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OXIDATIVE PHOSPHORYLATION
AND
ELECTRON TRANSPORT CHAIN
Dipesh Tamrakar
MSc. Clinical Biochemistry
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Overview
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introduction
• Oxidative phosphorylation is the culmination of energy-yielding
metabolism in aerobic organisms.
• It is the final stage of cellular respiration in degradation of
carbohydrates, fats and amino acids in which the energy of oxidation
drives the synthesis of ATP.
• Photophosphorylation is the means by which photosynthetic organisms
capture the energy of sunlight and harness it to make ATP.
• In eukaryotes, oxidative phosphorylation occurs in mitochondria,
photophosphorylation in chloroplasts.
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introduction
• Oxidative phosphorylation involves the reduction of 02 to H2O with
electrons donated by NADH and FADH2; it occurs equally well in Iight or
darkness.
• Photophosphorylation involves the oxidation of H2O to 02, with NADP+
as ultimate electron acceptor; it is absolutely dependent on the energy
of light.
• Despite their differences, these two highly efficient energy-converting
processes have fundamentally similar mechanisms
• Our current understanding of ATP synthesis in mitochondria and
chloroplasts is based on the hypothesis, introduced by Peter Mitchell in
1961
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• Each electron donor will pass electrons to a more electronegative acceptor,
which in turn donates these electrons to another acceptor, a process that
continues down the series until electrons are passed to oxygen, the most
electronegative and terminal electron acceptor in the chain.
• Passage of electrons between donor and acceptor releases energy, which is
used to generate a proton gradient across the mitochondrial membrane by
actively "pumping" protons into the intermembrane space, producing a
thermodynamic state that has the potential to do work.
• The entire process is called oxidative phosphorylation, since ADP is
phosphorylated to ATP using the energy of hydrogen oxidation in many steps.
• A small percentage of electrons do not complete the whole series and
instead directly leak to oxygen, resulting in the formation of the free-
radical superoxide, a highly reactive molecule that contributes to oxidative
stress and has been implicated in a number of diseases and aging.
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REDOX POTENTIAL
• In reactions involving oxidation and reduction, the free energy change
is proportionate to the tendency of reactants to donate or accept
electrons.
• Can be expressed as an oxidation-reduction or redox potential (E’0)
• Usually compared with the potential of H electrode (0.0 V at pH 0.0)
• However for biologic systems, is expressed at pH 7.0 at which
hydrogen electrode is -0.42V.
• Negative redox potential: lower affinity for electrons than hydrogen
• Positive redox potential: higher affinity for electrons than hydrogen
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Some Redox Potentials of Special Interest in Mammalian Oxidation Systems
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Electron Transport Chain
• An electron transport chain (ETC) is a series of complexes
that transfer electrons from electron donors to electron
acceptors via redox (both reduction and oxidation occurring
simultaneously) reactions, and couples this electron transfer with the
transfer of protons (H+ ions) across a membrane.
• This creates an electrochemical proton gradient that drives the synthesis
of adenosine triphosphate (ATP), a molecule that stores energy
chemically in the form of highly strained bonds.
• The molecules of the chain include peptides, enzymes and others.
• The final acceptor of electrons in the electron transport chain
during aerobic respiration is molecular oxygen although a variety of
acceptors other than oxygen such as sulfate exist in anaerobic
respiration. 9

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Biochemical anatomy of a
mitochondrion
• Most eukaryotic cells
have mitochondria, which produce
ATP from products of the citric acid
cycle, fatty acid oxidation, and amino
acid oxidation.
• At the mitochondrial inner
membrane, electrons
from NADH and FADH2 pass through
the electron transport chain to
oxygen, which is reduced to water.
The electron transport chain
comprises an enzymatic series of
electron donors and acceptors
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NADH GENERATION
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Organization of the chain
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The Respiratory Chain Consists of Four
Complexes: Three Proton Pumps
• Complex I : NADH-Q oxidoreductase
catalyzes transfer of e- s from NADH to CoQ.
• Complex II : Succinate-Q reductase
transfers e- s from succinate to CoQ.
• Complex III : Q-cytochrome C oxidoreductase
transfers e- s from ubiquinol ( reduced form of CoQ) to cytochrome C
• Complex IV : cytochrome C oxidase
transfers e- s from Cytochrome C to O2
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The 4 complexes consist of several different electron carriers.
• Flavoproteins – Prosthetic group FMN or FAD
• Heme containing proteins; Cytochromes
subclassified – cytochrome b,c1,c,a & a3.
a & a3 can directly react with molecular oxygen and are associated
with copper in Complex IV.
• Non-heme iron proteins; Iron-sulfur proteins
contained bound inorganic Fe and S.
• Copper
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Two mobile e- carriers in ETC
• Ubiquinone (Co Q)
UQ is lipid-soluble can accept electrons from FMNH2 / FADH2 &
transfer them to cytochrome.
• Cytochrome C
Cytochrome C is a water-soluble mobile electron carrier of the outer
face of the inner membrane
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PATHWAY OF ETC
 Complex I :Electrons transferred from
NADH to FMN, yielding FMNH2 .
 Then from FMNH2 to iron sulfur
centers.
 Eventually 2 electrons are transferred to
ubiquinone.
 Electron movement accompanied by a
net movement of protons from the
matrix to the intermembrane space.
 During oxidation of 1 NADH and
transfer of 2 e-
s to UQ by complex I , 4
protons are also pumped across the
mitochondrial membrane.
 UQ is lipid-soluble and shuttles
electrons between ETC complexes along
the inner mitochondrial membrane
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• Complex II ( succinate-UQ reductase/ succinate dehydrogenase)
contains 4 pp chain; the 1st two constitute the SDH; a Krebs cycle
enzyme that catalyzes:
Succinate + FAD Fumarate + FADH2
SDH contains Fe-S centre and covalently bound FAD.
eventually e-
s are transferred to UQ
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• Complex III (cytochrome b1 complex) transfers electrons from reduced
UQ (UQH2) to cytochrome c.
• Complex III contains b & c1 type of cytochromes and Fe-S protein called
Rieske’s centre.
UQH2  Cyt b Fe-S  Cyt c1  Cyt c
• Cytochrome c is a water-soluble mobile electron carrier of the outer face
of the inner membrane.
• The transfer of e-
s through Complex III is best explained by Q cycle
mechanism in which 4 H+ are translocated across the IMM per 2 e-
s
transferred from UQH2 to Cyt c.
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• Complex IV catalyzes the transfer of electrons from Cyt c to O2 one at a
time.
• Complex IV contains Cyt a,a3 and 2 copper centres; CuA and CuB
• Oxygen is tightly bound between heme a3 and copper during its
reduction and released only after its complete reduction to water.
Cyt c CuA  cyt a  a3-CuB.
• Finally four electrons and four protons (from matrix) are passed to O2 to
form H2O.
• In addition to protons req. for water formation, cytochrome oxidase also
pumps additional protons across the membrane; during the transfer of 2
electrons to O2 , 2 H+
s are released to the cytosol for every 4 H+
s taken
up from the matrix.
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Flow of electrons through the respiratory chain complexes, showing
the entry points for reducing equivalents from important substrates.
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Release of free energy during electron transfer
• The e- transfer from e- donor to e- acceptor is accompanied by the
release of free energy.
• e-
s can be transferred in different forms, e.g. as hydride ions (:H- ) to
NAD+ , as hydrogen atoms (.H) to FMN, CoQ & FAD or as electron (.e- )
to cytochromes.
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Redox pairs/ Redox couple
• The reduced and oxidised forms of the same carrier are together
referred to as the redox pairs. E.g. NADH/ NAD+ , CoQH2/ CoQ etc.
• Redox pairs differ in their tendency to lose e-
s .This is a characteristic
of a particular redox pair & quantitatively specified by a constant E0 ,
the standard reduction potential ( in Volts).
• The e-
s always flow from the –ve to +ve redox potential.
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Oxidative phosphorylation
• The energy released during the electron transfer reactions is used to
pump protons across the IMM.
• This creates a proton & a charge gradient ( inside becomes more
alkaline and more –vely charged)  electrochemical gradient.
• The energy stored in electrochemical gradient is termed “ proton
motive force” which is used to drive the ATP synthesis by movement of
protons down the electrochemical gradient through the ATP synthase.
• The process called chemiosmosis, was proposed originally by Prof.
Peter Mitchel ( NP 1978)
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• The chemiosmotic coupling theory explains how oxidative
phosphorylation links the ETC and ATP synthesis.
 The Chemiosmotic Theory
1. As electrons pass through the ETC, protons are pumped
into the intermembrane space, generating proton motive
force
2. Protons move back across the membrane through ATP
synthase driving ATP formation
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Machinery for ATP synthesis; ATP synthase
(Complex V)
 ATP synthase is a multiprotein enzyme
 Utilizes the electrochemical gradient across the IMM for ATP
synthesis
 Also c/a ATPase – isolated enzyme is capable of catalyzing
hydrolysis of ATP ADP + Pi
 Structure
 Consists of 2 domains
: F1 unit (ATP synthase) and F0 unit (transmembrane channel)
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Structure of ATP synthase
• F1 domain:
- Peripheral enzyme complex
bound to IMM.
- Involved in catalysis of ATP
synthesis
- Consists of 5 non-identical
subunits (α,β,ϒ,δ &ε)
α,β binding site for ATP, ADP
ϒ  forms central core
δ  attach F1 to IMM
• F0 domain
- Embedded in the membrane
- contains a transmembrane
channel ( Proton pumped across
the IMM during electron transfer
flow back into the matrix
through this.
- 3 subunits : a,b,c
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Synthesis of the ATP occurs on the surface of F1 domain
• Rxn
enz:ADP + Pi enz:ATP
• The results of isotopic-exchange experiments unexpectedly revealed that
enzyme-bound ATP forms readily on the surface of F1 domain even in
the absence of a proton-motive force.
• However, ATP does not leave the catalytic site unless protons flow
through the enzyme.
• Thus, the role of the proton gradient is not to form ATP but to release it
from the synthase.
• For the continuous synthesis of ATP, the enzyme must cycle between a
form that binds ATP very tightly and a form that releases ATP.
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Binding change mechanism for ATP synthesis
• Suggests that the 3 β subunits of the ATP synthase adopt different
conformations that change during catalysis with only one β subunit
acting as the catalytic site.
• Conformations adopted by β subunit
Open conformation (O) with a low affinity for ligands & is empty.
Loose conformation (L) with a low affinity for ligands & is inactive.
Tight conformation (T) with a high affinity for ligands & is active.
ATP synthesis occurs on the surface of the β subunit in the T
conformation.
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Rotation catalysis is the key to binding change mechanism for
ATP synthesis
• Paul Boyer proposed the rotational catalysis mechanism in which the 3
active sites (β) of F1 take turns catalyzing ATP synthesis.
• Passage of the protons through the F0 “pore” causes the cylinder of c
subunits & the attached ϒ subunits to rotate about the long axis of ϒ
which is perpendicular to the plane of the membrane.
• With each 1200 rotation, β adopts an another conformation. (β-
empty β-ADP β-ATP)
OR
( OLT)
In L conformation β binds to ADP & Pi
In T conformation there is synthesis of ATP from ADP & Pi
In O conformation there is release of ATP 34

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FIGURE 19–24 Binding-change model for ATP synthase.
At any given moment, one of these sites is in
the β–ATP conformation, a second is in the
β-ADP conformation, and a third is in the β-
empty conformation.
The proton motive force causes rotation of
the central shaft—the subunit, shown as a
green arrowhead—which comes into contact
with each subunit pair in succession.
This produces a cooperative conformational
change in which the β-ATP site is converted
to the β-empty conformation, and ATP
dissociates; the β-ADP site is converted to
the β–ATP conformation, which promotes
condensation of bound ADP Pi to form ATP;
and the β-empty site becomes a β-ADP site,
which loosely binds ADP Pi entering from the
solvent.

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Transporters on IMM
• Transport cytosolic ADP and Pi across the IMM to mitochondrial
matix.
& newly synthesized ATP to the cytosol.
Adenine nucleotide translocase :
Catalyses 1:1 exchange of ATP for ADP
Phosphate translocase:
transports cytosolic phosphate into the matrix along with a proton in
1:1 ratio
Both require the proton gradient to transport these molecules.
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ATP production and P:O ratio
P- referring to a high energy phosphate bond being synthesized.
O- referring to an atom of O being reduced.
P:O ratio is defined as the number of ATPs generated during the transfer
of 2 electrons through a segment of ETC.
3 energy conserving segments on ETC ( Phosphorylation sites)
This results in the release of sufficient energy for synthesis of ATP
Site I : between NADH & UQ
Site II: between UQH2 & Cytc
Site III: between Cyt c & O2
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3 sites correspond to Complex I, III & IV of the ETC.
Since complex I is bypassed when substrate is succinate ( with FADH2 )
only 2 ATPs are generated ( P:O ratio=2)
for NADH P:O ratio =3
However, since 10 protons are pumped across IMM during transfer of 2
electrons from NADH to O2 & 4 protons are req. for synthesis of ATP.
So,
Actual P:O ratio for NADH = 2.5
for FADH2 = 1.5
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Regulation of the oxidative phosphorylation
• Oxidative phosphorylation is regulated by cellular energy demands.
• The intracellular [ADP] and the mass-action ratio [ATP]/([ADP][Pi]) are
measures of a cell’s energy status. Normally this ratio is very high, so
the ATP-ADP system is almost fully phosphorylated.
• When the rate of some energy-requiring process (protein synthesis, for
example) increases, the rate of breakdown of ATP to ADP and Pi
increases, lowering the mass-action ratio.
• With more ADP available for oxidative phosphorylation, the rate of
respiration increases, causing regeneration of ATP. This continues until
the mass-action ratio returns to its normal high level, at which point
respiration slows again.
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Many poisons inhibit the respiratory chain
• Barbiturates such as amobarbital inhibit electron transport via
Complex I by blocking the transfer from Fe-S to Q. At sufficient
dosage, they are fatal in vivo.
• Antimycin A and dimercaprol inhibit the respiratory chain at Complex
III
• The classic poisons H2S, carbon monoxide, and cyanide inhibit
Complex IV and can therefore totally arrest respiration
• Malonate is a competitive inhibitor of Complex II.
• Atractyloside inhibits oxidative phosphorylation by inhibiting the
transporter of ADP into and ATP out of the mitochondrion
• The antibiotic oligomycin completely blocks oxidation and
phosphorylation by blocking the flow of protons through ATP
synthase
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Electron Transport Inhibitors
 Several molecules specifically inhibit the
electron transport process
 When electron transport is inhibited, O2
consumption is reduced or eliminated
 Important for understanding the correct
order of ETC components
• CN– = cyanide ; CO = carbon monoxide; H2S =
hydrogen sulfide ; NaN3 = sodium azide ; FMN =
flavin mononucleotide ;FAD = flavin adenine
dinucleotide ; CoQ = coenzyme Q; Cyto =
cytochrome
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Uncouplers
• Oxidation & phosphorylation are coupled processes. They can be
uncoupled from each other by certain compunds called uncouplers.
• Primary action of the uncouplers - increase permeability of the IMM to
protons.
• As a result, relatively free movement of the protons across the IMM
occurs, which prevent building of the electrochemical gradient stops
ATP generation.
• E.g. Of uncouplers DNP : lipophilic ( freely move across IMM)
can carry a proton along with, so prevent builing up of proton gradient.
Other e.g. Pentachorophenol,
dinitrocresol,
trifluorocarbonylcyanide
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Significance of uncoupling
• Uncoupling proteins (UCP) occur in the inner mitochondrial membrane
of mammals, including humans.
• Brown adipose tissue which are specialised to carry out oxidation
uncoupled from phosphorylation.
• When fats are oxidised in the brown adipose tissue, the energy
liberated is not trapped as ATP instead released as heat.
• UCP1, also called thermogenin is responsible for the activation of fatty
acid oxidation and heat production in the brown adipocytes of
mammals.
• Brown fat, unlike the more abundant white fat, wastes almost ninety
percent of its respiratory energy for thermogenesis in response to
cold, at birth, and in hibernating animals.
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Inherited defects in oxidative phosphorylation
• 13 of the approximately 100 polypeptides required for oxidative
phosphorylation are coded for by mitochondrial DNA
• whereas the remaining mitochondrial proteins are synthesized in the
cytosol and transported into mitochondria.
• mtDNA has a mutation rate about ten times greater than that of nuclear
DNA.
• mtDNA is maternally inherited because mitochondria from the sperm cell
do not enter the fertilized egg.
• Tissues with the greatest ATP requirement (for example, CNS, skeletal and
heart muscle, kidney, and liver) are most affected by defects in oxidative
phosphorylation.
• Mutations in mtDNA are responsible for several diseases, including some
cases of mitochondrial myopathies - Leber's hereditary optic neuropathy,
a disease in which bilateral loss of central vision occurs as a result of
neuroretinal degeneration, including damage to the optic nerve.
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Thank-you
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