Oxidative phosphorylation is a process that generates ATP through the transfer of electrons from NADH or FADH2 to oxygen, occurring in the mitochondria of eukaryotes. The process involves an electron transport chain and the chemiosmotic theory, where proton movement creates an electrochemical gradient that drives ATP synthesis via ATP synthase. This mechanism is responsible for about 90% of ATP production in cells during aerobic respiration.

1. OXIDATIVE
PHOSPHORYLATION
Dr. Divya Sharma
Assistant Professor
A Biodiction (A Unit of Dr. Divya Sharma)

2. RESPIRATION
▪Respiration: A process where cells derive energy
with a controlled reaction between H+ and O2; &
the end product being water.
▪Aerobic organisms are able to capture a far greater
proportion of the available free energy of
respiratory substrates than anaerobic organisms.
▪Objective of respiration: To produce ATP

3. ▪In the form of electrons, energy released from
oxidation reactions.
▪Electrons are shuttled by electron carriers (eg.
NAD+) to an Electron Transport Chain (ETC
reaction).
▪Electron energy is converted to ATP in the electron
transport chain.

4. METABOLISM
▪Metabolism: Sum of the chemical reactions in an
organism.
▪Catabolism: Energy releasing processes
▪Anabolism: Energy using processes
▪Catabolism provides the building blocks and
energy for anabolism.

5. Anabolism and Catabolism Reaction

6. Anabolism and Catabolism Coupling Reaction

7. ❑ Oxidative phosphorylation is the process of ATP
formation, when electrons are transferred by
electron carriers from NADH or FADH2 to oxygen.
❑ Oxidation coupled with phosphorylation is called
Oxidative phosphorylation.
❑ Mitochondria are the site of oxidative
phosphorylation in eukaryotes.
❑ During transfer of electrons through the ETC
energy is produced.
OXIDATIVE PHOSPHORYLATION

8. This energy is coupled to the formation of
ATPfromADP.
By an enzyme F0F1 ATPase.
A. Oxidation step: Electron transport chain
NADH + H+ + O2 --------→ NAD+ + H2O
FADH2 + O2 ---------→ FAD + H2O
B. Phosphorylation step
ADP + Pi --------→ ATP

9. MITOCHONDRIA
▪Mitochondria: “Powerhouse of the cell” since the
final energy release takes place in the
mitochondria only.
▪Mitochondria is the site of oxidative
phosphorylation in eukaryotes.

10. The NADH and FADH2 , formed during glycolysis, ᵝ-
oxidation of fatty acids and the TCA cycle, give up
their electrons to reduce molecular O2 toH2O.
Electron transfer occurs through a series of protein
electrons carriers, the final acceptor being O2; the
pathway called as the Electron Transport Chain
(ETC).
Function of ETC: To facilitate the controlled release of
free energy that was stored in reduced cofactors
during catabolism.

11. Energy is released when electrons are
transported from higher energy
NADH/FADH2 to lower energy O2.
This energy is used to phosphorylate ADP.

12. 3 sites of the chain that can give enough energy
forATP synthesis these sites are:
1. Site I between FMN and coenzyme Q at
enzyme complex I.
2. Site II between cyt a and cyt C1 at enzyme
complex III.
3. Site III between cyt a and cyt a3 at enzyme
complex IV.

15. Because energy generated by the transfer of electrons
through the electron transport chain to O2 is used in the
production of ATP.
The overall process is known as Oxidative
Phosphorylation.
Oxidative phosphorylation is responsible
for 90% of total ATP synthesis in thecell.

16. TWOWAYSTOSYNTHESIZE ATP
Oxidative Phosphorylation:
The phosphorylation of ADP to ATP coupled to
electron transfer.
Substrate Level Phosphorylation:
Direct transfer the phosphate from chemical
intermediate (also called substrate) to ADP or GDP
forming ATP or GTP, independent of electron transfer
chain.

17. SUBSTRATE LEVEL
PHOSPHORYLATION

18. MECHANISM OF OXIDATIVE
PHOSPHORYLATION

19. MECHANISM OF
OXIDATIVE PHOSPHORYLATION
Several hypotheses have been put forth to explain the
process of oxidative phosphorylation .
The most important among them namely:
▪ Chemical Coupling Hypothesis
▪ Chemiosmotic Theory

20. OXIDATIVE PHOSPHORYLATION

21. CHEMICALCOUPLING
HYPOTHESIS
This hypothesis was put forth by Edward Slater
(1953).
According to this hypothesis, during the course of
electron transfer in respiratory chain, a series of
phosphorylated high – energy intermediates are
first produced which are utilized for the synthesis
of ATP.
These reactions are believed to be analogous to the
substrate level phosphorylation that occurs in glycolysis
or citric acid cycle.
Eventually, this hypothesis lacks of experimental
evidences.

22. Mechanism of Oxidative
Phosphorylation:
CHEMIOSMOTIC
THEORY
Peter Mitchell
(1920-1992)

23. CHEMIOSMOTIC THEORY
This hypothesis is proposed by Peter Mitchell in
1961.
Toexplain the oxidative phosphorylation.
Nobel prize, in 1978
According to chemiosmotic hypothesis the electron
transport chain is organized so that protons move
outward from the mitochondrial matrix to inter-
membrane space (in eukaryotes; Fig. 24.6) and from
cytoplasm to periplasmic space passing across the
plasma membrane (in prokaryotes; Fig. 24.7).

26. It suggests that the transfer of electrons through the
ETC causes protons to be translocated (pumped out)
from the mitochondrial matrix to the intermembrane
space at the three sites of ATP production.
It results in an electrochemical potential difference
across the inner mitochondrial membrane.
The electrical potential difference is due to accumulation
of the H+ ions outside the membrane and the chemical
potential difference in pH, being more acidic outside the
membrane.
This electrochemical potential difference drives ATP
synthase to generate ATP from ADP and inorganic
phosphate.

28. CHEMIOSMOTIC THEORY
Basic 3 principles follows:
1. Pumping of protons via electron carrier proteins
2. Generation of electrochemical potential
a. Membrane potential
b. Proton gradient (Chemical potential)
3. Electron transport flow back to matrix through
ATPase.

29. CHEMICAL THEORY
❑ Suggests that there is a direct chemical coupling
of oxidation and phosphorylation through high-
energy intermediate compounds. This theory is
not accepted, as the postulated high-energy
intermediate compounds were never found.

30. 1. Generation of Proton Motive Force
(PMF):
❑ When O2 is reduced to H2O after accepting electrons
transferred from electron transport chain, it requires
proton (H+) from the cytoplasm to complete the reaction.
❑ These protons originate from the dissociation of water
into H+ and OH–. The use of H+ in the reduction of O2 to
H2O and the extrusion of H+ outside the membrane
during electron transport chain (Fig. 24.8) cause a net
accumulation of OH– on the inside of the membrane.

31. ❑ Despite their small size, because they are charged, neither
H+ nor OH– freely passes through the membrane, and so
equilibrium cannot be spontaneously restored on both
sides of membrane.
❑ This non-equilibrium state of H+ and OH– on opposite sides
of the membrane results in the generation of a pH gradient
and an electrochemical potential across the membrane,
with the inside of the membrane (cytoplasm side)
electrically negative and alkaline, and the outside of the
membrane electrically positive and acidic.
❑ This pH gradient and electrochemical potential cause the
membrane to be energized. The energised state of a
membrane, which is referred to as proton motive force
(PMF) and is expressed in volts, is used directly to drive
the formation of ATP, ion transport, flagellar rotation, and
other useful work.

33. 2. Proton Motive Force and ATP
Synthesis:
❑ Proton motive force-derived ATP synthesis involves a
catalyst, which is a large membrane enzyme complex
called ATP synthase or ATPase for short.

35. ATPase contains two major parts:
(1) A multi-subunit head piece called F1 located on
mitochondrial matrix side (in eukaryotes) and on
cytoplasmic side (in prokaryotes).
(2) A proton conducting channel called F0 that resides in the
inner membrane of mitochondrion (in eukaryotes) and in
plasma membrane (in prokaryotes) and spans the
membrane.

36. ➢ The ATP synthesis takes place at the F1/F0 ATPase,
which is the smallest known biological motor.
➢ F1, is the catalytic complex responsible for the inter
conversion of ADP + Pi (inorganic phosphate) and
ATP, and consists of five different polypeptides
present as an α3 β3 ϒƐδ complex.
➢ F0 is integrated in the membrane and consists of
three polypeptides in an ab2 c12 complex. 3, 3, 2 and
12 denote the numerical numbers of α, β, b and c,
respectively.

37. ❑ According to the current model of how the ATPase
functions in Escherichia coli (Fig. 24.9), subunit ‘a’ is
responsible for channeling protons (H+ ) across the
membrane while subunit b protrudes outside the
membrane and forms, along with b2 and δ subunit,
the stator. Protein movement through ‘a’ submit of
F0 drives rotation of the c proteins generating a torque
that is transmitted to F1, by the ϒε subunits.
❑ As a result, energy is transferred to F1through
coupled rotation of yε subunits causing
conformational changes in the β subunits. The
conformational changes in the β subunits allow for
binding of ADP + Pi and these are converted to ATP
when the β subunits return to their original
conformation.

38. Inhibition of Oxidative
Phosphorylation (ATP Synthesis):
❑ Many chemicals inhibit the synthesis of ATP and can
even kill cells to sufficiently high concentrations. Two
such classes of chemicals are known inhibitors and un-
couplers. Inhibitors directly block electron transport
chain.
❑ The antibiotic piericidin competes with coenzyme Q; the
antibiotic antimycin. A blocks electron transport between
cytochromes b, and c, and both carbon monoxide (CO)
and cyanide (CN–) bind tightly to certain cytochromes
and prevent their functioning.

39. ❑ The un-couplers, in contrast, prevent ATP synthesis
without affecting electron transport chain itself. Normally
the electron transport chain is tightly coupled with
oxidative phosphorylation, and the un-couplers
disconnect oxidative phosphorylation from electron
transport chain.
❑ Therefore, the energy released by the chain is given off
as heat rather than as ATP. Many lipid soluble un-
couplers (e.g., dinitrophenol, dicumarol, valinomycin)
make membranes leaky allowing free passage of
protons through the membrane without activating
F1/F0 ATPase. In this way they destroy the proton motive
force and its ability to drive ATP synthesis.

40. ATPSynthesis
▪ ∆G○ for transfer of 2 electrons from NADH to O2 is –
220 kJ/ mol.
This is sufficient to synthesize 7 molecules of ATP. (∆Go’
for ATP synthesis is 31 kJ/mol).
▪ However, a significant amount of energy is used
up to pump H+ out of the mitochondria. Only a third is
used for ATP synthesis

41. The number of ATP generated depends on the site at
which the substrate is linked to the respiratory chain:
➢ If the substrate is linked to the chain through NAD+ , 3
ATP are formed for each molecule oxidized.
➢ If the substrate is linked to the chain through FAD , 2
ATP are formed.

43. P/ORATIO
It is the ratio of the number of molecules of ADP
converted to ATP to the number of oxygen atoms
utilized by respiratory chain.
It is a measure to the efficiency of oxidative
phosphorylation.
It is 3/1 if NADH+H+ is used and 2/1 if FADH2 is
used.

44. Difference# Oxidative
Phosphorylation: (in plants)
1. It takes place during aerobic respiration (a catabolic
process) on cristae in mitochondria.
2. It occurs during terminal oxidation of reduced
coenzymes generated in glycolysis and Krebs’ cycle
with molecular oxygen.
3. It is independent of light.
4. It is associated with mitochondrial electron transport
system and is of only one type.
5. Ultimate source of energy for oxidative phosphorylation
is respiratory substrate.
6. It is inhibited by 2, 4—dinitro phenol

45. A Biodiction (A Unit of Dr. Divya Sharma)