Biological oxidation (part - III) Oxidative Phosphorylation - Mechanism of Oxidative Phosphorylation -- Chemiosmotic theory -P:O Ratio  Substrate Level Phosphorylation  Shuttle Systems for Oxidation of Extramitochondrial NADH

1. ADP + Pi
ATP
b2
δ
α
α
α
β
β β
H+
Fo
F1
( Part –III )

2. Definition
➢The synthesis of ATP from ADP
(phosphorylation), that occurs when
NADH and FADH2 are oxidized by
through electron transport chain
(respiratory chain)
➢Oxidation coupled with phosphorylation
is called Oxidative phosphorylation
➢Mitochondria are the site of oxidative
phosphorylation in eukaryotes
Oxidative Phosphorylation

3. During transfer of electrons through the
ETC energy is produced.
This energy is coupled to the formation of
ATP from ADP.
By an enzyme F0F1 ATPase.

5. ✓ 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.

6. Example of
Substrate level phosphorylation
Glycolysis Phosphoglycerate
kinase
1,3-bisphosphoglycerate
3-phosphoglycerate
ADP
ATP

7. Phosphoenolpyruvate
ADP
ATP
Pyruvate
Pyruvate kinase
Succinyl CoA
GDP
GTP
succinate
succinyl CoA
synthetase
Glycolysis
TCA cycle

9. Several hypotheses have been put forth to explain the
process of oxidative phosphorylation.
The most important among them-namely,

10. 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.
However, this hypothesis lacks experimental evidence.

11. CHEMIOSMOTIC
THEORY
Peter Mitchell
1920-1992

12. This hypothesis is the most accepted theory.
proposed by Peter Mitchell in 1961.
To explain the oxidative phosphorylation.
Nobel Prize, in 1978
It explains how the transport of electrons through
the respiratory chain (ETC) is effectively utilized to
produce ATP from ADP + Pi.

13. There are three basic principles of the theory.
1. Pumping of protons via electron carrier proteins
2. Generation of electrochemical potential.
i. Membrane potential
ii. Proton gradient (chemical potential)
3.Electron transport flow back to matrix through
ATPase.

14. The inner mitochondrial membrane, is
impermeable to protons & hydroxyl ions.
The transport of electrons through ETC is
coupled with the translocation of protons (H+)
across the inner mitochondrial membrane from
the matrix to the intermembrane space.
This results in an electrochemical or proton
gradient.

15. Electrochemical
gradient

17. 10H+
Complex
III
4H+
2H+
4H+
4H+4H+
2H+
NADH+ +H+
NAD+
O2
H2O
FoF1
Negative
(Alkaline)
Positive
(Acidic)
Outer mitochondrial membrane
10H+
2.5ADP
+2.5Pi
2.5
ATP
Chemiosmotic theory

18. Complex I and complex III
pumps 4 protons each
Complex IV pumps 2 protons
To inter-membranous space
10 protons are pumped by the
electron transport chain
The transfer of two electrons
from NADH+ H+ to O2 is
accompanied by the outward
pumping of 10 H+
10 protons are pumped out per NADH
1. 4 must flow in to produce 1 ATP
2. The proton-based P/O ratio is 2.5 for NADH as the electron donor and 1.5
(6/4) for succinate

19. ATP generation (old and new values)

20. In summary :
The oxidative phosphorylation process is as follow
Electron transport down the respiratory chain from
NADH or FADH2
Complex Ⅰ,Ⅲ,Ⅳ Cause protons be pumped out of the
mitochondrial matrix into the intermembrane space
The pumping out of H + generates a higher conc. of H+ and an
electrical potential , thus an electrochemical proton gradient is formed.
The H + flow back into the mitochondrial matrix through ATP synthase
and the electrochemical proton gradient drives ATP synthesis

21. Proposed by Paul Boyer in 1964
(Nobel Prize, 1997)

22. Fo F1 ATPase
F0
F1
Also called ATP
synthase.
Embedded in the
inner membrane
ADP + Pi
ATP
b2
δ
α
α
α
β
β β
H+
Fo
F1

23. ⚫Also called complex Ⅴ
⚫It is the enzyme that actually
synthesize ATP
⚫It located in the inner
mitochondrial membrane
⚫It utilizes energy from the proton
gradient to promote phosphorylation of
ADP forming ATP
ADP + Pi
ATP
b2
δ
α
α
α
β
β β
H+
Fo
F1

24. ❑Spherical projections from the inner membrane.
❑Is composed of two major components part,
❑F1 unit or called F1 ATPase :
❑The spheres of the ATP synthase &
point outward
❑F0 unit :
❑Spans the inner mitochondria membrane
❑So the ATP synthase is also called F0F1 ATPase
❑The stalk between F0 and F1 contains several
additional polypeptide

25. F1 unit :
Contains 5 types of polypeptides
Arranged in α3β3γδε.
F0 unit:
Made of abc polypeptides,
Is proton channel / proton transport.
F1 with F0 together can synthesize ATP
Only F1 components have ATPase activity, so also
called ATPase,
ADP + Pi
ATP
b2
δ
α
α
α
β
β β
H+
Fo
F1

26. ETC and Oxidative phosphorylation

29. P:O ratio
The P:O ratio refers to the number of inorganic
phosphate molecules incorporated into ATP for
every atom of oxygen consumed.
When a pair of electrons from NADH reduces an
atom of oxygen (½ O2), 2.5 mol of ATP are formed
per 0.5 mol of O2 consumed.
This results in conversion of energy required for
production of only 3 ATP from NADH and 2 ATP from
FADH2

30. The mitochondrial oxidation of NADH with a
classical P : O ratio of 3 can be represented by
the following equation :
NADH + H+ + ½ O2+ 3ADP + 3Pi
NAD+ + H2O+ 3ATP

31. Current Concept, Energetics of ATP Synthesis
According to the estimated free energy of synthesis, it was
presumed that around 3 protons are required per ATP
synthesized.
Hence when 1 NADH transfers its electrons to oxygen, 10
protons are pumped out.
This would account for the synthesis of approximately 3 ATP.
Similarly the oxidation of 1 FADH2 is accompanied by the
pumping of 6 protons, accounting for 2 molecules of ATP.
However, Peter Hinkle recently proved that the actual energy
production is less, because there is always leakage of protons.
This results in harnessing of energy required for the
production of 2.5 ATP from NADH and 1.5 ATP from FADH2.

32. Complex-I
Ⅱ
Complex-IV
F0
F1
Complex-III
Cyt c
Q
NADH+H+
NAD+
Fumarate
Succinate
H+
1/2O2+2H+
H2O
ADP+Pi ATP
H+
H+
H+
Intermembrane space
matrix
+ + + + + + + + + +
- - - - - - - - -
Coupling sites for ATP synthesis.

33. Generate ATP sums
2.5 ATP are synthesized per NADH oxidized
through the NADH respiratory chain
1.5 ATP are synthesized per FADH2 oxidized
through the FADH2 respiratory chain

34. Why ATP synthesize from FADH2 respiratory chain is
less than NADH respiratory chain?
NADH respiratory have 3 H+ pump, complex
Ⅰ,Ⅲ,Ⅳ
 FADH2 respiratory only have 2 H+ pump,
complex Ⅲ,Ⅳ
So the ATP made from FADH2 is less than from
NADH

36. Uncouplers

37. Electron transport is normally tightly coupled to
ATP synthesis
Electrons do not flow through the ETC to O2
unless ADP is simultaneously phosphorylated to
ATP.
Also, ATP is not synthesized unless
electron transport is occurring to provide the
proton gradient
The transport of electrons is tightly coupled with
oxidative phosphorylation (ATP synthesis).

38. There are certain compounds that can uncouple (or
delink) the electron transport from ATP synthesis.
Such compounds, called as uncouplers.
 the permeability of inner mitochondrial
membrane to H+.
The result is that ATP synthesis does not occur.
The energy is dissipated as heat.

39. What is uncouplers?
There are certain compounds that can
uncouple (or delink) the electron
transport from ATP synthesis.
2,4-dinitrophenol
(DNP)
Thermogenin
(Natural uncoupling
protein)

40. 2,4-dinitrophenol (DNP)

41. ?

42. ?
◆ electron transport occurs and pump out H+ ions
across the inner mitochondrial membrane to build the
H+ gradient .
◆But DNP in the same membrane carriers the H+ ions
back into the mitochondrion , preventing formation of
a proton gradient.
◆Since no proton gradient forms, so no ATP can be
made by oxidative phosphorylation
◆the energy derived from electron transport is
released as heat.

43. There is brown adipose tissue in the body,
 This tissue is rich in mitochondria, the
inner mitochondrial membranes of which
contains a protein called uncoupling
protein or thermogenin.

44. How uncoupling protein work ?
Uncoupling protein can be seen as a H+ passageway,
allows H+ to flow back into mitochondria without
having to enter the ATP synthase, thus
 Preventing formation of a proton gradient , so
uncouples electron transport & oxidative
phosphorylation.
Energy derived from electron transport is released
as heat

45. mechanism of uncoupling protein
（brown adipose tissue mitochondrial ）
Ⅲ
Ⅰ Ⅱ
F0
F1
Ⅳ
Cyt c
Q
Intermembrane space
Matrix
Uncoupling
protein
Heat energy
H+
H+
ADP+Pi ATP

46. ◆The production of heat by uncoupling is called
non-shivering thermogenesis.
◆It is important in certain biological situation ,
◆For example ,the brown adipose tissue is found in
sensitive body areas of some new brown animals
(including human ), where the heat production
provides protection from cold condition
◆In addition, thermogenesis by brown adipose
tissue plays a important role in maintaining body
temperature in hibernating animals

51. The availability of ADP regulates the process.
When ATP level is low and ADP level is high,
oxidative phosphorylation proceeds at a rapid rate.
This is called respiratory control or acceptor
control.

52. This mechanism ensures that electrons flow down
the chain only when ATP synthesis is needed.
If the level of ATP is high, ADP is low,
no electron transport occurs
NADH and FADH2 build up,
so does excess citrate
citric acid cycle & glycolysis are all inhibited

53. Over all
ADP high
Oxidative phosphorylation rises
Oxygen consumption rises
ADP low
Oxidative phosphorylation falls
Oxygen consumption falls

54. ATP

55. Adenosine triphosphate: ATP
Glycosidic
bond
N
O
CH2O
OHOH
N
N
N
NH2
P
O
OH
OP
O
OH
OP
O
OH
OH
ATP
Ester bond
αβr
Ribose
Adenine

56. Production and application of ATP
ATP
ADP
oxidative
Phosphorylation
~P
~P
Mechnism energy
Osmotic energy
Chemical energy
Electric energy
Hot energy
substrate level
Phosphorylation

58. The inner membrane of mitochondria
impermeable to some molecule and ions
Permeable to :
pyruvate, succinate, Citrate, α-
ketoglutarate, malate, Glu etc
Impermeable to :
H+, NADH, NADPH, oxaloacetate, etc

59. ◆The inner mitochondrial membrane is
impermeable to NADH.
◆Therefore NADH produced in the cytoplasm
during glycolysis go into the mitochondria through
the membrane shuttle , then in the mitochondria go
into the respiratory chain .
◆The membrane shuttle is a combination of enzyme
reaction that bypass this impermeability barrier

60. Which reaction of glycolysis produce NADH
Glyceraldehyde 3-phosphate
1,3-bisphosphoglycerate
Glyceraldehyde 3-phosphate
Dehydrogenase
NAD+
NADH
The reaction take place in the cytosol

61. There is two shuttle system in the
mitochondrial membrane

62. NADH+H+
Glycerol
3-phosphate
DH
FADH2
NAD+
FAD
inner
Membrane
outer
Membrane
Intermembrane
Space
matrix
electron
chain
DHAP
Glycerol
3-P
Glycerol 3-phosphate shuttle
Glycerol
3-phosphate
DH
DHAP
Glycerol
3-P

63. Note :
◼ The shuttle does not allow cytoplasm NADH to
enter the mitochondrion,
◼ But transports the two electrons from NADH into
the mitochondria
◼ Feed the electron into the FADH2 electron transport
chain .
◼ So synthesize 2ATPs

64. NADH
+H+
NAD+
-OOC-CH2-C-COO-
O
-OOC-CH2-C-COO-
OH
H
NADH
+H+
NAD+
glutamate –
aspartate
carrier
malate
α-ketoglutarate
carrier
-OOC-CH2-C-COO-
OH
H
malate
-OOC-CH2-C-COO-
O
oxaloacetate
-OOC-CH2-CH2-C-COO-
O
-OOC-CH2-CH2-C-COO-
O
-
OOC-CH2
-CH2
-C-COO
-
H3
N
+
H
glutamate
malate
dehydrogenase
aspartate
aminotransferase
intermembrane space
inner
memebrane
matrix
-
OOC-CH2
-C-COO
-
H3
N
+
H
asparate
-
OOC-CH2
-C-COO
-
H3
N
+
H
-
OOC-CH2
-CH2
-C-COO
-
H3
N
+
H
α-ketoglutarate
malate
α-ketoglutarate
glutamate

65. this cycle of reactions is to transfer the electrons from
NADH in the cytosol to NADH in the mitochondrial
matrix ,
The NADH in the mitochondria is then reoxidized by the
NADH electron transport chain
So synthesize 3ATPs
Note

66. In summary :
Cytosol NADH go into the mitochondria by this two shuttele
• glycerol 3-phosphate shuttle
in the mitochondria go into the FADH2 respiratory chain.
so produce 2 ATPs
Malate-asparate shuttle:
in the mitochondria go into the NADH respiratory chain.
so produce 3ATPs