1. The respiratory chain: a strategy to recover energy The mitochondrial electron transport chain functioning and control Oxidative phosphorylation Russian National Research Medical University Maxim A. Abakumov Moscow, 2014

2. Energy flow in cell Glicolysis TCA cycle Electron transfer chain Oxidative phosphorilation

3. Outer membrane Inner membraneIntermembrane space Cristae Matrix

4. TCA total energy outcome Acetyl-CoA + 3 NAD + + Q(FAD) + GDP + P i +2 H 2 0  HS-CoA + 3NADH + QH 2 (FADH 2 )+ GTP + 2 CO 2 + 2 H + Isocitrate Dehydrogenase 1 NADH=2.5 ATP α-ketoglutarate Dehydrogenase 1 NADH=2.5 ATP Succinyl-CoA Synthetase1 GTP=1 ATP Sunccinate Dehydrogenase1 QH 2 =1.5 ATP Malate Dehydrogenase1 NADH=2.5 ATP NADH and FADH 2 is not an ATP (no cash energy) – further conversion is needed

5. NADH and NAD + NAD – Nicotine Adenine Dinucleotide Produced from niacine (vitamine B 3 ) NADH – reduced form NADH – high energy (HE) electron carrier NAD + – oxidized form NAD + + H + + 2e - → NADH + H +

6. NADH and NAD + + H + + 2e - Reduction Oxidation

7. FADH 2 and FAD FAD – Flavine Adenine Dinucleotide Techically not a dinucleotide FADH 2 – reduced form (oxidizer) FADH 2 – high energy (HE) electron carrier FAD – oxidized form (reductor) FAD + 2H + + 2e - → FADH 2

8. FADH 2 and FAD Flavin Ribitol ADP

9. FADH 2 and FAD FADFADH 2

10. Electron transfer chain Electron transfer chain (ETC) – group of enzymes located on inner membrane of mitochondria Convert energy from NADH/FADH 2 electrons into energy of proton gradient on inner membrane Proton gradient is needed for further ADP to ATP conversion Consists of four complexes

11. Oxidative phosphorilation ADP + P i → ATP Reaction is catalized by ATP-synthase. ATP-synthase is intramembrane multisubunit enzyme (nanorotor) ATP-synthase uses energy of proton gradient

12. Electron transfer chain Consists of four complexes A lipid soluble coenzyme (UQ, CoQ) and a water soluble protein (cyt c) shuttle between protein complexes Fe-S, Hem, FMN, Cu atoms acts as a cofactors in different complexes Electrons generally fall in energy through the chain - from complexes I and II to complex IV

13. Electron transfer chain

14. Coenzyme Q10 (CoQ) Lipid soluble molecule Transfer e - from complex I and II to complex III through membrane

15. FMN FMN –Flavin Mono Nucleotide Accepts e - from NADH and transfers them to QH 2

16. Complex I NADH-CoQ Reductase. Electron transfer from NADH to CoQ More than 30 protein subunits - mass of 850 kD Transfers 4H +

17. Complex I NADH-CoQ Reductase NADH + H + FMN Fe 2+ S CoQ NAD + FMNH 2 Fe 3+ SCoQH 2

18. Complex I - NADH-CoQ Reductase.

19. Complex II Succinate-CoQ Reductase Succinate dehydrogenase (from TCA cycle!) Membrane bound enzyme Accepts e - from succinate and pass through FADH 2 and Fe-S clusters to CoQ Not a proton pump

20. Complex II

21. Complex III CoQ in membrane passes electrons to cyt c in a unique redox cycle known as the Q cycle 4 H + are released into intermembrane space The principal transmembrane protein in complex III is the b cytochrome - with hemes b L and b H Cytochromes, like Fe in Fe-S clusters, are one- electron transfer agents CoQ-Cytochrome c Reductase

22. Complex III UQH 2 is lipid soluble e - carrier Cyt c is water soluble e - carrier

23. Complex IV Cytochrome c oxydase Electrons from cyt c are used in a four-electron reduction of O 2 to produce 2H 2 O Oxygen is the terminal acceptor of electrons in the electron transport pathway Cytochrome c oxidase utilizes 2 hemes and 2 copper sites Complex IV also transports 2H +

24. Complex IV

25. ETC

26. ETC summary 1 NADH gives energy for 10 H + transport into intramembrane space 1 FADH 2 gives gives energy for 6 H + transport into intramembrane space How proton gradient energy is converted into ATP energy?

27. ETC summary http://www.learnerstv.com/animation/animat ion.php?ani=177&cat=biology http://www.learnerstv.com/animation/animat ion.php?ani=177&cat=biology

28. How is H + gradient energy used for ATP synthesis?

29. ATP synthase – nanoscale rotating motor H + movement through the channel in ATP synthase leads to ATP synthesis 3H + leads to one full circle 3H + reguired for 1 ATP synthesis

30. ATP-synthase

32. ATP is synthesized in mitochondria matrix It must be transported to cytosol 1H + is spend for each ATP transfer 3H + is spend for each ATP synthesis Total 4H + is spend for 1 ATP synthesis ATP-ADP Translocase

34. P/O ratio How many ATP is made per 2e - 1 ATP – 4H + NADH – 10H + P/O NADH = 10/4 = 2,5 FADH 2 = 6H + P/O FAD = 6/4 = 1,5

35. ETC and OP inhibitors Rotenone inhibits Complex I Cyanide, azide and CO inhibit Complex IV, binding tightly to the ferric form (Fe 3+ ) Oligomycin and DCCD (Dicyclohexyl carbodiimide) are ATP synthase inhibitors

36. ETC and OP inhibitors Rotenone -inhibits the transfer of electrons from iron-sulfur centers in complex I to ubiquinone

37. ETC and OP inhibitors Oligomycin –blocks proton channel on ATP- synthase (F o subunit)

38. e - /H + transfer uncoupling Uncoupling of e - /H + leads to no proton gradient No ATP synthesis Affected by lipid soluble H + acceptors, such as dinitrophenol

39. NADH mitochondria/cytosol transport Most NADH used in electron transport is cytosolic and NADH doesn't cross the inner mitochondrial membrane Malate-aspartate shuttle system for NADH transport without actuall NADH transfer

40. NADH mitochondria/cytosol transport

41. Free radicals Contain unpaired electron on valent level Highly reactive Reactive Oxygen Species (ROS) Reactive Nitrogen Species (RNS)

42. Reactive oxygen species

43. Reactive nitrogen species

44. Lipid peroxides

45. Phagosome NADPH oxidase

46. Antioxidants http://sbxsupplements.com/blog/Complementary-Treatment-of- Schizophrenia-with-Antioxidants

47. Antioxidants Enzymatic  Superoxide dismutase  Catalase  Glutathione peroxidase  Glutathione reductase Non-enzymatic  Vitamins  C and E  Carotenoids  Flavonoids  Minerals  Manganese  Copper  Zinc  Selenium

48. 2H 2 O 2 O 2 + 2H 2 O Antioxidant enzymes Superoxide Dismutase 2O 2 - + 2H + O 2 +H 2 O 2 + 2H 2 O Catalase

49. Glutathione (GSH) – tripeptide 2x GSH-reduced form GSSG-oxidized form Antioxidant enzymes

51. Antioxidants Enzymatic  Superoxide dismutase  Catalase  Glutathione peroxidase  Glutathione reductase Non-enzymatic  Vitamins  C and E  Carotenoids  Flavonoids  Minerals  Manganese  Copper  Zinc  Selenium

52. Vitamin E α-Tocopherol (vitamin E) Tocopheroxyl free radical (Oxidized vitamin E) Free radicals

53. Cell oxidative status