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Biological oxidation (part - III) Oxidative Phosphorylation
The document discusses the processes of oxidative phosphorylation and substrate-level phosphorylation in ATP synthesis, primarily occurring in the mitochondria. It explains the roles of the electron transport chain and proton gradients in ATP generation, detailing hypotheses including chemiosmotic theory and experimental findings on proton leakage affecting ATP production. Additionally, it covers the mechanisms of uncouplers, ATP synthesis regulation by ADP and ATP levels, and the shuttle systems for transporting NADH into mitochondria.
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Biological oxidation (part - III) Oxidative Phosphorylation
- 1. ADP + Pi ATP b2 δ α α α β ββ H+ Fo F1 ( Part –III )
- 2. Definition ➢The synthesis ofATP from ADP (phosphorylation), that occurs when NADH and FADH2 are oxidized by through electron transport chain (respiratory chain) ➢Oxidation coupled with phosphorylation is called Oxidative phosphorylation ➢Mitochondria are the site of oxidative phosphorylation in eukaryotes Oxidative Phosphorylation
- 3. During transfer ofelectrons through the ETC energy is produced. This energy is coupled to the formation of ATP from ADP. By an enzyme F0F1 ATPase.
- 5. ✓ Oxidative phosphorylation: the phosphorylation of ADP to ATP coupled to electron transfer ✓ Substrate level phosphorylation : direct transfer the phosphate from chemical intermediate (also called substrate ) to ADP or GDP forming ATP or GTP, independent of electron transfer chain.
- 6. Example of Substrate levelphosphorylation Glycolysis Phosphoglycerate kinase 1,3-bisphosphoglycerate 3-phosphoglycerate ADP ATP
- 7. Phosphoenolpyruvate ADP ATP Pyruvate Pyruvate kinase Succinyl CoA GDP GTP succinate succinylCoA synthetase Glycolysis TCA cycle
- 9. Several hypotheses havebeen put forth to explain the process of oxidative phosphorylation. The most important among them-namely,
- 10. This hypothesis wasput forth by Edward Slater (1953) According to this hypothesis, during the course of electron transfer in respiratory chain, a series of phosphorylated high-energy intermediates are first produced which are utilized for the synthesis of ATP. These reactions are believed to be analogous to the substrate level phosphorylation that occurs in glycolysis or citric acid cycle. However, this hypothesis lacks experimental evidence.
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- 12. This hypothesis isthe most accepted theory. proposed by Peter Mitchell in 1961. To explain the oxidative phosphorylation. Nobel Prize, in 1978 It explains how the transport of electrons through the respiratory chain (ETC) is effectively utilized to produce ATP from ADP + Pi.
- 13. There are threebasic principles of the theory. 1. Pumping of protons via electron carrier proteins 2. Generation of electrochemical potential. i. Membrane potential ii. Proton gradient (chemical potential) 3.Electron transport flow back to matrix through ATPase.
- 14. The inner mitochondrialmembrane, is impermeable to protons & hydroxyl ions. The transport of electrons through ETC is coupled with the translocation of protons (H+) across the inner mitochondrial membrane from the matrix to the intermembrane space. This results in an electrochemical or proton gradient.
- 15.
- 17. 10H+ Complex III 4H+ 2H+ 4H+ 4H+4H+ 2H+ NADH+ +H+ NAD+ O2 H2O FoF1 Negative (Alkaline) Positive (Acidic) Outer mitochondrialmembrane 10H+ 2.5ADP +2.5Pi 2.5 ATP Chemiosmotic theory
- 18. Complex I andcomplex III pumps 4 protons each Complex IV pumps 2 protons To inter-membranous space 10 protons are pumped by the electron transport chain The transfer of two electrons from NADH+ H+ to O2 is accompanied by the outward pumping of 10 H+ 10 protons are pumped out per NADH 1. 4 must flow in to produce 1 ATP 2. The proton-based P/O ratio is 2.5 for NADH as the electron donor and 1.5 (6/4) for succinate
- 19. ATP generation (oldand new values)
- 20. In summary : Theoxidative phosphorylation process is as follow Electron transport down the respiratory chain from NADH or FADH2 Complex Ⅰ,Ⅲ,Ⅳ Cause protons be pumped out of the mitochondrial matrix into the intermembrane space The pumping out of H + generates a higher conc. of H+ and an electrical potential , thus an electrochemical proton gradient is formed. The H + flow back into the mitochondrial matrix through ATP synthase and the electrochemical proton gradient drives ATP synthesis
- 21. Proposed by PaulBoyer in 1964 (Nobel Prize, 1997)
- 22. Fo F1 ATPase F0 F1 Alsocalled ATP synthase. Embedded in the inner membrane ADP + Pi ATP b2 δ α α α β β β H+ Fo F1
- 23. ⚫Also called complexⅤ ⚫It is the enzyme that actually synthesize ATP ⚫It located in the inner mitochondrial membrane ⚫It utilizes energy from the proton gradient to promote phosphorylation of ADP forming ATP ADP + Pi ATP b2 δ α α α β β β H+ Fo F1
- 24. ❑Spherical projections fromthe inner membrane. ❑Is composed of two major components part, ❑F1 unit or called F1 ATPase : ❑The spheres of the ATP synthase & point outward ❑F0 unit : ❑Spans the inner mitochondria membrane ❑So the ATP synthase is also called F0F1 ATPase ❑The stalk between F0 and F1 contains several additional polypeptide
- 25. F1 unit : Contains5 types of polypeptides Arranged in α3β3γδε. F0 unit: Made of abc polypeptides, Is proton channel / proton transport. F1 with F0 together can synthesize ATP Only F1 components have ATPase activity, so also called ATPase, ADP + Pi ATP b2 δ α α α β β β H+ Fo F1
- 26. ETC and Oxidativephosphorylation
- 29. P:O ratio The P:Oratio refers to 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. This results in conversion of energy required for production of only 3 ATP from NADH and 2 ATP from FADH2
- 30. The mitochondrial oxidationof NADH with a classical P : O ratio of 3 can be represented by the following equation : NADH + H+ + ½ O2+ 3ADP + 3Pi NAD+ + H2O+ 3ATP
- 31. Current Concept, Energeticsof ATP Synthesis According to the estimated free energy of synthesis, it was presumed that around 3 protons are required per ATP synthesized. Hence when 1 NADH transfers its electrons to oxygen, 10 protons are pumped out. This would account for the synthesis of approximately 3 ATP. Similarly the oxidation of 1 FADH2 is accompanied by the pumping of 6 protons, accounting for 2 molecules of ATP. However, Peter Hinkle recently proved that the actual energy production is less, because there is always leakage of protons. This results in harnessing of energy required for the production of 2.5 ATP from NADH and 1.5 ATP from FADH2.
- 32. Complex-I Ⅱ Complex-IV F0 F1 Complex-III Cyt c Q NADH+H+ NAD+ Fumarate Succinate H+ 1/2O2+2H+ H2O ADP+Pi ATP H+ H+ H+ Intermembranespace matrix + + + + + + + + + + - - - - - - - - - Coupling sites for ATP synthesis.
- 33. Generate ATP sums 2.5ATP are synthesized per NADH oxidized through the NADH respiratory chain 1.5 ATP are synthesized per FADH2 oxidized through the FADH2 respiratory chain
- 34. Why ATP synthesizefrom FADH2 respiratory chain is less than NADH respiratory chain? NADH respiratory have 3 H+ pump, complex Ⅰ,Ⅲ,Ⅳ FADH2 respiratory only have 2 H+ pump, complex Ⅲ,Ⅳ So the ATP made from FADH2 is less than from NADH
- 36.
- 37. Electron transport isnormally tightly coupled to ATP synthesis Electrons do not flow through the ETC to O2 unless ADP is simultaneously phosphorylated to ATP. Also, ATP is not synthesized unless electron transport is occurring to provide the proton gradient The transport of electrons is tightly coupled with oxidative phosphorylation (ATP synthesis).
- 38. There are certaincompounds that can uncouple (or delink) the electron transport from ATP synthesis. Such compounds, called as uncouplers. the permeability of inner mitochondrial membrane to H+. The result is that ATP synthesis does not occur. The energy is dissipated as heat.
- 39. What is uncouplers? Thereare certain compounds that can uncouple (or delink) the electron transport from ATP synthesis. 2,4-dinitrophenol (DNP) Thermogenin (Natural uncoupling protein)
- 40.
- 41.
- 42. ? ◆ electron transportoccurs and pump out H+ ions across the inner mitochondrial membrane to build the H+ gradient . ◆But DNP in the same membrane carriers the H+ ions back into the mitochondrion , preventing formation of a proton gradient. ◆Since no proton gradient forms, so no ATP can be made by oxidative phosphorylation ◆the energy derived from electron transport is released as heat.
- 43. There is brownadipose tissue in the body, This tissue is rich in mitochondria, the inner mitochondrial membranes of which contains a protein called uncoupling protein or thermogenin.
- 44. How uncoupling proteinwork ? Uncoupling protein can be seen as a H+ passageway, allows H+ to flow back into mitochondria without having to enter the ATP synthase, thus Preventing formation of a proton gradient , so uncouples electron transport & oxidative phosphorylation. Energy derived from electron transport is released as heat
- 45. mechanism of uncouplingprotein （brown adipose tissue mitochondrial ） Ⅲ Ⅰ Ⅱ F0 F1 Ⅳ Cyt c Q Intermembrane space Matrix Uncoupling protein Heat energy H+ H+ ADP+Pi ATP
- 46. ◆The production ofheat by uncoupling is called non-shivering thermogenesis. ◆It is important in certain biological situation , ◆For example ,the brown adipose tissue is found in sensitive body areas of some new brown animals (including human ), where the heat production provides protection from cold condition ◆In addition, thermogenesis by brown adipose tissue plays a important role in maintaining body temperature in hibernating animals
- 51. The availability ofADP 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.
- 52. This mechanism ensuresthat electrons flow down the chain only when ATP synthesis is needed. If the level of ATP is high, ADP is low, no electron transport occurs NADH and FADH2 build up, so does excess citrate citric acid cycle & glycolysis are all inhibited
- 53. Over all ADP high Oxidativephosphorylation rises Oxygen consumption rises ADP low Oxidative phosphorylation falls Oxygen consumption falls
- 54.
- 55.
- 56. Production and applicationof ATP ATP ADP oxidative Phosphorylation ~P ~P Mechnism energy Osmotic energy Chemical energy Electric energy Hot energy substrate level Phosphorylation
- 58. The inner membraneof mitochondria impermeable to some molecule and ions Permeable to : pyruvate, succinate, Citrate, α- ketoglutarate, malate, Glu etc Impermeable to : H+, NADH, NADPH, oxaloacetate, etc
- 59. ◆The inner mitochondrialmembrane is impermeable to NADH. ◆Therefore NADH produced in the cytoplasm during glycolysis go into the mitochondria through the membrane shuttle , then in the mitochondria go into the respiratory chain . ◆The membrane shuttle is a combination of enzyme reaction that bypass this impermeability barrier
- 60. Which reaction ofglycolysis produce NADH Glyceraldehyde 3-phosphate 1,3-bisphosphoglycerate Glyceraldehyde 3-phosphate Dehydrogenase NAD+ NADH The reaction take place in the cytosol
- 61. There is twoshuttle system in the mitochondrial membrane
- 62.
- 63. Note : ◼ Theshuttle does not allow cytoplasm NADH to enter the mitochondrion, ◼ But transports the two electrons from NADH into the mitochondria ◼ Feed the electron into the FADH2 electron transport chain . ◼ So synthesize 2ATPs
- 64.
- 65. this cycle ofreactions is to transfer the electrons from NADH in the cytosol to NADH in the mitochondrial matrix , The NADH in the mitochondria is then reoxidized by the NADH electron transport chain So synthesize 3ATPs Note
- 66. In summary : CytosolNADH go into the mitochondria by this two shuttele • glycerol 3-phosphate shuttle in the mitochondria go into the FADH2 respiratory chain. so produce 2 ATPs Malate-asparate shuttle: in the mitochondria go into the NADH respiratory chain. so produce 3ATPs