Oxidative Phosphorylation PDF

Oxidative Phosphorylation Tultul Nayyar, Ph.D., MSCI Associate Professor and Director Master of Health Sciences Program West Basic Science, Room 3210 Phone: 615-327-6898 (O) 615.944.0824 (C) email: [email protected] Oxidative Phosphorylation *  Glycolysis and the citric acid cycle yield NADH and...

Oxidative Phosphorylation Tultul Nayyar, Ph.D., MSCI Associate Professor and Director Master of Health Sciences Program West Basic Science, Room 3210 Phone: 615-327-6898 (O) 615.944.0824 (C) email: [email protected] Oxidative Phosphorylation *  Glycolysis and the citric acid cycle yield NADH and FADH2  These electron carriers are energy-rich molecules  Oxidative phosphorylation is the process of converting the energy stored in NADH and FADH2 into energy-rich ATP molecules (respiration), occurs inside the mitochondria [Only a small portion of the energy of glucose has been converted to ATP as a result of glycolysis, and the citric acid cycle through substrate level phosphorylation. At this point, most of the usable energy is contained in which of the following: NADH and FADH2, NAD+ and FAD, Pyruvate, Acetyl coenzyme A or Carbon dioxide?] The electron transport chain (ETC) is a series of proteins and organic molecules found in the inner membrane of the mitochondria. Electrons are passed from one member of the ETC to another in a series of redox reactions. Energy released in these reactions is captured as a proton gradient, which is then used to make ATP in a process called chemiosmosis. Delivery of electrons by NADH and FADH2: Reduced electron carriers (NADH and FADH2) transfer their electrons. In the process, they turn back into NAD+ and FAD, which can be reused in other steps of cellular respiration. A. Oxidation step: *  involves electron transport chain which transfers the electrons from NADH and FADH2 finally to oxygen, the electron acceptor, to produce water NADH + H+ + FADH2 + 1 2 1 2 O2 O2 NAD+ + H2O FAD + H2O During electron transfer, released energy is used to transport H+ (protons) across the inner mitochondrial membrane to create an electrochemical gradient (proton gradient) * B. Phosphorylation step : ADP + Pi ATP (Endergonic) ATP is synthesized by proton gradient formed in the mitochondrion by ATP synthase : The “chemiosmotic hypothesis” Mitochondria     Outer Membrane Intermembrane space Inner membrane (ETC is here) Matrix Electron Transport * Four protein complexes (I-IV) in the inner mitochondrial membrane (They are stationary because membrane bound) • Complex I: NADH-Q oxidoreductase • Transfers electrons from NADH to coenzyme Q (also called ubiquinone or Co Q or UQ) • Complex II: succinate-Q-reductase • Transfers electrons from FADH2 to Co Q • Complex III: Q-cytochrome c oxidoreductase • Passes the electrons from CoQ to cytochrome c • Complex IV: Cytochrome c oxidase • Passes the electrons from cytochrome c to molecular oxygen Oxygen is reduced to water * * CoQ or QU (lipid soluble) and cytochrome c (water soluble) are mobile and thus shuttle between protein complexes Remember: Coenzyme Q is involved in electron transport as a lipid-soluble electron carrier and cytochrome C as water-soluble electron carrier Flow of electrons through complexes results in pumping of protons from the matrix into the intermembrane space That means: Electrons moves through complexes Protons gets pumped by the complexes Transfer of electrons through the protein complexes in the ETC, involves series of reactions that include the following components: • FMN (or FAD) • Components of complex I, II • Transfers: • 2 electrons to form FADH2 • 1 electron to form semiquinone • Iron-sulfur proteins (Fe-S proteins) • Components of complex I, II, III • Transfers: • 1 electron Complex I: NADH-CoQ Reductase Electron transfer from NADH to CoQ NADH + Q + 5H+matrix → NAD+ + QH2 + 4H+ QH2 diffuses through the membrane, delivering electrons to complex III (QH2→Q), then returns to complex I for more • Four H+ pumped out of mitochondrial matrix per 2 e- Complex II: Succinate-CoQ Reductase (aka succinate dehydrogenase) Electron transfer from FADH2 to CoQ • Entry point for electrons from FADH2 formed from oxidation of succinate (from TCA cycle!) • Electrons transferred to coenzyme Q • Path: succinate FADH2 QH2 …(QH2 Q in complex III) • No protons are pumped REMEMBER! * Complex III: CoQ-Cytochrome c Reductase Electrons transfer from QH2 to cytochrome c: Q cycle • Cytochrome c is one- electron transfer agent: whereas Q carries 2 electrons. Thus, oxidation of one QH2 is coupled to the reduction of 2 molecules of cytochrome c via the Q cycle • Four H+ pumped out Complex IV: Cytochrome c Oxidase Electrons transfer from reduced cytochrome c to molecular oxygen • Oxygen is thus the terminal electron acceptor in the electron transport pathway - the end! • Two H+ pumped out per 2e- from NADH or FADH2 molecule by complex IV * Coupling e- Transport and Oxidative Phosphorylation Chemiosmotic hypothesis: a proton gradient across the inner membrane drives ATP synthesis * The flow of electrons through complex I, III and IV act as proton pump that draw protons (H+) from matrix to the inter-membrane space Negative on the matrix side and positive on intermembrane space A proton motive force thus created by the electrochemical potential difference H+ ions flow down their concentration gradient via the proton channel in ATP synthase (complex V) embedded in the inner membrane to make ATP from ADP and Pi (endergonic) What powers the ATP synthase???? Thus, oxidative phosphorylation generates ATP by * phosphorylating ADP, powered by proton gradient which is formed by removal of electrons from NADH and FADH2 What cellular conditions favor increased activity of the electron transport chain and oxidative phosphorylation? High NADH, high ADP or high oxygen? Which of the following would be expected to increase the amount of ATP produced through oxidative phosphorylation: administration of oxidative phosphorylation uncouplers, Decreasing NADH levels, Decreasing the pH in intermembrane space, Increasing the permeability of the inner mitochondrial membrane to protons or Increasing the proton concentration in the mitochondrial matrix? Glucose degradation under aerobic condition, which is correct? A. Oxygen is the final electron acceptor B. Oxygen is necessary for all ATP synthesis C. Net water is consumed D. The proton-motive force is necessary for all ATP synthesis When ADP levels rise and the demand for ATP synthesis increases, electron flow through the ETC also increases. A. NADH and FADH2 will be oxidized to NAD+ and FAD less rapidly. B. The flow of protons through ATP synthase will decrease. C. Oxygen consumption will increase. D. Fewer protons will be pumped across the inner mitochondrial membrane. Cyanide causes histotoxic hypoxia. As a result, ATP production during respiration is significantly reduced. A. Cyanide inhibits the transport of pyruvate across the mitochondrial membrane. B Cyanide inhibits the transfer of electrons to the final acceptor in the electron transport chain. C. Cyanide inhibits the enzymatic breakdown of glucose during glycolysis. D. Cyanide inhibits the reduction of NAD+ and FAD during the Krebs cycle. • ETC plays a critical role in forming and utilizing the proton gradient to synthesize ATP • Protons are “pumped” from the matrix into the intermembrane space • ATP is synthesized in the matrix, as protons flow back through the membrane ATP-ADP Translocase ATP must be transported out of the mitochondria • ATP out, ADP in - through a "translocase" enzyme • ATP out and ADP in is net movement of a negative charge out - equivalent to a H+ going in • So one ATP transported out costs one H+ • One ATP synthesis costs about 3 H+ • Thus, making and exporting 1 ATP costs 4H+ P/O (Phosphate/Oxygen) Ratio i.e., How many ATP made per electron pair through the ETC? • ETC yields 10 H+ pumped out per electron pair from NADH to 0.5 mol of oxygen (starting from complex I) 4 H+ (complex I) + 4 H+ (complex III) + 2 H+ (complex IV) =10 H+ • 4 H+ flow back into matrix per ATP to exit into cytosol 2.5 ATP for electrons entering as NADH (P/O Ratio) ** • 10/4 = * • For electrons entering as succinate (FADH2), about 6 H+ pumped per electron pair to 0.5 mol of oxygen • 4 H+ (complex III) + 2 H+ (complex IV) = 6 H+ 1.5 ATP for electrons entering as FADH2 (P/O Ratio) • 6/4 = So, relative to FADH2, at what energy state does NADH enter the electron transport chain? Higher or lower or same? * Shuttle Systems for electrons transport Glycolysis yields NADH in the cytosol (Reaction step # 6) Mitochondrial membrane is impermeable to cytosolic NADH "Shuttle systems" accept electrons from cytosolic NADH and enter mitochondria, and give up electrons to electron acceptors in the ETC * Two different “shuttles” are commonly encountered: • Glycerophosphate shuttle stores electrons in glycerol-3-P, which transfers electrons to FAD • Malate-aspartate shuttle uses malate to carry electrons across the membrane What you know from GLYCOLYSIS Reaction 5: isomerization Triose Phosphate Isomerase (TIM) catalyzes: dihydroxyacetone-P  glyceraldehyde-3-P What you know from GLYCOLYSIS Reaction 6: oxidation This is the only step in Glycolysis in which NAD+ is reduced to NADH Glyceraldehyde-3-phosphate Dehydrogenase catalyzes: glyceraldehyde-3-P + NAD+ + Pi  1,3-bisphosphoglycerate + NADH + H+  Glycerol 3-phosphate shuttle: (skeletal muscle and brain) * 1. Dihydroxyacetone phosphate (DHP) + NADH glycerol-3-phosphate + NAD+ by glycerol-3-phosphate dehydrogenase (cytosolic form) 2. glycerol-3-phosphate +FAD DHP+FADH2 by isozyme of glycerol-3-phosphate dehydrogenase (mitochondrial membrane bound) 3. FADH2 transfers electrons to Q via ETC with the formation of QH2 +FAD • Thus, in muscle and brain though two NADH are produced by glycolysis, actually, two FADH2 are available for entry into ETC (So how many ATP will be formed here?....3) Glycerol 3-phosphate shuttle: (NADH 2e- FADH2) * 33  Malate-aspartate shuttle: (Heart and liver) * • Oxaloacetate (TCA cycle Reaction # 8), formed inside the mitochondria, impermeable to mitochondrial membrane, but needs to come out to cytosol for gluconeogenesis or goes back to mitochondria for oxidation and energy formation 1. Oxaloacetate + glutamate α-Keto glutarate + aspartate by transaminase (mitochondria) 2. Aspartate comes out from mitochondria to cytosol 3. Aspartate + α-Keto glutarate Oxaloacetate + glutamate (cytosol) 4. Oxaloactate goes for gluconeogenesis OR 5. Oxaloacetate + NADH Malate + NAD+ (cytosol) by malate dehydrogenase 6. Malate enters mitochondria 7. Malate + NAD+ Oxaloacetate + NADH (mitochondria) (TCA cycle Reaction # 8) TCA cycle….ETC • Malate-aspartate shuttle: Gluconeogenesis 35 Net Yield of ATP from one molecule of Glucose * It depends on which shuttle is used! • 30 ATP (glycerol-3-P shuttle used) • 32 ATP (malate-asp shuttle used) Why cytosolic NADH can yield potentially less ATP than mitochondrial NADH? A. Cytosolic NADH always loses energy when transferring electrons. B. Once NADH enters the mitochondrial matrix from the cytosol, it becomes FADH2. C. Electron transfer from cytosol to mitochondrial matrix can take more than one pathway. D. There is an energy cost for bringing cytosolic NADH into the matrix. * * A: ATP from glycolysis Net gain: 2 2 * B: ATP from Citric Acid Cycle Net gain 2 2 * C: ATP from Oxidative Phosphorylation Net gain So net yield of ATP (A + B + C) : 26 28 30 32 Uncoupling oxidative phosphorylation * • ATP synthesis can be “uncoupled,” if the proton gradient is prematurely dissipated or impeded • Uncoupling serves to generate body heat • Certain inhibitors of electron transport act at specific sites to stop electron flow • Reactive oxygen species (ROS): Toxic derivatives of molecular oxygen may be _ _ formed by partial reduction O2 → O2 → O2 2 Cells protect itself against these ROS by • use of superoxide dismutase and catalase _ • 2O2 + 2H+ → O 2 + H2O2 • 2H2O2 → O2 + 2H2O Summary ***** • Energy released from oxidation of carbohydrates, fat and proteins, makes reducing equivalents (NADH, FADH2 ) that are funneled through ETC inside the mitochondria, reacts with oxygen and form water • Electron transfer coupled with oxidative phosphorylation (OP) produces ATP • The ETC consists of 4 protein complexes, 3 proton pumps • Proton gradient, created by flow of electrons, powers (potential energy) the synthesis of ATP • Regulation of OP, is primarily by the need for ATP Which of the following directly provides the energy needed to form ATP in the mitochondrion? A. Electron transfer in the electron transport chain B. An electrochemical proton gradient C. Oxidation of acetyl-CoA During electron transport, protons are pumped out of the mitochondrion at each of the major sites except for: A. Complex I B. Complex II C. Complex III D. Complex IV E. Complex V • Which shuttle system employs an intermediate of glycolysis rather than the TCA cycle? Aspartate shuttle, Citrate shuttle , Cytochrome shuttle, Malate shuttle or Glycerol phosphate shuttle? • Human produce a natural uncoupling protein that can uncouple the proton gradient from ATP synthesis. Under which of the following conditions, you think that body produces this protein ? After a large meal, After extensive fasting, During physical exercise, After excessive fluid loss or In extremely cold weather? Streptococcus mutans, an anaerobic bacterium often found in the oral cavity, is strongly associated with the formation of dental cavities. S. mutans metabolizes glucose and other dietary sugars remaining in the mouth after a meal, producing lactic acid as a byproduct. Over time, high levels of lactic acid can erode tooth enamel, eventually leading to the formation of dental cavities. Which of the following drugs would be most likely to prevent cavities caused by S. mutans? A. drug that prevents the movement of oxygen across the cell membrane B. A drug that inhibits the formation of a proton gradient across the cell membrane C. A drug that inhibits the production of NADH and FADH2 during the Krebs cycle D. A drug that prevents the conversion of glucose into pyruvate The enzyme isocitrate dehydrogenase (IDH) catalyzes the conversion of isocitrate to αketoglutarate (step 3). IDH is allosterically activated by ADP at high concentrations. Which of the following best describes how this interaction helps regulate the Krebs cycle? • Low levels of ADP stimulate the Krebs cycle, leading to the production of excess ATP that is stored for later use • High levels of ADP stimulate the Krebs cycle, leading to the increased conversion of ADP to ATP during oxidative phosphorylation. • High levels of ADP inhibit the Krebs cycle because ADP prevents IDH from catalyzing the conversion of isocitrate to α\alphaαalpha-ketoglutarate. • Low levels of ADP inhibit the Krebs cycle because IDH requires ADP as a substrate for the reaction it catalyzes. Cyanide acts as a poison because it inhibits complex IV, making it unable to transport electrons. How would cyanide poisoning affect 1) the electron transport chain and 2) the proton gradient across the inner mitochondrial membrane? A. The electron transport chain would speed up, and the gradient would become stronger B. The electron transport chain would stop, and the gradient would decrease C. Both the electron transport chain and the gradient would stay the same D. The electron transport chain would be re-routed through complex II, and the gradient would become weaker Dinitrophenol (DNP) is a chemical that acts as an uncoupling agent, making the inner mitochondrial membrane leaky to protons. It was used until 1938 as a weight-loss drug. How would DNP affect the amount of ATP produced in cellular respiration? Why do you think it is now off the market? It would increase ATP production, but could also cause dangerously high body temperature It would decrease ATP production, but could also cause dangerously high body temperature It would decrease ATP production, but could also cause dangerously low body temperature It would increase ATP production, but could also cause dangerously low body temperature A patient has been exposed to a toxic compound that increases the permeability of mitochondrial membranes to protons. Which of the following metabolic changes would be expected in this patient? A. Increased ATP levels * B. Increased oxygen utilization C. Increased ATP synthase activity D. Decreased pyruvate dehydrogenase activity The increased permeability of the inner mitochondrial membrane allows the proton-motive force to be dissipated. Therefore, ATP synthase is less active and is forming less ATP. The body will attempt to regenerate the proton-motive force by increasing fuel catabolism, eliminating choice D. This increase in fuel use requires more oxygen utilization in the electron transport chain.