The document summarizes electron transport chain and oxidative phosphorylation. It discusses: 1) The four complexes of the electron transport chain located in the inner mitochondrial membrane that facilitate the transfer of electrons from NADH and FADH2 to oxygen. This creates a proton gradient used by ATP synthase to generate ATP. 2) The enzymes, electron carriers like cytochromes and iron-sulfur proteins, and redox reactions involved in electron transport. 3) How the proton gradient is used by ATP synthase to drive ATP synthesis via chemiosmosis. 4) Inhibitors and uncouplers that disrupt the proton gradient or electron transport.

1. ELECTRON TRANSPORT CHAIN
PROF. (DR.) V.P.ACHARYA

2. Biological Oxidation
Oxidation- removal of electrons
Reduction- gain of electrons
Electron donator- reducing agent/ reductant;
gets oxidized itself
Fe++ (reduced)  Fe+++ (oxidized) + e-
Electron acceptor- oxidizing agent/ oxidant; gets
reduced itself
Two important e- carriers in metabolism: NAD+
& FAD

3. NAD+, Nicotinamide Adenine Dinucleotide,
is an electron acceptor in catabolic
pathways.
The Nicotinamide ring, derived from the
vitamin niacin, accepts 2 e- & 1 H+ (a
hydride) in going to the reduced state,
NADH.
NADP+/NADPH is similar except for Pi.
NADPH is e- donor in synthetic pathways.

4. The electron transfer reaction may be
summarized as :
NAD+ + 2e- + H+  NADH
It may also be written as:
NAD+ + 2e- + 2H+  NADH + H+

5. Redox couple
• When a substance exists both in the reduced
and oxidized state, the pair is called a redox
couple
• Redox potential- Electromotive force
measured by (EMF)
• Positive redox potential- higher affinity for e
than H+
• Negative redox potential- lower affinity for e
than H+

6. Redox potential
Analogous expression of standard free energy
Eo’
Redox couple
Electron flows from one redox couple to
another in the direction of more positive
system
↑negativity- ↑tendency to lose electrons
(more affinity towards H)
↑positivity- ↑ tendency to accept electrons

7. The more negative redox potential represents a greater tendency to
lose electrons

8. Substrate level phosphorylation
• Energy from a high energy compound is
directly transferred to nucleoside
diphosphate to form NTP
• 3 steps
• 1,3 –BPG (Glycolysis)
• Phosphoenolpyruvate (Glycolysis)
• Succinyl CoA (TCA cycle)

9. Do not pretend- be
Do not promise- act
Do not dream- realise

10. Electron Transport and
Oxidative Phosphorylation
It all reduces down to water.

11. • Primary metabolism
• Secondary metabolism
• Tertiary metabolism

12. Biological oxidation
• Transfer of electrons from the reduced
co-enzymes through the respiratory
chain to oxygen
• Electrons flow from electronegative
potential (-0.32) to electropositive
potential (+0.82)- unidirectional flow

13. Oxidative Phosphorylation
• Energy released during biological
oxidation- trapped to form ATP
• Oxidation + phosphorylation= Oxidative
phosphorylation

14. How it all happened???
• Eugene Kennedy & A.
Lehninger – Discovered that
mitochondria is the site of
Ox. Phosphorylation
Helmut Beinert - FeS
proteins-
John E Walker-
Crystallographic str of F1
structure

15. Electron transport chain

16. Why Electron transport chain?
• Gradual flow of electrons through a sequence
of dehydrogenases- Electron transport chain
• NADH→ H2O; ∆ G0’= 53 Kcal/ mol
• The amount of energy is so huge that if
produced at one stretch then body may not be
able to use it
• Through ETC this energy is released in small
increments- trapped in Chemical bond energy-
ATP

17. Location of enzymes in mitochondria
Mitochondria outer membrane:
MAO
Acyl CoA synthatase
Phospholipase A2
Inter membrane space:
Adenylate kinase
Creatine kinase
Inner membrane outer surface:
Glycerol-3- P dehydrogenase
Inner membrane inner surface:
Succinate dehydrogenase
Enzymes of ETC
Soluble matrix:
TCA cycle enzymes
Beta oxidation of FA enzymes

18. • Groups of redox proteins
– Found on inner mitochondrial membrane
– Binding sites for NADH and FADH2
• On matrix side of membrane
• Electrons transferred to redox proteins
But NADH is impermeable to
mitochondrial membrane !!!

19. How NADH crosses the
mitochondrial membrane??
• Malate - aspartate shuttle- liver, kidney & heart
• Glycerol-3- Phosphate shuttle

20. Glycerol-3-P shuttle- operates in skeletal
mm and brain
• Cytoplasmic NADH is carried as FADH2 !!

21. Creatine phosphate shuttle
• Carries active phosphate from mitochondria
to extra-mitochondrial sites
• Skeletal mm & Heart
• MtCK - mitochondrial CK involved in the
shuttle
• Ubiquitous mtCK (umtCK) & Sarcomeric mtCK
(smtCK)

22. Enzymes, coenzymes and electron carriers
Oxidoreductases (Enzymes)
1. Oxidases- removal of H using O as a H acceptor; cyt
oxidase, tyrosinase, MAO
2. Dehydrogenases- Removal of H but O not as an
acceptor; Require co-enzymes NAD, NADP, FMN, FAD
3. Hydroperoxidases- Peroxidases & Catalases;
Hydrogen peroxide or organic peroxide are
eliminated
4. Oxygenases- Addition of 1 or both of the atoms of O2
a) Monooxygenases (mixed function oxidases)
b) dioxygenases (true oxygenases)

23. Dehydrogenases
• NAD+ dependent dehydrogenase- ADH,
Glycerol-3-P dehydrogenase
• NADP+ dependent dehydrogenase- HMG CoA
reductase, Enoyl reductase
• FMN dependent dehydrogenase- NADH
dehydrogenase
• FAD dependent dehydrogenase- Succinate
dehydrogenase, Acyl CoA dehydrogenase
• Cytochromes- all except cytochrome oxidase
(aa3)

24. Oxygenases
Mono-oxygenases- Incorporate one atom of oxygen (1/2
O2)
• NADPH provides the reducing equivalents
• Ex- Cyt P450 monooxygenase system in microsome –
drug metabolism (morphine, aniline, aminopyrine)
• Biosynthesis of steroid hormones
Dioxygenases- Incorporates both atoms of oxygen
Ex- Homogentisate oxidase, L-Tryptophan pyrrolase

25. Electron carriers
• Cytochromes - Fe containing electron
transferring proteins
• 3 classes- a, b , c
• Cytochrome oxidase (Cyt aa3)- oxidase enz
• Rest all- Dehydrogenase
• ETC b→ c1 → c → aa3
• Cyt c- water soluble
• Cytochromes are also found in ER

26. ETC carriers: Cytochromes
Heme proteins
Cytochrome c is
a soluble
peripheral
protein
Most are integral
proteins

27. Iron-sulfur protein (Fe4S4)
• Fe not in heme form
• 8 Fe-S proteins participate
• One electron transfer and 2 H+ transferred
• Fe+++
3, Fe++
1 (oxidized) + 1 e-  Fe+++
2, Fe++
2
• (reduced)
• About 6 FeS proteins are discovered
• Mechanism not very clear
• Associated with FMN→ CoQ and Cyt b and C1
Helmut Beinert

28. Ubiquinone
O
O
CH3O
CH3CH3O
(CH2 CH C CH2)nH
CH3
OH
OH
CH3O
CH3CH3O
(CH2 CH C CH2)nH
CH3
2 e-
+ 2 H+
coenzyme Q
coenzyme QH2
FAD FeS
FeS
FeS
FMN
NAD+
ubiquinone
Cyt b
ubiquinone

29. • Coenzyme Q (CoQ, Q or Ubiquinone) is lipid-
soluble.
• Accepts 2e- via complex I & II
• Q→ QH2
• It stays dissolved in the hydrocarbon core of a
membrane.
• Only electron carrier not bound to a protein.
• it can accept/donate 1 or 2 e-

30. If an idea presents itself to us, we must
not reject it simply because it does not
agree with the logical deductions of a
reigning theory.
—Claude Bernard

31. ETC- oxidizes reducing equivalents and acts a proton
pump

32. NAD+
FMN
FeS
ubiquinoneFAD FeS
Cyt b
FeS Cyt c1 Cyt c Cyt a Cyt a3
1/2 O2
ubiquinone
I
II
III IV
NADH Dehydrogenase
Succinate
dehydrogenase
Cytochrome Oxidase
CoQ-cyt c Reductase

33. 4 protein complexes in ETC

34. Also mentioned as….
1. NADH-CoQ reductase/ NADH
dehydrogenase complex
2. Succinate –Q-reductase
3. Cytochrome reductase
4. Cytochrome oxidase
5. ATP synthase

35. Order and Reduction Potentials

36. Complex I (NADH→ Ubiquinone)
• NADH dehydrogenase complex
• Has NADH binding site
– NADH reductase activity
• NADH - NAD+
– NADH ---> FMN--->FeS --> ubiquinone
– Ubiquinone ---> Ubiquinone H2
– 4 H+ pumped/NADH
 25 different proteins
 Energy released- 12 Kcal/mol
 Energy utilised to pump out protons
 1 ATP generated

37. Substrates for complex I
• Glyceraldehyde - 3 P
• Isocitrate
• Malate
• Glutamate
• Beta hydroxy acyl CoA
• Pyruvate
• Alpha keto glutarate lipoate→ FAD

38. Complex II
Succinate-Q-Reductase
• Succinate →FAD → FeS → CoQ
– FADH2 binding site
• FAD reductase activity
• FADH2 -- FAD
Substrates are- 1. Succinate
2. Acyl CoA
3. Glycerol-3- P

39. FADH2 bypasses complex I
• FADH2 doesn’t produce enough energy
• 1 ATP less produced than NADH

40. Complex III- Cytochrome reductase
• Cluster of FeS proteins, Cyt b
& c1
• CoQ → FeS → Cyt b c1 →
Cyt C
• FeS- Rieske’s FeS (bound to
His residues instead of 2Cys
residues)
• Free energy change- -10 Kcal
/ Mol
• 1 ATP synthesised
• 4H+ pumped out

41. Complex IV-Cytochrome oxidase
• Reduction of oxygen
• Cytochrome oxidase
• Cyt a+a3 red → oxidized state
• oxygen → water
– 2 H+ + 2 e- + ½ O2 -- 2 H2O
– transfers e- one at a time to oxygen
• Pumps 2H+ out
– Total of 10 H+ / NADH
– Total of 6 H+ / FADH2
1 ATP generated

42. 3 subunits,

43. Proton gradient

44. Complex V- ATP Synthase complex
• ATP synthase
• Chemiosmotic theory
• Proton gradient-
electrochemical potential
• pH outside- 1.4 units lower
• Outside is positive relative
to the inside- +0.14 v
• Only 40% energy trapped

45. Organisation of ATP synthase complex
• 2 units
• Fo and F1
• Fo – o for Oligomycin
• Fo embedded in the inner mitochondrial
membrane
• F1- towards matrix
• 3α and 3β, 1 γ and 1 δ subunit

46. Binding change mechanism
• Water driven hammer minting
coins
• F1 has 3 confirmations- O, L & T
• O- doesn’t bind to substrates /
products
• L- Loosely binding
• T- Tightly binding
Paul D Boyer
N.P. 1997

47. Protons pumped to inter-membrane space
↓
Inner membrane impermeable to H+
↓
Protons induce rotation of γ subunits
↓
Induces conformational changes in β subunits
↓
ADP and Pi bind in L conformation
↓
L changes to T and ATP is formed
↓
T changes to O and ATP is released

48. An energy dependent process
2.5 ATP generated from NADH and 1.5 ATP from FADH2

50. O2 consumption as a measure of electron transport
 An oxygen electrode can measure O2 consumption in respiring mitochondria.

51. Inhibitors and uncouplers of ETC

53. Babies do not shiver- Brown fat contains
Thermogenin (Non-shivering thermogenesis)
Thermogenin acts as a channel and allows H+
↓
Proton gradient disrupted
↓
Phosphorylation uncoupled; Energy dissipated as
heat

54. Other inhibitors of Oxidative
phosphorylation
• Atractyloside – Inhibition of ATP- ADP
exchange, inhibits translocase
• Doxorubicin- cardiotoxic; inhibits Ox-Phos
and also damages Mitochondria by free
radical generation

55. Valinomycin is an ionophore
• Works by changing K+
gradient
• Disrupts the gradient
across the membrane

56. MtDNA is maternally inherited
• OXPHOS diseases
• Lack of introns in MtDNA makes it 10 times more
susceptible to mutation
• Occurs in tissues having high rate of Ox-Phos
• CNS, skeletal mm, cardiac mm, Liver

57. • Lethal infantile mitochondrial
ophthalmoplegia/ myopathy
• Leber’s hereditary optic neuropathy (LHON)-
complex I defect
• Leigh syndrome (SA necrotizing
encephalopathy)-

58. • Myoclonic epilepsy and ragged
red fibre disease (MERRF)-
proteins for tRNA synthesis are
not synthesised properly
• MELAS (Mito. Encephalo-
myopathy lactic acidosis stroke
like episodes)
• DM- Mutations in the
mitochondrial lysyl-tRNA gene

60. Mitochondria is involved in Apoptosis and
oxidative stress
• MPTP- Mitochondrial permeability transition
pore- escape of Cyt C and activating caspase 9
• Free radical damage by superoxide formation

61. …Forward, ever forward, without
fear and without hesitation

62. For more ppt on Medical
Biochemistry please visit my
website
www.vpacharya.com