ELECTRON TRANSPORT

AND
OXIDATIVE PHOSPHORYLATION
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Slide 2
 Respiration
Food-to-Energy
 THE ELECTRON TRANSPORT CHAIN

The
Mitochondrion Electron
Inhibitors and
transport chain
as a major uncouplers of
and oxidative
centre for the ETC
phosphorylation
respiration
Objectives
➢ Understand how the energy from electrons in
glucose is converted to chemical energy in ATP
during oxidative phosphorylation.
➢ Explain the role of oxygen in cellular respiration
➢ Explain how the H+ gradient across the inner
mitochondrial membrane is generated and how it
drives ATP synthesis
➢ Predict the effects of various drugs on oxidative
phosphorylation.
 Cellular respiration breaks down energy-rich molecules to
CO2 & water, extracting their energy.
Light
energy
ECOSYSTEM

Photosynthesis
Low CO2 + H2O
in chloroplasts
Organic+ O High
molecules 2
energy Cellular respiration
in mitochondria
energy

C-H bond
“burned” with O2
ATP to H2O + CO2
ATP powers most cellular work

Heat 6
energy
 Overview
• 1st Law of Thermodynamics:
• Potential (stored) energy is
neither created or destroyed
• It can be transferred and
transformed to other forms
(chemical, electrical,
mechanical, light, or heat).
• Cells transform energy to
perform work
• ATP is the primary energy
Potential Energy in Food Is Converted to
currency of cells Kinetic Energy in a Hummingbird’s Body
 CELLS STORE POTENTIAL ENERGY WITH GRADIENTS

ATP + H2O =ADP + Pi Go’ =-30.5KJ/mole

ADP + H2O= AMP + Pi Go’ = -28.4KJ/mole

• Cells couple energetically favorable & unfavorable reactions

• Nucleotide triphosphates store energy for immediate use
• The amount of potential energy stored in an ion gradient can
be expressed as an electrical potential
 Oxidation-Reduction Potential
✓ High-energy electrons and redox potentials are of
fundamental importance in oxidative
phosphorylation.
✓ In oxidative phosphorylation, the electron transfer
potential of NADH or FADH2 is converted into the
phosphoryl transfer potential of ATP.
✓ The corresponding expression for the electron
transfer potential is E´0, the reduction potential (also
called the redox potential or oxidation-reduction
potential).
✓ A negative reduction potential means that the reduced
form of a substance has lower affinity for electrons than
does H2.
✓ A (more) positive reduction potential means that the
reduced form of a substance has higher affinity for
electrons than does H2.
✓ Thus, a strong reducing agent (such as NADH) is poised
to donate electrons and has a negative reduction
potential, whereas a strong oxidizing agent (such as O2 )
is ready to accept electrons and has a positive reduction
potential.
✓ Electrons tend to pass from the most negative carrier
to the most positive carrier (oxygen). This help stepwise
flow of electrons.”
✓ The standard free-energy change G°´ is related to the
change in reduction potential  E´° by:
Δ G°´= - n f Δ E°´
Where:
ΔG°´ = standard free energy
n = number of electrons
F = is Faraday constant (23.04 cal/volt)
ΔE°´ = the difference in the standard reduction potentials &its in volt
E´º in volts is measured by a responsive electrode placed in
solution containing both the electron donor and its
conjugate electron acceptor at standard conditions: 1 M
concentration, 25ºC and pH 7
Example

• The free-energy change of an oxidation-reduction reaction

can be readily calculated from the reduction potentials of
the reactants. For example, consider the reduction of
pyruvate by NADH, catalyzed by lactate dehydrogenase.
The reduction potential of the NAD+:NADH couple, or
half-reaction, is -0.32 V, whereas that of the
pyruvate: lactate couple is -0.19 V.

To obtain reaction a from reactions b and c, we need to

reverse the direction of reaction c so that NADH
appears on the left side of the arrow. In doing so, the
sign of E´0 must be changed
 • To obtain reaction a from reactions b and c, we
need to reverse the direction of reaction c so
that NADH appears on the left side of the arrow.
In doing so, the sign of E’0 must be changed.

For reaction b, the free energy can be calculated with

n = 2.
 Likewise, for reaction d,

Thus, the free energy for reaction a is given by

REDOX POTENTIAL UNDER NON-STANDARD CONDITIONS
(NERNST EQUATION)
• Under standard conditions:
Δ G°= -n f Δ E°
• If we not operating under standard conditions we know
that Δ G = Δ G° + RT lnKeq
Since :
ΔG=-nfE and Δ G°= -n f E°
These can be combined to give

-n f E = -n f E° +RT lnKeq
 E = E° - RT / nf ln [ oxidant]/ [reductant]
Or
E = E° - RT/nf 2.303 log [ oxidant]/ [reductant]

Nernst equation is used to calculate redox potential E,

at any concentration of oxidant and reductant from Eº

When a system is at equilibrium, ∆E = 0.

We have: ∆E◦ = RT/nf ln Keq

Thus, the equilibrium constant and ∆E° are related

• The transfer of electrons down the respiratory chain is
energetically spontaneous because
• NADH is a strong electron donor
• Oxygen is strong electron acceptor
• Since ATP is too unstable,
• C-H bonds in sugars are used for energy storage
 MITOCHONDRIA STRUCTURE AND FUNCTION
• Double membrane
• Outer membrane
• Inner membrane
• Highly folded cristae*
• Fluid-filled space between membranes =
intermembrane space
• Matrix
 LOCALIZATION OF METABOLIC FUNCTIONS IN MITOCHONDRION
COMPARTMENT METABOLIC FUNCTION
Freely permeable to small molecules and ions. Contains porins
Outer with 10,000 Dalton limit. Phospholipid synthesis. Fatty acid
Membrane Desaturation & Elongation.
Protein rich (4:1 protein: lipid). Impermeable. Contains ETR, ATP
Inner synthase, transporters. Electron Transport, Metabolite
membrane transport Pyruvate oxidation, TCA Cycle

Cristae Highly folded inner membrane structure. Increase surface area

Protein rich (500 mg/ml) Contains TCA cycle enzymes, Pyruvate
Matrix D’ase, FA and AA oxidation pathway, DNA, Ribosomes, Beta
oxidation, transcription and translation

Intermembrane Composition similar to cytosol

Space
GENERATION OF ATP IN AEROBIC OXIDATION OF GLUCOSE
Reactions Moles of ATP
Pathway Methods of
Catalyzed by formed per mol
ATP production
of glucose

Glycolytic Glyceraldehyde 3-phosphate

dehydrogenase Respiratory chain Oxidation of 2 NADH
pathway
Phosphoglycerate kinase Phosphorylation at substrate level
Pyruvate kinase Phosphorylation at substrate level
Allow for consumption of ATP by reactions catalyzed by hexokinase and phosphofructokinase

Production of Pyruvate dehydrogenase Respiratory chain Oxidation

acetyl CoA complex of 2 NADH
Tricarboxylic Isocitrate dehydrogenase Respiratory chain Oxidation of 2 NADH
acid cycle Alpha-ketoglutarate
Respiratory chain Oxidation of 2 NADH
Dehydrogenase complex
Succinyl CoA synthetase Phosphorylation at substrate level
Succinate dehydrogenase Respiratory chain Oxidation of 2 FADH2
Malate dehydrogenase Respiratory chain Oxidation of 2 NADH

By convention 1NADPH= 2.5/3ATP; 1FADH2=1.5/2

Total per mole of glucose under aerobic conditions: …. or …..ATPs
SUMMARY OF CELLULAR RESPIRATION
 General course of biological oxidation
Glycogen Triglyceride Protein

Glucose Fatty acid + Amino Acids I

Glycerol

Acetyl CoA

II III
TCA
CO ADP+Pi ATP
2
2 Respiratory chain H2
H
 SHUTTLING ELECTRON CARRIERS INTO
THE MITOCHONDRION

Inner membrane is impermeable to NADH. e-s carried by

NADH that are created in the cytoplasm must be shuttled
into the matrix before they can enter the ETC.

The 3-GP and the Apartate shuttle systems also assist in

the creation of ATP but the former is inefficient.

Note that NADH and FADH2 are electron carriers and are
not actual component
MALATE/ASPARTATE SHUTTLE
 DHAP/G3P
Shuttle/Glycerol/Shuttle System
 SUBSTRATE LEVEL
PHOSPHORYLATION
 THE FALLING WATER ANALOGY
• Visualize a dam - stores water at height
• Holds Potential based on difference in height.
• The greater the height difference the larger the difference in
potential energy (PE)
• PE converted to Kinetic energy (KE) by permitting water to flow
through a channel in the dam using force of flowing water to drive
turbines. Be careful not to over extend the principle.
• The force is the effect of gravity in the dam
• In cells membranes/pores act as channels with the force based
on concentration differences.
• High-energy electrons and ion gradients are examples of short-
term potential energy
Potential energy (ion gradient)
used for ATP formation
• Glycolysis → 2 ATP
• Kreb’s cycle → 2 ATP

• Life takes a lot of energy to run, need to

extract more energy than 4 ATP!

There’s got to be a better way!

 THERE IS A BETTER WAY! ELECTRON TRANSPORT CHAIN
❖Sequence of molecules built into the inner mitochondrial
membrane
❖Sequential transport proteins forming the ETC
❖Act as electron carriers
❖Transport of electrons down ETC is linked to ATP synthesis
❖Yields ~34 ATP from 1 glucose! That sounds more
like it! More
Energy
❖Only in presence of O2 (aerobic)
❖ETC facilitates the controlled release of free energy
stored in reduced cofactors/proteins during catabolism.
 What is the electron
transport chain?
A chain of protein complexes embedded in the inner
mitochondrial membrane. Transports electrons and
pumps hydrogen ions into the intermembrane space
to create a gradient.
 COMPONENTS OF THE ETC
➢The series of oxidation-reduction reactions requires four
membrane-bound multi-protein complexes called
complexes I, II, III and IV
➢Each complex consist of multiple proteins and Fe-S, heme
or copper prosthetic groups.
➢Complexes I, III and IV are also proton pumps
➢NADH passes electrons to the ETC
➢H+ cleaved off NADH & FADH2 releases electrons
➢Electrons passed from one electron carrier to next in the
mitochondrial membrane (ETC)
 COMPONENTS OF THE ETC
➢Transport proteins in membrane pump H+ across inner
membrane to intermembrane space

➢ In the ETC, the electron carriers are arranged such that

the flow of electrons is spontaneous.

➢ Each acceptor has sequentially greater electron affinity

(greater ΔE0') than the electron donor.
 OXIDATION-REDUCTION REACTIONS
➢Definition: Chemical reactions that involve transfer of
electrons from one substance to another
➢Oxidation refers to the loss of electrons to any electron
acceptor - not just to oxygen.
➢Uses exergonic flow of electrons through ETC to pump H+
➢Redox potential = relative tendency to gain or lose e-
(O2 = +0.82 V) readily gains electrons;
(NADH =-0.32 V) readily gives up electrons)
➢Reduced Coenzymes Conserve Energy from Biological
Oxidations: NAD(P), FAD, FMN
 ELECTRONS FLOW DOWNHILL
Electrons move in steps from carrier to carrier downhill to O2
– Each carrier more electronegative
– Spontaneous
– Controlled oxidation
– Controlled release of energy

Standard reduction potentials of the major respiratory electron carriers.

 COMPLEX I
• NADH-CoQ Reductase
• Electron transfer from NADH to CoQ
• More than 30 protein subunits - mass of 850 kD
• 1st step is 2 e- transfer from NADH to FMN
• FMNH2 converts 2 e- to 1 e- transfer
• Four H+ transported out per 2 e-
 COMPLEX II
• Succinate-CoQ Reductase - aka Succinate Da’se
(TCA cycle!)
• Four subunits
• Two largest subunits contain 2Fe-S proteins
• Other subunits involved in binding succinate
dehydrogenase to membrane and passing e- to
Ubiquinone
• FAD+ accepts 2 electrons and then passes 1 e- at a time
to Fe-S proteins
• No protons pumped from this step
 COMPLEX III
• CoQ-Cytochrome c Reductase
• CoQ passes electrons to Cytochrome C (and pumps H+)
in a unique redox cycle known as the Q cycle
• Cytochromes, like Fe in Fe-S clusters, are one- electron
transfer agents
• Cytochrome C is a water-soluble electron carrier
• 4 protons pumped out of mitochondria (2 from UQH2)
 COMPLEX IV
• Cytochrome c Oxidase
• Electrons from cyt c are used in a four-electron
reduction of O2 to produce 2H2O
• Oxygen is thus the terminal acceptor of electrons in
the electron transport pathway - the end!
• Cytochrome c oxidase utilizes 2 hemes (a and a3) and 2
copper sites
• Complex IV also transports H+ (2 protons)
 pyruvate from cytoplasm inner membrane

H+ electron
transport
Coenzymes e− system
NADH give up
acetyl-CoA electrons,
hydrogen (H+) e−
NADH to transport H+
TCA system
H+
cycle FADH2
As electrons pass
e−
through system,
H+ is pumped out
carbon dioxide
from matrix

ATP Oxygen accepts

2 ATP
synthesized electrons, joins with
Pi ADP 2H+, forms water
oxygen
H+

H+ INTERMEMBRANE
MATRIX H+ flows in H+ space
H+
BUT WHAT “PULLS” THE ELECTRONS DOWN THE ETC?

electrons flow The electron transport chain pumps protons against the
downhill to concentration gradient; builds up a high H+ concentration in
O2 intermembrane space.
 WHY THE BUILD UP H+?
• ATP synthase enzyme in inner
membrane of mitochondria
ADP + Pi → ATP
• Only channel permeable to H+
• H+ flow down concentration
gradient
• Provides energy for ATP synthesis
✓ Molecular Power Generator!
✓ Flow Like Water Over Water
Wheel
✓ Flowing H+ Cause Change In
Shape Of ATP Synthase Enzyme
✓ Powers Bonding Of Pi To ADP ATP Synthase: An Electrical
Mechanochemical Molecular Complex
✓ “Proton-motive” force
 ATP SYNTHESIS
• Chemiosmosis couples ETC to ATP synthesis
• Build up of H+ gradient just so H+ could flow through ATP
synthase enzyme to build ATP

So that’s
the point!
 CHEMIOSMOTIC THEORY
HOW ELECTRON TRANSPORT COUPLED WITH ATP SYNTHESIS

In Mitchell’s chemiosmotic theory, H+ are driven across

the membrane from the matrix to the intermembrane
space and cytosol by the events of electron transport.
This mechanism stores the energy of electron transport in
an electrochemical potential.
As protons are driven out of
the matrix, the pH rises &the
matrix becomes negatively
charged with respect to the
cytosol.
 CHEMIOSMOTIC THEORY
➢ Chemiosmotic coupling is the mechanism for coupling
electron transport to oxidative phosphorylation- requires
a proton gradient across the inner mitochondrial memb.
➢ Proton pumping thus creates a pH & an electrical
(proton and electrochemical gradients) gradient across
the Inner membrane, both of which tend to attract
protons back into the matrix from the cytoplasm.
➢ Flow of protons down this electrochemical gradient, an
energetically favorable process, then drives ATP synthesis.
 CHEMIOSMOTIC THEORY
➢ Electron transport is coupled with Oxidative
Phosphorylation
➢ The ATP synthase molecules are the only place that will
allow H+ to diffuse back to the matrix.
➢ This exergonic flow of diffusion of H+ is used by the
enzyme to generate ATP.
➢ The Chemiosmotic Theory states that coupling of
electron transfer to ATP synthesis is indirect, via a H+
electrochemical gradient:

Slide 49
 SUMMARY OF CELLULAR RESPIRATION
C6H12O6 + 6O2 → 6CO2 + 6H2O + ~36 ATP
• Where did the glucose come from? From food eaten
• Where did the O2 come from? From the atmosphere
• Where did the CO2 come from? Oxidized carbons
cleaved off of the sugars
• Where did the H2O come from? From O2 after it accepts
electrons in ETC
• Where did the ATP come from? Mostly from ETC
• What else is produced that is not listed in this equation?
NAD, FAD, heat!
• Why do we breathe?
 GENERATION OF ATP IN AEROBIC
OXIDATION OF GLUCOSE

Pathway Reactions Methods of Moles of ATP formed

Catalyzed by ATP production per Mol of glucose

Glycolytic Glyceraldehyde 3-phosphate Respiratory chain Oxidation of 2 NADH

pathway dehydrogenase Phosphorylation at substrate level
Phosphoglycerate kinase
Phosphorylation at substrate level
Pyruvate kinase
Allow for consumption of ATP by reactions catalyzed by hexokinase and phosphofructokinase

Production Pyruvate dehydrogenase Respiratory chain Oxidation

of A CoA complex of 2 NADH

Tricarboxylic Isocitrate dehydrogenase Respiratory chain Oxidation of 2 NADH

acid cycle Alpha-ketoglutarate
Respiratory chain Oxidation of 2 NADH
Dehydrogenase complex
Succinyl CoA synthetase Phosphorylation at substrate level
Succinate dehydrogenase Respiratory chain Oxidation of 2 FADH2
Malate dehydrogenase Respiratory chain Oxidation of 2 NADH
By convention 1NADPH= 2.5/3ATP; 1FADH2=1.5/2
• What is the final electron acceptor in
Taking
electron transport chain?
it beyond…
O2
▪ So what happens if O2 unavailable?
▪ ETC backs up
▪ ATP production ceases
▪ cells run out of energy
▪ and you die!
 INHIBITORS OF OXIDATIVE PHOSPHORYLATION
• Many potent and lethal poisons exert their effect by
inhibiting OP at one of a number of different location.
• Two major classes of mitochondrial inhibitor : respiration
inhibitors and phosphorylation inhibitors.
• Respiration inhibitors prevent oxygen uptake.
• Phosphorylation inhibitors allow electron transport but
prevent ATP manufacture
• Remember the concept of an energetically favourable
electron transport system, which in some way powers an
energetically unfavourable phosphorylation system.
 INHIBITORS OF OXIDATIVE PHOSPHORYLATION
• The free energy available from the redox reactions was
used to drive ATP synthesis.
• Respiration inhibitors such as cyanide ions block the
electron transport chain and were always effective in
suppressing oxygen uptake.
• Phosphorylation inhibitors such as oligomycin, which
prevented ATP manufacture, could only block respiration if
the coupling were intact.

http://www.bmb.leeds.ac.uk/illingworth/oxphos/poisons.htm
Slide 54
 INHIBITORS OF OXIDATIVE PHOSPHORYLATION
• Rotenone & Pericidine A inhibits Complex I - and helps
natives of the Amazon rain forest catch fish!
• Poorly absorbed
• Non-toxic inhibitor of Electron transport chain.
• Binds at Complex I between Fe-S protein and Ubiquinone.

• Cyanide, Azide and CO inhibit Complex IV, binding tightly

to the ferric form (Fe3+) of a3
• Oligomycin and DCCD are ATP synthase inhibitors
 INHIBITORS OF OXIDATIVE PHOSPHORYLATION
Inhibitor Action
Rotenone-Insecticide
Amytal/Amobarbital -
barbiturate
Blocks complex I
Methylphenylpyridinium ion
(MPP+)
Pericidin - Antibiotic
II: 3-nitropropionate Blocks complex II
Antimycin A,
Thenoyltrifluoroacetone
Blocks complex III
(TTFA)
Dimecraprol
Cyanide (CN), Azide (N3) , H2S Blocks complex IV
Carbon monoxide (CO),
Oligomycin Blocks the proton
Dicyclohexylcarbodiimide channel (Fo) in ATP’ase
(DCCD)
Atractyloside ATP/ADP Antiporter
 UNCOUPLERS OF OXIDATIVE PHOSPHORYLATION
• Coupling of ETC and OP can be uncoupled.
• Uncouplers carry protons across the inner mitochondrial
membrane, down their concentration gradient.
• Uncouplers are hydrophobic molecules with a dissociable
proton.
• In their presence , electron transport proceeds in a
normal fashion, but ATP is not formed by mitochondrial
ATP synthase,
• NADH and oxygen are consumed, but energy is released
as heat
 CHEMICAL & BIOLOGIC UNCOUPLERS
Free energy change associated with respiration is
dissipated as heat. This "non-shivering thermogenesis" is
costly in terms of respiratory energy unavailable for ATP
synthesis, but it provides valuable warming of the organism
• 2,4 Dinitrophenol
• Dicoumarol
• Chlorocarbonylcyanide phenylhydrazine [CCP]
• Salicylate
• Thermogenin
• CaCl2
 CHEMICAL & BIOLOGIC UNCOUPLERS
❖ Thermogenin is produced in brown adipose tissue of
newborn mammals and hibernating mammals.
❖Dinitrophenol once used as diet drug, people ran 107oF
temperatures. Russian Soldiers………
❖Uncouplers of oxidative phosphorylation inhibit
coupling between the ETC and phosphorylation reactions
and thus inhibit ATP synthesis without affecting the
respiratory chain and ATP synthase.
❖Blocks/dissipates proton/electrochemical gradient,
thereby stimulating respiration.
INHIBITORS & UNCOUPLERS

By What mechanisms do
Atractyloside
the following operate?
• Oligomycin?
• DNP? oligomycin
• CaCl2 ?
• Atractyloside?
DNP
• Valinomycin?

Ca2+
 COMPONENTS OF THE ETC
Complex Name No. of Prosthetic Groups
Proteins
Complex I NADH 46 FMN, 9 Fe-S centers
Dehydrogenase
Complex II Succinate-CoQ 5 FAD, cyt b560, 3Fe-S
Reductase centers
Complex III CoQ-cyt c Reductase 11 cyt bH, cyt bL, cyt c1,
Fe-SRieske
Complex IV Cytochrome Oxidase 13 cyt a, cyt a3, CuA, CuB
 Terminal Electron Acceptors
Different e- acceptors are used sequentially in microbial
ecosystems.
O2 ∆G = -479 kJ mol-1
NO3- ∆G = -453 kJ mol-1
Mn4+ ∆G = -349 kJ mol-1
Fe3+ ∆G = -114 kJ mol-1
SO42- ∆G = -77 kJ mol-1

Electron donors {[CH2O], H2, H2S, CH4, Fe2+, etc.}

 MITOCHONDRIAL INHERITANCE/DISEASES
Mitochondrial traits are inherited in a non-Mendelian fashion because they are
carried on mitochondrial DNA. They have the following characteristics:

The disease is inherited only maternally, since only the mother contributes
mitochondrial DNA to the progeny.

Both males and females can be affected by the disease. All offspring of an affected
female are affected, whereas there is no inheritance of the disease from an
affected male.

Mitochondrial diseases are often expressed as neuropathies and myopathies

because brain and muscle are highly dependent on oxidative phosphorylation.
Mitochondrial genes code for some of the components of the electron transport
chain and oxidative phosphorylation, as well as some mitochondrial tRNA
molecules.
 Think …………. Energy harvest.
What is the yield of ATP when each of the following
substrates is completely oxidized to CO2 by a mammalian
cell homogenate? Assume that glycolysis, the citric acid
cycle, and oxidative phosphorylation are fully active.

(a) Pyruvate (d) PEP

(b) Lactate (e) Galactose
(c) F 1,6-BP (f) Dihydroxyacetone
 Think ………….
➢ What is the mechanistic basis for the observation that the
inhibitors of ATP synthase also lead to an inhibition of the
electron-transport chain?
➢ How does the inhibition of ATP-ADP translocase affect the citric
acid cycle? Glycolysis?
➢ Years ago, uncouplers were suggested to make wonderful diet
drugs. Explain why this idea was proposed and why it was
rejected. Why might the producers of antiperspirants be
supportive of the idea?
➢ It has been noted that the mitochondria of muscle cells often
have more cristae than the mitochondria of liver cells. Provide an
explanation for this observation.
http://www.bmb.leeds.ac.uk/illingworth/oxphos/poisons.htm

IT IS
FINISHED !!!!!