Oxidative Phosphorylation

1. Oxidative Phosphorylation Lecture for MBBS by, Dr. Nivedita L. Rao Prof., Biochemistry

2. LEARNING OBJECTIVES • To know how NADH and FADH2are oxidized by molecular oxygen using the electron transport chain in mitochondria. • To understand oxidative phosphorylation, the mechanism by which living organisms utilize redox energy to synthesize ATP.

3. Introduction • Mitochondria contain the system called electron transport chain (Respiratory chain). • Transfer of electrons from NADH and FADH2 through electron transport chain to O2 is called biological oxidation (cellular respiration). • Energy is released during this process which is trapped as ATP. • The coupling of respiration to generation of ATP is called oxidative phosphorylation.

4. Biological Importance • Oxidative phosphorylation causes capture of greater proportion of free energy from respiratory substrates in aerobic organisms than anaerobic organisms • Oxidation of substrates in mitochondria, -generates heat; maintains body temperature -producesreactive oxygen species-ROS (egs. superoxide and hydrogen peroxide) involved in apoptosis (programmed cell death) necessary for tissue differentiation

5. Medical Importance • A number of drugs and poisons inhibit oxidative phosphorylation, usually with fatal consequences. • Generation of ROS leads to propagation of free radicals, damage to cells, contributing to disease and, possibly, aging (senescence) • Inherited defects of respiratory chain have been reported. • Patients present with myopathy,encephalopathy and often have lactic acidosis.

6. Why do living organisms requirecontinuous input of free energy? For: • active transport of molecules and ions across cell membrane (40% of total energy need) 2) muscle contraction & other cellular movements 3) synthesis of macromolecules & other biomolecules from simple precursors 4) maintenance of body temperature

7. Energy Capture - an Overview Food Carbohydrate Fat • Protein Fuel molecules present in blood Fatty acids Amino acids • Glucose Ketone bodies chemical energy (combustible energy) resides in C–H bonds containing electrons with high transfer potential Cells oxidation of fuel molecules Reducing equivalents: 1 reducing equivalent  1 electron  1 hydrogen atom CO2, water and Energy

8. The energy yield in the body per gram (calorific value) Carbohydrate and Protein= 4 kcal Fat = 9 kcal

9. Energy Capture -- an Overview • Body cannot directly use the energy released from oxidation of fuel molecules. • The energy is captured in high-energy phosphate bonds of Adenosine triphosphate (ATP)

10. Energy Capture -- an Overview ATP a high-energy compound on hydrolysis gives 7.3 kcal of energy per mole The energy in ATP is in readily usable form ATP is the immediate source of energy for most of the energy-requiring reactions ATP + H2O • ADP + Pi G o= – 7.3 kcal/mole

11. ATP: Universal currency of energy CH2OH P P • P

12. Energy Capture -- an Overview • Cells cannot directly oxidize the fuel molecules for synthesis of ATP First, Oxidation of Fuel molecules obtained from Carbohydrates, Fat, Protein NADH, FADH2 NAD+, FAD and then Mitochondrial electron transport chain Heat H2O O2 Oxidative Phosphorylation ADP + Pi ATP

14. Sources of ATP Oxidative phosphorylation: • is the coupling of oxidation of NADH/FADH2 through mitochondrial electron transport chain to ATP synthesis • is responsible for 90% of total ATP synthesis in cell Substrate level phosphorylation: • ATP is directly produced from a high-energy intermediate of a metabolic pathway (two in glycolysis and one in TCA cycle) • is a minor source of energy

15. Oxidative Phosphorylation Definition It is a mitochondrial process under aerobic condition, whereby the free energy released by oxidation of NADH and FADH2 through electron transport chain is coupled to the phosphorylation of ADP to form ATP.

16. Oxidative Phosphorylation Is explained under following headings: • Sources of NADH and FADH2 • Electron transport chain (Respiratory chain)– organization, ATP sites and transport of electrons • Mechanism of coupling of phosphorylation of ADP to oxidation of NADH and FADH2 - Chemiosmotic theory of oxidative phosphorylation • Regulation of oxidative phosphorylation • Shuttle mechanisms for transport of reducing equivalents of cytosolic NADH to mitochondrial matrix • Transport of ADP and ATP • Inhibitors and uncouplers of oxidative phosphorylation

17. Sources of NADH and FADH2 Reactions catalyzed by NAD- and FAD- dependant dehydrogenases of: • Glycolysis (cytosol) • Oxidative decarboxylation of pyruvate (mitochondria) • TCA cycle (mitochondria) • -oxidation (mitochondria)

18. Sources of NADH and FADH2 • NAD-dependant dehydrogenases: • Glyceraldehyde-3-phosphate dehydrogenase (Glycolysis) • Pyruvate dehydrogenase complex • Isocitrate, -keto glutarate and malate dehydrogenases (TCA cycle) • 3-hydroxy acylCoA dehydrogenase (β –oxidation) • FAD-dependant dehydrogenases: • Succinate dehydrogenase (TCA cycle) • AcylCoAdehydrogenase (β – oxidation)

19. Electron Transport Chain (ETC) Syn: Mitochondrial Electron Transport ChainRespiratory chain • Organization • ATP Sites • Transport of electrons

20. Organization of ETC- Location • Components of the ETC are highly organized and located on the inner mitochondrial membrane • The folded cristae provide • large surface area • The inner membrane is • highly impermeable and • requires specific • transporters

21. Organization of ETC- Location ETC • inner membrane outer membrane Matrix Intermembrane space

22. Organization of ETC- Components The following components of ETC are embedded in the inner mitochondrial membrane: • 4 protein complexes – complex I, II, III and IV • coenzyme Q (CoQ) or ubiquinone • cytochrome c All these components, except CoQ, are enzymes catalyzing the transfer of electrons serially from one component to other.

23. Electron transport chain – Organization Cytosol • Outer mitochondrial membrane Intermembrane space • Cyt c Inner membrane Complex I Complex III • Complex IV • CoQ NADH Dehydrogenase • Cytochrome c • reductase Cytochrome c oxidase FMN Fe-S Matrix Complex II Succinatedehydrogenase Complex I • Protein complex + • (NADH dehydrogenase) 2 prosthetic groups: • flavin mononucleotide (FMN) • non-heme iron • iron-sulfur centers (Fe-S)

24. Iron-sulfur centers (Fe-S)

25. Electron transport chain – Organization Cytosol • Outer mitochondrial membrane Intermembrane space • Cyt c • CoQ Complex III Inner membrane Complex I • Complex IV FMN Fe-S Matrix Complex II FAD Fe-S Complex II • Fe-S • and Succinatedehydrogenase • FAD

26. Electron transport chain – Organization Cytosol • Outer mitochondrial membrane Intermembrane space • Cyt c Inner membrane Complex I Complex III • Complex IV • CoQ Fe-S FMN Fe-S • Cyt b Cytc1 Matrix Complex II FAD Fe-S Complex III • Cyt b • Cyt c1 has 2 cytochromes (heme proteins) • Cytochrome c reductase cytochrome b and cytochrome c1 Fe-S and Fe-S centers

27. Electron transport chain – Organization Cytosol • Outer mitochondrial membrane Intermembrane space • Cyt c Inner membrane Complex I Complex III • Complex IV • CoQ Fe-S Cyt a Cyt a3 Cu Cu FMN Fe-S • Cyt b Cytc1 Matrix Complex II FAD Fe-S • Complex IV has 2 cytochromes, Cyt a Cu Cytochrome c oxidase • cytochrome a and cytochrome a3 Cyt a3 Cu each associated with a copper (Cu) atom

28. Answer this: ??? • Where is the ETC located? • Ans: • Inner mitochondrial membrane

29. Electron transport chain – Organization Cytosol • Outer mitochondrial membrane Intermembrane space • Cyt c Inner membrane Complex I Complex III • Complex IV • CoQ NADH Dehydrogenase • Cytochrome c • reductase Cytochrome c oxidase Matrix Complex II Succinatedehydrogenase

30. Electron transport chain – Organization Cytosol • Outer mitochondrial membrane Intermembrane space Cyt c Inner membrane Complex I Complex III • Complex IV Fe-S • CoQ Cyt a Cyt a3 Cu Cu • Cyt b Cyt c1 FMN Fe-S Complex II Matrix FAD Fe-S Coenzyme Q Cytochrome c lipid soluble a heme protein structure is similar to vitamin K situated between complex III and IV situated between complex I, II and III CoQ and Cyt c are Mobile electron carriers

32. Structure of Heme protein • Fe++ • Fe++ • Heme Protein prosthetic group A Heme protein (Porphyrin + Fe++) • (Eg:Hemoglobin, Cytochromes)

33. Electron transport chain – Organization Cytosol • Outer mitochondrial membrane Intermembrane space • Cyt c Complex III • Complex IV Inner membrane Complex I Fe-S • CoQ Cyt a Cyt a3 Cu Cu FMN Fe-S • Cyt b Cytc1 Matrix Complex II FAD Fe-S The components of ETC are arranged such that the redox potential (electron affinity) of electron carriers becomes greater from complex I to complex IV

34. Electron transport chain – ATP sites Cytosol • Outer mitochondrial membrane ATP Site 3 ATP Site 2 Intermembrane space ATP Site 1 • Cyt c Complex I Complex III • Complex IV Inner membrane Fe-S • CoQ Cyt a Cyt a3 Cu Cu FMN Fe-S • Cyt b Cytc1 Matrix Complex II FAD Fe-S ATP site 1, 2 and 3 situated in complex I, III & IV respectively They support synthesis of ATP

35. Electron transport chain- Transport of Electrons Cytosol • Outer mitochondrial membrane ATP Site 3 ATP Site 2 Intermembrane space ATP Site 1 • Cyt c Complex I Complex III • Complex IV Inner membrane NADH Dehydrogenase Fe-S • CoQ Cytochrome c reductase • Cytochrome c • oxidase Cyt a Cyt a3 Cu Cu FMN Fe-S 2e- • Cyt b Cyt c1 Matrix Complex II 2e- 2e- FAD Fe-S NADH + H+ Succinatedehydrogenase ½O2 + 2H+ H2O NAD + As electrons flow, Iron atoms of cytochromes oscillate between ferrous (Fe++) and ferric (Fe+++) forms components oscillate between reduced and oxidized forms

36. Electron transport chain- Transport of Electrons Cytosol • Outer mitochondrial membrane ATP Site 3 ATP Site 2 Intermembrane space ATP Site 1 • Cyt c Complex I Complex III • Complex IV Inner membrane NADH Dehydrogenase Fe-S • CoQ Cytochrome c reductase • Cytochrome c • oxidase Cyt a Cyt a3 Cu Cu FMN Fe-S 2e- • Cyt b Cyt c1 Matrix Complex II 2e- Succinatedehydrogenase FAD Fe-S Succinate • Fumarate ½O2 + 2H+ H2O

37. Answer this: ??? • The second mobile component of ETC which shuttles between protein complexes is • Ans: • cytochrome c

38. Mechanism of oxidative phosphorylation Chemiosmotic theory of oxidative phosphorylation proposed by P. Mitchell explains the mechanism of coupling of oxidation of NADH and FADH2 to phosphorylation of ADP

39. Chemiosmotic theory of oxidative phosphorylation F1 protein F0 protein H+ H+ H+ H+ (ATP Synthase) H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ Intermembrane space H+ H+ Complexes I, III & IV H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ ADP + Pi act as H+ • I H+ NADH H+ H+ proton pumps H+ H+ H+ H+ H+ • III H+ H+ FADH2 ATP H+ H+ H+ H+ H+ ½ O2 + 2H+ H+ • IV H+ H+ H+ H+ H+ H+ H+ H+ H+ H2O H+ H+ H+ H+ Mitochondrial matrix H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ Inner membrane H+ H+ H+ Outer membrane H+ H+ H+ H+

40. Mitchell’s Chemiosmotic Theory of Mechanism of Oxidative Phosphorylation The theory postulates that the energy from transfer of electrons along ETC is used to translocate protons from inside to outside of inner mitochondrial membrane – complex I, complex III and complex IV acting as proton pumps

41. Mitchell’s Chemiosmotic Theory ofMechanism of Oxidative Phosphorylation This creates a potential energy in the form of proton gradient across the inner membrane, proton concentration being higher on the outside of the membrane. [combination of electrostatic (chemical) and osmotic gradient – hence chemiosmotic]

42. Mitchell’s Chemiosmotic Theory ofMechanism of Oxidative Phosphorylation This gradient makes the protons leak back spontaneously to the mitochondrial matrix through proton channels (F0 proteins) embedded in inner mitochondrial membrane

43. Mitchell’s Chemiosmotic Theory ofMechanism of Oxidative Phosphorylation Attached to F0 protein on the matrix side is the F1 protein or ATP synthase ATP synthase catalyzes the formation of ATP from ADP and Pi Kinetic energy due to the proton inflow through the proton channels drives the endergonic reaction of ATP synthesis

44. Overall Reaction of Oxidative Phosphorylation NADH + H+ NAD+ + 2.5 ATP + ½ O2 + 2.5 ADP + 2.5 Pi + 3.5 H2O

45. Regulation of • Oxidative Phosphorylation ADP • NADH/FADH2 Availability of substrates • O2 need for ATP for the cell most important availability of ADP ATP utilized for energy ADP produced rate of ATP synthesis

46. Number of ATP Molecules Produced from • NADH and FADH2 2.5 ATP molecules NADH as electrons from NADH pass through all the 3 ATP sites FADH2 1.5 ATP molecules electrons from FADH2 bypass the ATP site 1

47. Energy Efficiency • of Oxidative Phosphorylation about 35% Efficiency of the system That is, about 35% of energy evolved during oxidation of NADH and FADH2 in the ETC is captured in the high energy phosphate bond of ATP Rest of the energy is evolved as heat which helps in the maintenance of body temperature (The efficiency of automobile engines is 30%)

48. Answer this: ??? • What kind of gradient is created by the electron transport chain? • Ans: • A chemical and osmotic (chemiosmotic) gradient

49. P.O. Ratio Number of molecules of ATP produced per atom of oxygen consumed • For NADH • 2.5 FADH2 1.5

50. Shuttle Mechanisms • for Transport of Reducing Equivalents • of Cytosolic NADH to the Mitochondria Glucose Glycolysis Glyceraldehyde-3-P NAD Glyceraldehyde -3- P Dehydrogenase NADH 1, 3-bisphsophoglycerate Malate-aspartate shuttle Glycerophosphate shuttle Pyruvate (in skeletal muscle and brain) (in heart and liver) cytosol • mitochondria ETC reducing equivalents ATP