Biological oxidation and Electron transport chain (ETC).pptx

Biological oxidation
Electron Transport chain
ATP synthesis
ATP synthesis inhibitors
Redox potential
High energy compounds

1. Biological Oxidation & Electron Transport Chain Dr Anurag Yadav MBBS, MD Associate Professor Department of Biochemistry Instagram page –biochem365 Email: [email protected]

2. Stages of oxidation of food First stage Second stage Third stage

3. Redox potential It is a system of electron transfer potential E . Oxidation is defined as loss of electrons Reduction is defined as gain of electron. When substance exists both in reduced state and oxidised state, the pair is called as redox couple.

4. Redox potential type Negative redox potential When substance has lower affinity for electron than hydrogen. Positive redox potential When substance has higher affinity for electron than hydrogen. NADH is strong reducing agent with negative redox potential and oxygen a strong oxidant with positive redox potential.

5. Substrate level phosphorylation Here energy from a high energy compound is directly transferred to nucleoside diphosphate to form a triphosphate without the help of electron transport chain; a. Bisphosphoglycerate kinase b. Pyruvate kinase c. Succinate thiokinase

6. Biological oxidation The transfer of electrons from the reduced co-enzymes through the respiratory chain to oxygen is known as biological oxidation. Energy released during this process is trapped as ATP. This coupling of oxidation with phosphorylation is called oxidative phosphorylation. In the body, this oxidation is carried out by successive steps of dehydrogenations.

7. ELECTRON TRANSPORT CHAIN The electron flow occurs through successive dehydrogenase enzymes, together known as electron transport chain (ETC). The electrons flow from electronegative potential (-0.32) to electropositive potential (+ 0.82).

8. Enzymes and Co-Enzymes All the enzymes involved in this process of biological oxidation belong to the major class of oxidoreductases. They can be classified into the following 5 headings:

9. Enzymes and Co-Enzymes

10. 1. Oxidases These enzymes catalyze the removal of hydrogen from substrates, but only oxygen can act as acceptor of hydrogen, so that water is formed. AH2 + ½ O2 A + H2O

11. 2. Aerobic dehydrogenases These enzymes catalyze the removal of hydrogen from a substrate, but oxygen can act as the acceptor. These enzymes are flavoproteins and the product is usually hydrogen peroxide. AH2 + O2 A + H2O2 Contain either FMN or FAD.

12. 3. Anaerobic Dehydrogenases These enzymes catalyze the removal of hydrogen from a substrate but oxygen cannot act as the hydrogen acceptor. They therefore require co-enzymes as acceptors of the hydrogen atoms. When the substrate is oxidized, the coenzyme is reduced.

13. a. NAD+ linked dehydrogenases : When the NAD+ accepts the two hydrogen atoms, one of the hydrogen atoms is removed from the substrate as such. The other hydrogen atom is split into one hydrogen ion and one electron. The electron is also accepted by the NAD+ so as to neutralize the positive charge on the co-enzyme molecule. The remaining hydrogen ion is released into the surrounding medium 3. Anaerobic Dehydrogenases

14. H2 H + H+ + e– AH2 + NAD+ → A + NADH + H+ The NAD+ linked dehydrogenases are: i . Glyceraldehyde-3-phosphate dehydrogenase ii. Isocitrate dehydrogenase iii. Malate dehydrogenase iv. Glutamate dehydrogenase v. Beta hydroxyacyl CoA dehydrogenase vi. Pyruvate dehydrogenase vii. Alpha ketoglutarate dehydrogenase 3. Anaerobic Dehydrogenases

15. b. NADP+ linked dehydrogenases NADPH cannot be oxidized with concomitant production of energy. NADPH is used in reductive biosynthetic reactions like fatty acid synthesis and cholesterol synthesis. 3. Anaerobic Dehydrogenases

16. c. FAD linked dehydrogenases : When FAD is the coenzyme, (unlike NAD+), both the hydrogen atoms are attached to the flavin ring. Examples: i . Succinate dehydrogenase ii. Fatty acyl CoA dehydrogenase iii. Glycerolphosphate dehydrogenase 3. Anaerobic Dehydrogenases

17. d. Cytochromes : All the cytochromes , except cytochrome oxidase, are anaerobic dehydrogenases . All cytochromes are hemoproteins having iron atom. Cytochrome b, cytochrome c1, and cytochrome c are in mitochondria while cytochrome P-450 and cytochrome b5 are in endoplasmic reticulum 3. Anaerobic Dehydrogenases

18. 4. Hydroperoxidases Peroxidase: Examples of peroxidases are glutathione peroxidase in RBCs (a selenium containing enzyme), leukocyte peroxidase and horse radish peroxidase. Peroxidases remove free radicals like hydrogen peroxide. H2O2 + AH2 (peroxidase) 2 H2O + A

19. 4. Hydroperoxidases Catalases : Catalases are hemoproteins. Peroxisomes are subcellular organelles having both aerobic dehydrogenases and catalase. 2 H2O2 (catalase) 2 H2O + O2

20. 5. Oxygenases Mono- oxygenases These enzymes are also called hydroxylases because OH group is incorporated into the substrate. A-H+ O2+ BH2--(hydroxylase)→ A-OH+ H2O+ B

21. i . Phenylalanine hydroxylase ii. Tyrosine hydroxylase iii. Tryptophan hydroxylase iv. Microsomal cytochrome P-450 mono- oxygenase is concerned with drug metabolism. v. Mitochondrial cytochrome P-450 mono- oxygenase . v. Nitric oxide synthase 5. Oxygenases

22. 5. Oxygenases b. Di-Oxygenases: They are enzymes which incorporate both atoms of a molecule of oxygen into the substrate, e.g. Tryptophan pyrrolase and homogentisic acid oxidase A + O2 AO2

23. High energy compounds These compounds when hydrolyzed will release a large quantity of energy, that is, they have a large ΔG0’

24. Organisation of ETC In the Electron transport chain, or respiratory chain, the electrons are transferred from NADH to a chain of electron carriers. The electrons flow from the more electronegative components to the more electropositive components. All the components of electron transport chain (ETC) are located in the inner membrane of mitochondria.

25. There are four distinct multi-protein complexes; these are named as complex-I, II, III and IV. These are connected by two mobile carriers, co-enzyme Q and cytochrome c. Organisation of ETC

26. Complex I It is also called NADH- CoQ reductase or NADH dehydrogenase complex. It contains a flavoprotein ( Fp ), consisting of FMN as prosthetic group and an iron- sulfur protein (Fe-S).

27. Complex II The electrons from FADH2 enter the ETC at the level of coenzyme Q.

28. Complex III This is a cluster of iron- sulfur proteins, cytochrome b and cytochrome c1, both contain heme prosthetic group.

29. Complex IV It contains different proteins, including cytochrome a and cytochrome a3. The Complex IV is tightly bound to the mitochondrial membrane.

30. P : O ratio The P:O ratio is defined as 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. In other words, the P:O ratio of NADH oxidation is 2.5; The P:O value of FADH2 is 1.5.

31. Site of ATP synthesis Traditionally, the sites of ATP synthesis are marked, as site 1, 2 and 3, as shown in Figure. But now it is known that ATP synthesis actually occurs when the proton gradient is dissipated, and not when the protons are pumped out

32. Chemiosmotic theory The coupling of oxidation with phosphorylation is termed oxidative phosphorylation. The transport of protons from inside to outside of inner mitochondrial membrane is accompanied by the generation of a proton gradient across the membrane. Protons (H+ ions) accumulate outside the membrane, creating an electrochemical potential difference

33. Chemiosmotic theory This proton motive force drives the synthesis of ATP by ATP synthase complex

34. ATP Synthase (Complex V)

35. It is a protein assembly in the inner mitochondrial membrane. It is sometimes referred to as the 5th Complex Proton pumping ATP synthase (otherwise called F1-Fo ATPase) is a multisubunit transmembrane protein. ATP Synthase (Complex V)

36. It has two functional units, named as F1 and Fo . It looks like a lollipop since the membrane embedded Fo component and F1 are connected by a protein stalk. ATP Synthase (Complex V)

37. Mechanism of ATP synthesis Translocation of protons carried out by the Fo catalyzes the formation of phospho -anhydride bond of ATP by F1. The binding change mechanism proposed by Paul Boyer (Nobel prize, 1997) explains the synthesis of ATP by the proton gradient.

38. Fo is the wheel; flow of protons is the waterfall and the structural changes in F1 lead to ATP coin being minted for each turn of the wheel. The F1 has 3 conformation states for the alpha-beta functional unit: O state—Does not bind substrate or products L state—Loose binding of substrate and products T state—Tight binding of substrate and products Mechanism of ATP synthesis

39. According to this theory, the three beta subunits (catalytic sites), are in three functional states: O form is open and has no affinity for substrates. L form binds substrate with sluggish affinity . T form binds substrate tightly and catalyzes ATP synthesis. Mechanism of ATP synthesis

40. Mechanism of ATP synthesis

41. Mechanism of ATP synthesis

42. Regulation of ATP synthesis 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. The major source of NADH and FADH2 is the citric acid cycle, the rate of which is regulated by the energy charge of the cell.

43. Inhibitors of ATP Synthesis

44. Inhibitors of ATP Synthesis

45. Uncouplers of Oxidative phosphorylation Uncouplers will allow oxidation to proceed, but the energy instead of being trapped by phosphorylation is dissipated as heat. This is achieved by removal of the proton gradient

46. Uncouplers of Oxidative phosphorylation Sometimes, the uncoupling of oxidative phosphorylation is useful biologically. In hibernating animals and in newborn human infants, the liberation of heat energy is required to maintain body temperature. In Brown adipose tissue, thermogenesis is achieved by this process. Thermogenin , a protein present in the inner mitochondrial membrane of adipocytes, provides an alternate pathway for protons. It is one of the uncoupling proteins (UCP). Thyroxine is also known to act as a physiological uncoupler .

47. Dr Anurag Yadav MBBS, MD Associate Professor Department of Biochemistry Instagram page –biochem365 Email : [email protected]