
CO2
(1 C)
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To summarise further:
food molecules containing
H atoms may be metabolised thus:
food
molecule
NAD+
or
FAD
reduced
anabolic
product
CO2
NADH + H+
or
FADH2
biosynthetic
precursor
or like this:
food
NAD+
molecule or
FAD
reduced
co-reactant
oxidised
co-reactant
H2O
etc
CO2
NADH + H+ oxidised
or
co-reactant
FADH2
reduced
co-reactant
TRS
O2
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2 A QUESTION OF LOCATION
For oxidation in the TRS of eukaryotic
cells, NADH and FADH2 must be in the
mitochondrial matrix.
They are mainly formed there, by the
pyruvate dehydrogenase-catalysed reaction;
citric acid cycle;
fatty acid  oxidation pathway
(Citric Acid Cycle Lecture 2;
Lipid Metabolism lectures).
However, a little NADH is made in the
cytosol, by the
glyceraldehyde 3-phosphate dehydrogenasecatalysed reaction of glycolysis
(Carbohydrates and Intermediary Metabolism
Lecture 4).
This NADH cannot cross the inner
mitochondrial membrane.
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Shuttles are used to move - not NADH
itself - but its reducing equivalents into
the matrix.
This is an example of such a shuttle:
outside
inner
mitochondrial
membrane
NADH
+ H+
dihydroxy
acetone
phosphate
NAD+
glycerol
3-phosphate
FADH2
FAD
FADH2 is now accessible to the TRS.
Its oxidation in the TRS generates, per
mol, less ATP than does oxidation of
NADH (Section 7),
so an energetic ‘price’ is paid for using
cytosolic reduced co-substrate in the TRS.
[This is relevant to part of the calculation in Tutorial 3.]
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3 THE ELECTRON CARRIERS OF THE
TRS
In eukaryotic cells, these are present in
the inner membrane of mitochondria.
In prokaryotic cells, they are membraneassociated.
They consist of four protein assemblies:
‘Complexes I, II, III, IV’.
Complex I,
oxidises NADH and passes the electrons to the
TRS.
Complex II
is the reaction of the citric acid cycle that oxidises
succinate and reduces FAD to FADH2
(Citric Acid Cycle lectures Section 12).
It oxidises the FADH2 back to FAD, and passes the
electrons to the TRS.
Complex III
has cytochrome electron carriers.
Like haemoglobin, they contain haem groups,
and are coloured.
[Why is brown fat (Section 9) brown?]
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Complex IV
is where the electrons finally reach O2
and reduce it to water.
Cyanide inhibits this final step
(Enzymes Lecture 4).
Two other TRS components are:
‘Q’ (aka ‘coenzyme Q’ or ‘ubiquinone’);
and
cytochrome c.
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In summary:
NADH feeds in electrons at Complex I;
FADH2 from the citric acid cycle feeds in
electrons at Complex II.
There is another electron feed-in point,
at Q,
that is used by FADH2 made
in the shuttle
(Section 2)
and
in fatty acid -oxidation (Lipid Metabolism lectures).
Further,
at Complexes I, III and IV,
ATP is made by oxidative phosphorylation
(Section 6).
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So, flow of ‘high-energy’ electrons,
originally part of food molecules,
through the TRS looks like this:
From cytosolic NADH + H+
via the glycerol 3-phosphate
shuttle (Section 2)
FADH2 FAD
Cytochrome c
H2O
I
Q
NADH+ NAD+
+ H+
FADH2
succinate fumarate FADH2 FAD
From citric
This is part of
acid cycle,
the citric acid
 oxidation
cycle
oxidation
and the
pyruvate dehydrogenasecatalysed reaction
IV
1
II
FAD
III
From 
oxidation
/2O2
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In each of the complexes,
a series of redox reactions,
not shown in the previous diagram,
occurs:
AH2
B
CH2
etc
A
BH2
C
as the ‘high-energy’ electrons move
towards O2.
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4
ENERGY IS ‘RELEASED’ AS
ELECTRONS PASS ALONG THE
TRS.
As the ‘high energy’ electrons, originally
part of food molecules, are passed along
the TRS,
they move down an energy gradient
towards electronegative O2.
As they do so,
their energy is systematically released in
‘packets’, and ‘saved’,
at particular points along the TRS
(in Complexes I, II and IV.)
This is oxidative phosphorylation.
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BI25M1
THE TERMINAL
RESPIRATORY SYSTEM
and
OXIDATIVE
PHOSPHORYLATION
LECTURE 2:
AIM:
To review
the way in which the potential energy of
food molecules is conserved in the synthesis
of ATP by oxidative phosphorylation.
Lehninger:
Instant Notes:
Chapter 19
Section L2
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5 CHEMIOSMOSIS AND THE
PROTON-MOTIVE FORCE
In eukaryotic cells, TRS components are
in the inner membrane of the
mitochondrion (Section 3).
As electrons pass through Complexes I, III
and IV,
protons are moved,
as part of the redox reactions,
from the matrix
to the outside of the inner mitochondrial
membrane.
These reactions therefore have
particular spatial directionality,
and are said to be ‘vectorial’.
During them, protons are taken from the
matrix, and protons are deposited on the
outside of the membrane.
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The overall effect is that protons are
‘transported’ from the inside to the
outside.
The process is called ‘chemiosmosis’.
The inner membrane is otherwise
impermeable to protons, so they can’t
move back in, along their concentration
gradient, to equalise concentrations on the
two sides.
Instead, the proton gradient stores,
temporarily, some of the energy ‘released’
as the electrons move along the TRS.
This is a particular example of an energy
transformation.
The protons, held at a higher
concentration on the outside of the
membrane, are like water stored in a dam
(Energy Transformations Lecture 1).
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Because of its altitude, the water has
potential energy, which is ‘released’ and
transformed into a form able to do work
when the water is allowed to flow
downwards.
Similarly, the proton gradient stores
potential energy
(the ‘proton-motive force’),
which is ‘released’ and transformed into a
form able to do work when protons are
allowed to flow down the gradient, back
into the matrix.
Photosynthesis and many other biological
energy transformations involve similar
proton gradients.
In fact, it has gradually been realised that
setting up proton-motive forces by
chemiosmosis underpins much of Biology.
In the present context, the work to be
done is ATP synthesis (oxidative
phosphorylation).
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6 HOW DOES ATP SYNTHESIS
ACTUALLY OCCUR?
Protons eventually flow
down their concentration gradient,
across the inner mitochondrial membrane,
and back into the matrix.
This only occurs at certain sites in the
membrane.
At the sites are protein complexes of
ATP synthase.
Each complex has a pore, through which
the protons pass.
As the protons pass through,
energy stored in the gradient is used by
the ATP synthase to convert ADP + Pi to
ATP.
So, potential energy held in the gradient is
transformed into potential energy held in
ATP structure.
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H+
outside
section of
inner mitochondrial
membrane
inside
ADP + Pi
ATP
ATP synthase
ATP synthase has two units
(each of which has sub-units):
F0 deals with proton movement;
F1 deals with ATP synthesis
Lehninger Edition 4 pp.708-713; Edition 5 pp.725-730
Instant Notes pp.380-381.
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As protons pass through the F0 pore,
the F0 cylinder and the F1 shaft rotate,
and the , part of F1 is held stationary.
The cylinder/shaft movement causes
sequential conformational changes
in the  sub-units.
These are:
from a form that binds ADP and Pi;
to a form that binds ATP;
(This change drives ADP
ATP.)
to a form that doesn’t bind ATP.
(This change causes ATP release.)
The result is synthesis of ATP.
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7 THE STOICHIOMETRY OF
OXIDATIVE PHOSPHORYLATION
In Section 2,
it was stated that FADH2 oxidation in the
TRS generates, per mol, less ATP than
does NADH oxidation.
The structure of the electron transport
chain (Section 3) shows why this is.
NADH feeds in electrons at Complex I,
and so they pass through all three sites
containing ATP synthase
(Complexes I, III and IV).
FADH2, however, feeds in electrons at
Complex II or at Q,
and so they pass through only two of these
sites
(Complexes III and IV).
In fact, about 2.5 and 1.5 mol ATP are made per
mol of NADH and FADH2 respectively.
These figures are rounded to 3 and 2 respectively
in the exercise in Tutorial 3.
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8 IN SUMMARY:
Food molecules containing ‘high-energy’ electrons
of electropositive H atoms
are catabolised through pyruvate production, the
pyruvate dehydrogenase-catalysed reaction, oxidation and the citric acid cycle, to CO2.
The H atom electrons are passed to, and reduce
co-reactants (NAD+ and FAD).
The reduced co-reactants pass the electrons to a
series of redox reactions (the TRS) and they
eventually reach electronegative oxygen,
forming H2O.
The food molecules are completely broken to CO2
and H2O.
Some of their potential energy is ‘saved’, having
been transformed through the potential energies of
the reduced co-reactants and the terminal
respiratory redox components and the protonmotive force, and is eventually used in making
ATP.
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9 COUPLING AND UNCOUPLING
When electron transport is accompanied
by ATP synthesis (Section 6), the two are
said to be coupled.
Any process that makes the mitochondrial
inner membrane leaky to protons
stops the proton gradient being set up.
When this happens, electron transport
still occurs, and O2 is reduced to H2O,
but no ATP is made.
The two processes are said to be
uncoupled.
Because the energy released as electrons
move along the TRS
can’t be used in ATP synthesis,
it is released as heat.
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Malignant hyperthermia is the result of a
genetic defect(s).
Susceptible people, exposed to the clinical
anaesthetic halothane,
experience a sudden, life-threatening
increase in body temperature.
Part of the reason is that halothane,
in susceptible people,
makes inner mitochondrial membranes in
muscle leaky to protons.
Some strains of pigs are ‘stress-prone’.
When shipped to market,
they experience large increases in body
temperature.
The stress
(by unclear mechanisms)
causes inner mitochondrial membranes
to become leaky to protons.
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Sometimes uncoupling occurs purposefully.
Brown fat occurs on the neck and upper
back of new-born babies,
(and in other young or hibernating
mammals).
Brown fat cells
(unlike white fat cells)
contain many mitochondria.
Why does this make them brown? (Section 3).
In the cold, nor-epinephrine is released.
This (indirectly) triggers the opening of a
channel in a protein, thermogenin,
that spans the mitochondrial inner
membrane of brown fat cells.
Protons move along their concentration
gradient back into the matrix, and energy
is released as heat.
This is important in thermo-regulation of
the animals.
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Some plants, by a different mechanism,
also ‘deliberately’ uncouple electron
transport from oxidative phosphorylation
to generate heat
that either directly,
or by aiding evaporation of strong
smelling molecules,
attracts pollinating insects.