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  1. 1. Electron Transport and Oxidative Phosphorylation M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
  2. 2. Electron Transport and Oxidative Phosphorylation *Introduction* stage 3 of respiration NADH & FADH oxidized, electrons are “carried” (ETS) energy in form of ATP (Ox/Phos)  aerobic acceptor = oxygen
  3. 3. Mitochondrion -- A. football shaped (1-2μ), 1-1000s in each cell B. electron transport and oxidative phosphorylation Cytosol
  4. 4. C. Outer membrane- permeable to small molecules D. Inner membrane- electron transport enzymes embedded;  also ATP synthase  Cristae increase area  Impermeable to small molecules Integrity required for coupling ETS to ATP synthesis Cytosol
  5. 5. E. Matrix TCA enzymes, other enzymes; also ATP, ADP, NAD+, NADH, Mg2+, etc.
  6. 6.  The Electron Transport System is the mechanism the cell uses to convert the energy in NADH and FADH2 into ATP.  Electrons flow along an energy gradient via carriers in one direction from a higher reducing potential (greater tendency to donate electrons) to a lower reducing potential (greater tendency to accept electrons).  The ultimate acceptor is molecular oxygen.
  7. 7. -- The overall voltage drop from NADH E = -(-0.32 V) to O Eº = +0.82 V is Eº = 1.14 V
  8. 8. -- This corresponds to a large free energy change of G = - nFE = -220 kJ/mole (n =2) -- Since ATP requires 30.5 kJ/mole to form from ADP, more than enough energy is available to synthesize 3 ATPs from the oxidation of NADH.
  9. 9. NADH Dehydrogenase- Complex I NADH-CoQ oxidoreductase Contains FMN/FMNH2 and an Iron Sulfur Center as Electron Carriers NADH is substrate Coenzyme Q is second substrate
  10. 10. NAD+/NADH NADP+/NADPH Never covalently bound- freely diffusible  Nicotinamide 
  11. 11. Flavin mononucleotide = FMN Flavin adenine dinucleotide = FAD Riboflavin = ring + ribitol isoalloxazine ring ribitol
  12. 12. 2H++2e Coenzyme Q Coenzyme Q Coenzyme Q = Ubiquinone a lipid in inner membrane  carries electrons  polyisoprene tail  moves freely within membrane CoQ CoQH2 (reduced form)
  13. 13. For NADH, one of two entry points into the electron transport chain: -- So the oxidation of one NADH results in the reduction of one CoQ -- Another important function of the enzyme will be mentioned later.
  14. 14. Succinate Dehydrogenase- Complex II Succinate:CoQ oxidoreductase Similar reaction can be written yielding CoQH2 Second entry into electron transport Substrate is succinate Contains Iron Sulfur Center  FAD is reduced, not FMN CoQH2 carries electrons to cytochrome b
  15. 15. Cytochromes - proteins in ETS Carry electrons Contain heme or heme-like group  carries electrons only: Fe(III) + e-  Fe(II)
  16. 16. -- Cytochromes in respiration are on inner mitochondrial membrane  cytochromes b, c1, c, a, a3 , relay electrons,one at a time, in this order 
  17. 17.  COMPLEX III = b, an Fe-S and c1.  Cytochrome c is mobile.  COMPLEX IV = a+a3 = cytochrome a-a3 = cytochrome c oxidase -- large protein. -- both a and a3 contain heme A and Cu -- a3 Cu binds to oxygen and donates electrons to oxygen cytochrome a3 - only component of ETS that can interact with O2
  18. 18. Cytochrome c oxidase Heme A and Cu act together to transfer electrons to oxygen Cu(II)  Cu(I)e- from cyt c to a
  19. 19. Sequence of Respiratory Electron Carriers Inhibitors in green
  20. 20. How is amount ATP synthesized measured? Quantify P/O ratio Definition: # Pi taken up in phosphorylating ADP per atom oxygen (½O2), in other words per 2e-. NADH 3 FADH2 2
  21. 21. Experimental, we know As electrons are passed through: NADH oxidized by CoQ Cytochrome b oxidized by cytochrome c1 Cytochrome a oxidized by O2 Each yields enough energy to synthesis about one ATP So oxidation of NADH yields about 3 ATPs Oxidation of FADH2 gives only 2 ATPs (succinate dehydrogenase & others)
  22. 22. What about energy and ATP stoichiometry? -- measured -- 220 kJ/mole from NADH oxidation -- Each ATP produced: ADP + Pi  ATP G = +30.5 kJ/mole [3 (30.5)/220] 100 = 41% efficiency
  23. 23. Oxidative Phosphorylation -- (ox-phos) Definition: Production of ATP using transfer of electrons for energy = coupled --for NADH, we know cyt b O2 NADHFMN-FeS  CoQ  FeScyt c  cyt aa3  cyt c1  ATP ATP ATP Complex I Complex III Complex IV Note: Several small energy steps
  24. 24. What are the requirements for coupling? -- Lehninger in the 50's and 60's  Intact mitochondria = intact inner membrane, respiratory chain  Pi  ADP  NADH or other reductant no other metabolites needed!
  25. 25. Acceptor Control Suspend intact mitochondria with NADH and Pi Add ADP add ADP O2 taken up time add ADP Requires ADP for oxygen uptake = coupling
  26. 26. How is this coupling accomplished? -- It was originally thought that ATP generation was somehow directly done at Complexes I, III and IV. -- We now know that the coupling is indirect in that a proton gradient is generated across the inner mitochondrial membrane which drives ATP synthesis.
  27. 27. ATP Synthetic Machinery = FoF1 ATP synthase Complex -- in inner mitochondrial membrane Matrix
  28. 28. -- knob-like projections on the matrix side called F1 spheres. -- responsible for ATP production since when removed by trypsin treatment, the resulting membranes still transport electrons but do not make ATP.
  29. 29. FoF1 ATP synthase -- ATP synthesized on matrix side. -- electron transport complexes and FoF1 ATP synthase arranged on the inner membrane of the mitochondrion facing in and lining the membranes bordering the cristae. *********************************************************
  30. 30. Chemiosmotic Theory --Peter Mitchell -- A proton gradient is generated using energy from electron transport. --The vectorial transport of protons (proton pumping) is done by Complexes I, III, IV from the matrix to intermembrane space of the mitochondrion.
  31. 31. -- The protons have a thermodynamic tendency to return to the matrix = Proton-motive force The proton move back into the matrix through the FoF1ATP synthase driving ATP synthesis.
  32. 32. The proton pumps are Complexes I, III and IV. Protons return thru ATP synthase
  33. 33. The return of protons “downhill” through Fo rotates Fo relative to F1, driving ATP synthesis. Note: Subunit  rotates through F1.
  34. 34. ATP synthesis at F1 results from repetitive comformational changes as  rotates  rotates 1/3 turn- energy for ATP release animation
  35. 35. Experimental corroboration  Uncoupling. The compound 2,4 dinitrophenol (DNP) allows proton through the membrane and uncouples. Blocking. The antibiotic oligomycin blocks the flow of H+ through the Fo, directly inhibiting ox-phos.
  36. 36. Respiratory Control -- Most mitochondria are said to be tightly coupled. That is there is no electron flow without phosphorylation and no phosphorylation without electron flow. -- Reduced substrate, ADP, Pi and O2 are all necessary for oxidative phosphorylation.
  37. 37. For example, in the absence of ADP or O2 electron flow stops, reduced substrate is not consumed and no ATP is made = acceptor control. Under certain conditions, coupling can be lost. -- A toxic, nonphysiological uncoupler, DNP, was described previously.
  38. 38. -- Brown adipose (fat) cells contain natural uncouplers to warm animals - cold adaptation and hibernation.
  39. 39. Shuttling Reducing Equivalents from Cytosolic NADH -- Electrons from NADH are shuttled across the mitochondrial membrane by carriers since NADH cannot cross inner membrane. -- reoxidation of cytosolic NADH leads to different energy yields depending on mechanism the cell uses to shuttle the reducing equivalents.
  40. 40. -- The dihydoxyacetone phosphate shuttle yields 2 ATP/NADH -- The malate shuttle yields 3 ATP/NADH
  41. 41. cytosolic malate dehydrogenase mitochondrial malate dehydrogenase
  42. 42. Review of the Energy Yield from Glycolysis, Pyruvate Dehydrogenase and the TCA Cycle Glycolysis: glucose 2pyruvate + 2NADH+2ATP 6-8 ATPs Pyruvate Dehydrogenase: pyruvate  acetyl CoA + NADH 6 ATPs TCA cycle: acetyl CoA 2CO2+3NADH+FADH2+GTP 2x12ATPs OVERALL yield from glucose 36-38 ATPs
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