1. Biological oxidation and Electron transport chain
Dr. Saidunnisa
Professor and chairperson
Biochemistry (3 lectures)

2. Learning objectives
At the end of the session the student shall be able to define, describe, enumerate, analyze and list:
Location of ETC
Components of ETC
Sites of ATP production
Oxidative phosphorylation
Chemiosmotic theory
Energetics of ETC
(Chapter 19 pages: in (Text Book of Biochemistry DM Vasudevan)

3. I. Case: A 35-year old male is rescued from enclosed fire.
Upon arrival in the emergency department, he is disoriented and in moderate distress. He is coughing up soot, and has difficulty breathing.
Initial vital signs:
BP 90/60, HR 120, RR 30, O2 sat 95%
On examination: burned nasal hair, soot around mouth, burns to face, arms and back.
What are the immediate concerns?

4. 1Case conti…
We have a tendency to focus on CO as the diagnosis in victims of smoke inhalation.
Pitfall –
CN exposure is frequently overlooked. Consider that CN can be produced from the combustion of paper, silk, wool, plastic, and cotton.
The probability of CN exposure in fires is therefore high.

5. 1. Case conti……
Lets us unfold the mystery of CO and CN poisoning on human health while studying respiratory chain.

6. Stages of oxidation of food stuffs
First stage:
Digestion in the GIT converts the macro molecules into small units. (carbohydrates into glucose, lipids into fatty acids, proteins into amino acids).
This is called primary metabolism.
Second stage: These products are absorbed in mitochondrial citric acid cycle to generate reducing equivalents NADH or FADH2.
This is called secondary or intermediary metabolism.
Third stage: These reducing equivalents enter into the electron transport chain or respiratory chain where energy is released (ATP).
This is tertiary metabolism or internal or cellular respiration.
The energy (ATP) is then used for body synthetic purpose.

7. ATP-Universal currency of energy in living cells
The energy released from the hydrolysis of ATP is utilized for
Mechanical -muscle contraction
Transport work -Sodium potassium ATPase pump
Biochemical work -Initial steps of Glycolysis
Anabolic pathways -TAG, DNA, Protein synthesis
Detoxification(urea cycle),formation of active intermediates like UDP glucose
HEAT

8. Chemistry of ATP hydrolysis
Two high energy bonds in ATP which are represented by a squiggle bond ( ˜ ) on hydrolysis each releasing -7.3kcal/ mole.
More than 90% of ATP is formed through ETC (Oxidative phosphorylation)
Remaining in creatine phosphate shuttle, and Substrate level phosphorylation

9. High energy compounds: ATP, GTP, UTP, PEP Carbamoyl phosphate cAMP
Any bond that can be hydrolyzed with the release of same energy as ATP hydrolysis is called as “high energy bonds”.
High energy compounds:
ATP, GTP, UTP,
PEP
Carbamoyl phosphate
cAMP
1-3 Bisphosphoglycerate
Creatine Phosphate
Acetyl Co A
SAM
Low energy compounds
AMP
Glucose-1 phosphate
Fructose-1-phosphate

10. Redox potentials Oxidation: loss of electrons
Reduction: gain of electrons
Oxidation is always accompanied by reduction.
Redox couple: when a substance exists both in the reduced and oxidized state.
Redox potentials: electron transfer potential E0’.

11. Expressing Redox reactions as half reactions
E.g. Fe Cu 2+ = Fe 3+ + Cu +
which can be expressed in the form of 2 half reactions
Fe 2+ = Fe 3+ + e / Fe 2+
Cu / Cu 2+ + e-
They together make a conjugate redox pair.

12. Substrate level phosphorylation
Energy from a high energy compound is directly transferred to ADP or GDP to form ATP or GTP without the help of electron transport chain.
Examples:
Bisphosphoglycerate kinase (glycolysis)
Pyruvate kinase (glycolysis)
Succinate thiokinase (TCA cycle)

13. Biological oxidation
Definition: Transfer of electrons from reduced coenzymes through ETC to oxygen.
Energy released during this process is trapped as ATP.
This coupling of oxidation with phosphorylation is called oxidative phosphorylation.
In the body this is carried out by dehydrogenations.

14. Oxidoreductases Oxidases Aerobic dehydrogenases
Anaerobic dehydrogenases
Hydro peroxidases
Oxygenases

15. Oxidases
Removal of hydrogen from substrates but only oxygen can act as acceptor of hydrogen so that water is formed.
Example: cytochrome oxidase (terminal component of ETC), MAO, tyrosinase, etc.

16. Aerobic dehydrogenases
Removal of hydrogen from substrates but only oxygen can act as acceptor of hydrogen product H2O2 (hydrogen peroxide) is formed.
Example: Flavoproteins (FMN FAD), Xanthine oxidase

17. Anaerobic dehydrogenases
NAD+ dehydrogenases
NADP+ dehydrogenases
FAD+ dehydrogenases
Cytochromes

18. NAD+ Linked dehydrogenases
NAD+ is derived from nicotinic acid a Vitamin B-complex.
The electron is also accepted by the NAD+ so as to neutralize the charge on the co- enzyme.
H H + H+ + e-
AH2 + NAD+
A + NADH + H+
When NAD+ accepts the two hydrogen atoms, one of the hydrogen atom is removed from the substrate as such the other is split into one hydrogen ion and one electron.

19. NAD+ Linked dehydrogenases
Examples:
Glyceraldehyde -3-phosphate dehydrogenase.
Isocitrate dehydrogenase
Glutamate dehydrogenase
Pyruvate dehydrogenase
Alpha ketoglutarate dehydrogenase
NADP+ Linked dehydrogenases takes part in reductive biosynthesis.

20. FAD Linked dehydrogenases
AH2 + FAD
FADH2
FAD is derived from riboflavin a Vitamin B- complex.
Examples:
Succinate dehydrogenase
Fatty acyl CoA dehydrogenase.
Both the hydrogen atoms are attached to the flavin ring.

21. Hydroperoxidases Includes 2 sets of enzymes : catalase and peroxidases
Peroxisomes are rich in oxidases and catalases
H2O2 + AH H2O + A
2 H2O2
2H2O
Peroxidases reduce H2O2 which is a free radical
Catalase uses H2O2 as electron acceptor & electron donor

22. Oxygenases Consists of two sets of enzymes
Dioxygenases : Incorporate both atoms of oxygen into the substrate:
A + O AO2
e.g. Homogentisic acid oxidase
Monooxygenases : Incorporates one atom of oxygen into the substrate & the other is reduced to water
A – H + O2 + ZH2
A – OH + H2O + Z
e.g. phenylalanine hydroxylase

23. Energetics of oxidative phosphorylation
Free energy change between NAD+ and water is equal to 53kcal/mol.
This is so great that if this much energy is released at one stretch body cannot utilize it hence the ETC assembly the total energy released in small increments so that energy can be trapped as chemical bond energy ATP.

24. Mitochondrial Organization

25. Electron Transport and Oxidative Phosphorylation
Note: Electrons ultimately combine with oxygen and protons to form water.

26. Components of ETC 5 Complexes
Enzyme complex I, (NADH dehydrogenase)
Enzyme complex II (Succinate dehydrogenase)
Enzyme complex (III) Cytochrome reductase
Enzyme complex (IV) Cytochrome oxidase.
ATP Synthase (V)

27. Components of ETC

28. Two mobile carriers
These are connected by two mobile carriers Coenzyme Q, Cytochrome C.
Coenzyme Q, connects complex-1 and 11.
Cytochrome C connects complex 111 and 1V
Electrons flow from more electronegative to electropositive components.

29. Complex-I NADH Dehydrogenase
NAD+ is reduced to NADH+H+ by dehydrogenases with the removal of two hydrogen atoms from the substrate (AH2).
AH2 + NAD A + NADH+H+
It has binding sites for NADH, FMN and Fe-S proteins and for Co Q.
FMN accepts two electrons and protons from NADH and pass electrons to Fe-S and pass to CoQ.

30. Complex-II-Succinate dehydrogenase
The coenzymes FAD is derived from vitamin riboflavin.
FAD accepts (2H+ and 2electrons) from Succinate fumarate, and beta oxidation to form FADH2.
They pass electrons to CoQ.

31. Iron sulfur proteins
Several iron atoms paired with sulfur atoms to make iron sulfur centers. They exist in the oxidized (Fe+3) or reduced (Fe+2) on accepting an electron.
Following FeS types are normally present:
FeS: Single Fe coordinated to the side chain SH groups of four cysteine residues
Fe2S2, Fe4S4.
One FeS participates in the transfer of electrons from FMN to coenzyme Q.
Other FeS proteins transfer electrons from Cyt.c, b1 to cyt,c.

32. Coenzyme Q
Is a Quinone derivative with a long isoprenoid tail. It is also called ubiquinone because it is ubiquitous in biologic systems. It is lipophilic electron carrier. It can accept hydrogen atoms both from FMNH2 and FADH2

33. Cytochromes Cytochrome C is a mobile component of ETC.
Are conjugated proteins containing heme group having porphyrin ring and iron atom.
Iron in cytochromes is alternatively oxidized (Fe+3) and reduced (Fe+2) in contrast to iron of hemoglobin and myoglobin which remains in (Fe+2) state.
The electrons are transported from coenzyme Q to cytochromes in the order b,c1,c, a and a3 during electron transport.

34. Complex-III (Cytochrome b-c1)

35. Complex- IV Cytochrome oxidase
Cytochrome a and a3 electron carrier that can react with molecular oxygen, protons and electrons to form water.
This also contain copper that undergoes oxidation- reduction (Cu Cu+)

36. Components of ETC

37. Complex-V ATP synthase
Enzyme that generates ATP sometimes referred as complex-V is a multi subunit having 9 poly peptide chains ( 3alpha, 3 beta,1 gamma,1sigma ,1 epsilon). The alpha chains have binding sites for ATP and ADP.
It has two functional subunits Fo (oha) portion embedded in the IMM where as F1 portion
protrudes into the mitochondrial matrix.

38. ATP synthase conti…..
The Fo contain a central pore (proton channel) this is because the IMM is impermeable to protons and so the extruded protons in the intermembrane space can reenter the mitochondrial matrix through this proton channel.
A proton pair attacks one Oxygen of Pi to form H2Oand an active form of pi which immediately combines with ADP to form ATP.

40. Chemiosmotic theory
Peter Mitchell proposed this theory to explain the oxidative phosphorylation.
The transport of electrons from inside to outside of IMM is accompanied by the generation of a proton gradient across the membrane.
Protons (H+) accumulate outside the membrane, creating an electrochemical potential difference.

41. Chemi-osmotic theory conti…
The proton pumps (complexes -I, III, IV) expels H+ from inside to outside of the membrane.
So there is high H+ concentration outside.
This causes H+ to enter into mitochondria through the channels (Fo – F1complex ) , this proton influx binds to oxygen of pi+ADP to form ATP

42. Chemi-osmotic theory conti….

43. Current concept of ATP synthesis
Proton gradient is created across the IMM till the electrons are transferred to oxygen to from water.
Energy of electron transfer is used to drive protons out of the matrix by the complexes 1, 111 and IV which are proton pumps.
This electrochemical potential of this gradient is used to synthesize ATP.

44. When 1 NADH transfers its electrons to oxygen, 10 protons are pumped out this accounts for approximately 3 ATP synthesis.
Around 3 protons are required per ATP synthesized.
Oxidation of 1 FADH2 is accompanied by the pumping of 6 protons accounting for 2 molecules of ATP.

45. Peter Hinkle proved that actual energy production is less because there is always leakage of protons.
According to recent findings:
1 NADH generates- 2.5ATP
1 FADH2 generates- 1.5 ATP

46. Recent concept of: Sites of ATP synthesis
Traditionally Between complex-I and coenzyme-Q –First site.
Between complex -III and cytochrome c
Second site.
At complex-IV – third site.
Now ATP synthesis occurs when proton gradient is dissipated and not when protons are pumped out.

47. Oxidative Phosphorylation
The process of synthesizing ATP from ADP and Pi coupled with the electron transport chain is known as oxidative phosphorylation.
The complex V of the IMM is the site of O.P

48. Inhibitors of oxidative phosphorylation
Oligomycin: an antibiotic, used as anti- fungal drug, prevents the cell from using the established H+-gradient, to make ATP.
Atractyloside: Plant toxin

49. Inhibitors of ETC
The inhibitors bind to one of the components of ETC and block the transport of electrons.
This causes the accumulation of reduced components before the blockade step and oxidized components after that step.
The synthesis of ATP is dependent on ETC.
Hence, all the site specific inhibitors of ETC also inhibit ATP formation.

50. Inhibitors of ETC ETC inhibitors prevent:
The reduction of oxygen to water,
The build-up of the H+-gradient and
Finally the synthesis of ATP

51. Site- of inhibitors Site –I Between complex-I and Co-Q:
Rotenone : a potent, plant-derived and widely used pesticide.
Amytal : a barbiturate sedative drug
Piercidin A : antibiotic

52. Site- of inhibitors Site-II cytochrome b and c1:
Antimycin A : antibiotic
BAL (British antilewisite): an antidote used against war-gas

53. Site- of inhibitors Site-III At complex-IV ( cytochrome oxidase):
Carbon monoxide: - odor-less, toxic gas frequently released during in-complete combustion reactions, e.g. in car engines or during coal gasification
Cyanide: most potent inhibitor of ETC, an extremely toxic compound; low doses are lethal to humans
Used by the Nazis during WWII in form of “Zyklon B” in the 1940s to commit mass murder of the Jewish population imprisoned in the concentration camps.
In 1984, in one of the worst industrial accidents in human history, the toxic cyanide derivative methyl isocyanate killed 3,000 people and injured more than 100,000 humans after a catastrophic gas leak in a chemical factory in Bhopal, India.
3. Hydrogen Sulfide:

54. Uncouplers
These increase the permeability of IMM to protons (H+).
Thus an Uncoupler allows ETC but blocks the establishment of proton gradient across the IMM.
Compounds that can uncouple or delink the electron transport chain from oxidative phosphorylation, such compounds are known as Uncouplers.
The result is that ATP synthesis does not occur.
The energy linked with the transport of electrons is dissipated as heat.

55. Chemical Uncouplers Physiological Uncouplers Thyroid hormones.
2,4-dinitrophenol ( has been extensively studied).
Dinitrocresol.
Pentachlorophenol
Tri fluoro carbonyl cyanide phenyl hydra zone (FCCP).
Aspirin (high doses)
Physiological Uncouplers
Thyroid hormones.
Long chain fatty acids.
Unconjugated Bilirubin.
These act as Uncouplers only at high concentration.

56. Significance of Uncoupling
Brown adipose tissue present in the upper back and neck portions and around kidney is rich in mitochondria and carry oxidation uncoupled from phosphorylation.
This causes liberation of heat when fat is oxidized in this tissue.

57. Significance of Uncoupling
Examples :
New born infant, (Non-shivering Thermogenesis)
Hibernating animals

58. However, research, that was published in the New England Journal of Medicine, is showing that brown adipose tissue helps adults burn more calories than white adipose tissue.
In certain individuals due to presence of this brown adipose tissue it is believed to protect them from becoming obese.
The excess calories consumed by this people are burnt and liberated as heat , instead of being stored as fat.
Brown adipose tissue is located in the neck area and is more physiologically active in the woman than in the man.

59. Disorders of Oxid.Phos.
DNA is present in mitochondria (mtDNA) and nucleus.
mtDNA is maternally inherited since mitochondria from the sperm do not enter the ovum.
mtDNA is about 10times more susceptible to mutations than nuclear DNA.
It is present in tissues like CNS, Skeletal , heart muscle and liver.

60. Diseases associated with oxida . phos
LHON : Lebers hereditary optic neuropathy
MELAS: Mitochondrial encephalopathy
Lactic Acidosis
Stroke

61. Case-1
Mr. X 26yrs old male noted heat intolerance, with profuse sweating . He is loosing weight in spite of good appetite . On physical Examination thyroid swelling was present and T3 and T4 are increased. Give biochemical explanation for above symptoms.

62. An excess thyroid hormones affect the efficiency of ATP production resulting in fewer ATP production.

63. What about NADH made in cytosol Can’t get into matrix of mitochondrion?
By 2 Shuttle pathways:
In muscle and brain
Glycerol phosphate shuttle
In liver and heart
Malate / aspartate shuttle

64. Glycerol Phosphate shuttle
In muscle and brain

65. Malate – Aspartate Shuttle
In liver and heart

66. Apoptosis
Cytochrome C is a mediator of apoptosis in response to oxidant stress due to ROS or free radicals.