Lecture 21 ETC and ATP with MCQs - Tagged PDF

Electron transport and oxidative phosphorylation 4BICH001W Biochemistry Dr Sarah K Coleman 1  Where we were: The TCA cycle is: 1) Final oxidative step in the catabolism of carbohydrates, fatty acids and amino acid...

Electron transport and oxidative phosphorylation 4BICH001W Biochemistry Dr Sarah K Coleman 1  Where we were: The TCA cycle is: 1) Final oxidative step in the catabolism of carbohydrates, fatty acids and amino acids. 2) It provides a flow of simple carbon compounds into anabolic processes. 3) It functions as a major source of energy compounds: generating 1 ATP by SLP, 3 NADH (7.5 ATP) and 1 FADH2 (1.5 ATP) for one turn of cycle. 2 Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous). 3  Substrate Level Phosphorylation If the glycerol-3 phosphate shuttle used - gives FADH2 - makes 3 ATP (not 5) Substrate Level Phosphorylation NADH gives 2.5 ATP and FADH2 gives 1.5 ATP 4  Cellular Respiration … Electron Transport Chain and Oxidative Phosphorylation to make ATP This is different from making of ATP via substrate-level phosphorylation (occurs in glycolysis). Remember OILRIG – oxidation is loss of electrons and reduction is the gain of electrons. – In electron transport chain NADH is oxidised (loses electrons) and these are passed to oxygen; which is reduced (to water). 5 6  The formation of ATP Substrate level ATP formation takes place in glycolysis Oxidative phosphorylation takes place as a product of the electron transfer chain following the TCA cycle 7 Location of Glycolysis, TCA Cycle and Oxidative Phosphorylation in a Eukaryotic Cell Inner mitochondrial membrane is impermeable 8  Electron Flow and Work Amount of work that can be carried out is given by the tendency of NADH to give up electrons and for oxygen to accept electrons. Tendency to give up or accept electrons is given by the standard reduction /oxidation (redox) potential E0’ E0’ is measured in volts: strong oxidising agents have a positive redox potential. E0’ (reduced to) volts oxygen (water) +0.82 FAD (FADH2) -0.12 OILRIG NAD+ (NADH) -0.32 9  Electron Transport is Exergonic The difference between the given values reflects the work that can be obtained (E0’ net). Amount of work is given by ΔG0’ = -nF E0’net where n = number of electrons transferred F = Faraday’s constant (96 kJ/mol) E0’net = net potential difference. 10  Electron Transport is Exergonic The difference between the given values reflects the work that can be obtained (ΔE0’). For example… Oxidation of NADH by O2 1) NAD+ + H+ + 2e- NADH E0’ = -0.315 V 2) ½O2 + 2H+ + 2e- H2O E0’ = +0.815 V Which of the above reactions has the greater standard reduction potential (greater affinity for electrons) ? 11  Electron Transport is Exergonic The difference between the given values reflects the work that can be obtained (ΔE0’). For example… Oxidation of NADH by O2 1) NAD+ + H+ + 2e- NADH E0’ = -0.315 V 2) ½O2 + 2H+ + 2e- H2O E0’ = +0.815 V Overall reaction is: ½O2 + NADH + H+ H2O + NAD + So net E0’ is: ΔE0’ = +0.815 V – (-0.315 V) = 1.130 V 12 Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous). 13 The Electron Transport Chain 4 protein complexes – within or on inner mitochondrial membrane NADH or FADH2 feed electrons into these complexes Electrons flow from high energy state to low energy state, eventually bind to oxygen Energy released is used to pump protons across the membrane 14  Nature of electron transport chain This is found in the inner mitochondrial membrane. Components are organised into large complexes in membrane. Components are vectorially organised – Complex I (NADH dehydrogenase) accepts e- from NADH – Complex II (succinate dehydrogenase) accepts e- from FADH2 – Complex III (ubiquinone-cytochrome c oxidoreductase) – Complex IV (cytochrome c oxidase) NONE of these complexes make ATP! 15  Electrons are shuttling through the complexes via redox centres 16  Nature of electron transport chain Components are organised into large complexes in membrane. Electrons flow through and between complexes Electrons shuttled between the Complexes by ‘mobile’ components Electrons from Complex I and II shuttled by Coenzyme Q to Complex III Electrons from Complex III shuttled by Cytochrome C to Complex IV 17 Nature of Electron Transport Chain Energy generated by electron flow (ΔG0’ ) used to pump protons across inner mitochondrial membrane – from matrix to intermembrane space. Against the concentration gradient. 18  Components of the ETC: Flavin ring Flavoproteins are proteins with FAD tightly bound to it. Complex I and II are a flavoproteins. The flavin ring accepts 2 electrons and 2 protons. 19  Components of the ETC: Iron-sulphur clusters Fe/S proteins (non-haem iron proteins) have an iron ion that can be oxidised or reduced (Fe 2+/Fe3+). The iron is bound with sulphur atoms. Complex I, II, III have these. 20  Components of the ETC: Quinones The most common is ubiquinone (Coenzyme Q). Mobile electron carriers in membrane bilayer Shuttles between Complex I, II to complex III Highly hydrophobic, so lipid soluble molecules They accept 2 electrons and 2 protons. 21  Components of the ETC: Cytochromes Cytochromes are haem proteins with a central iron ion. Classified based on the structure of the haem groups. (Types a,b,c,d) They undergo 1 electron reduction. In Complex II, III, cytochrome c and Complex IV. 22  Components of the ETC: Cytochrome oxidase Cytochrome oxidase; Complex IV Terminal oxidase, has cytochrome a/a3 and 2x copper centres. Last component of the chain. Delivers electrons to oxygen, reducing it. A structurally complex enzyme with many subunits. Pumps protons across membrane. 23 ETC and ATP Production in Mitochondria 24 Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous). 25 Net Yield of ATP from oxidation of one molecule of glucose… 1 molecule of NADH generates 2.5 ATP and 1 molecule of FADH2 generates 1.5 ATP. Oxidation of NADH causes 10 protons to be pumped out Oxidation of FADH2 causes 6 protons to be pumped out What do the protons do? Pumping protons is the reason for the ETC! 26  ATP Production in Mitochondria ATP synthase (ATPase) is separate from electron transport chain Enzyme which makes ATP; from ADP and phosphate (Alternatively, it can use ATP and hydrolyse it to release energy). Found on the inner face of the inner mitochondrial membrane as stalked particles. 27 Mitochondrial Matrix ATP Synthase Stalk is the F1 region. Catalytic site Part associated with the membrane is the Fo region. OXIDATIVE PHOSPHORYLATION synthesis of ATP Mitochondrial Intermembrane Space 28 How is electron flow coupled to ATP synthesis? Electron transport and oxidative phosphorylation are coupled by process of Chemiosmosis. Proposed by biochemist Peter Mitchell Nobel prize for Chemistry in 1978 "for his contribution to the understanding of biological energy transfer through the formulation of the chemiosmotic theory.” https://www.nobelprize.org/prizes/chemistry/1978/ mitchell/facts/ 1920 - 1992 29  Principles of Chemiosmosis ETC and ATP synthase are vectorially organised - they have a particular orientation in space. Inner mitochondrial membrane is impermeable to H+ ; it is essential for existence of H+ gradient. Primary energy conserving event is movement of H+ across the membrane. 30  Generating the proton gradient Proton gradient is also known as the proton motive force (PMF) Complexes I, III, IV of electron transport chain pump protons from matrix to inter-membrane space - as electrons are passed from NADH to oxygen. In plants photosynthesis can create the proton gradient. In some bacterial specific H+ pumping proteins. 31  Using the Proton Gradient The flow of H+ down the gradient (into matrix) drives ATP synthesis… Believed formation of ATP from ADP and Pi occurs spontaneously on ATP synthase headpiece. – So no major input of free energy Protein conformation changes allows release of ATP. Protein conformation change caused by rotation of subunits; this is driven by the H+ gradient. 32 33 Net Yield of ATP from oxidation of one molecule of glucose… Oxidation of NADH causes 10 protons to be pumped out Oxidation of FADH2 causes 6 protons to be pumped out 4 protons need to flow back in to make one ATP molecule Therefore: Oxidation of NADH is: 10 H+out / 4 H+in = 2.5 ATP Oxidation of FADH2 is: 6 H+out / 4 H+in = 1.5 ATP 34 Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous). 35  What is the evidence for chemiosmosis? Can detect protons as they are pumped out of the inner membrane. If a pH gradient is created across the membrane, ATP is synthesised. Evidence for vectorial organisation of the electron transport chain. – E.g. the ATPase is on inner side of membrane, cytochrome c is on other side. A closed compartment is necessary for ATP synthesis (to maintain the proton gradient). Ruptured mitochondria do not make ATP. Reconstitution experiments using different biological systems. Action of uncoupling agents. 36  Reconstitution experiments Using the proton pumping protein bacteriorhodopsin found in the purple membrane of bacterium halophile Halobacterium. Bacteriorhodopsin will pump protons when activated by light Isolate ATP synthase from cow heart tissue. Put the two proteins together in artificial membrane. The researcher controls what components present. 37  Reconstitution experiments Light Cow ATP synthase Bacterio- H+ ion gradient ATP rhodopsin When illuminated ATP is made. Conclusion reached is that the very, very different biological systems are linked by the proton gradient. 38  Action of uncoupling agents These are artificial IMS substances (e.g. DNP) that reversibly bind H+ and return it back through inner membrane and into matrix of mitochondrion. Breaks the proton Matrix gradient. 39  Natural uncoupling agent: Thermogenin Brown adipose tissue has specialised mitochondria containing thermogenin (UPC1) Thermogenin acts as uncoupling agent; uses proton gradient and generates heat. Important in newborn animals, hibernating animals and mammals adapted to the cold. Similar system in some plants allows them to melt the snow covering them, enabling them to receive more light. 40 41 Natural uncoupling agent: Thermogenin Thermogenin also known as UCP1 (Uncoupling Protein 1) 42  Other inhibitors of chemiosmosis… Cyanide: binds to complex IV (cytochrome oxidase) and stops electron transport. Rotenone: pesticide – binds to complex I and stops electron transport Mitochondrial diseases – reduced ATP production – Show in energy demanding tissues such as muscle and neurons – e.g. Cardiomyopathy, Sporadic myopathy affect Complex I and II – e.g. LHON, MELAS affect Complex I 43  Other uses of the proton gradient The proton gradient has a central role in many processes that involve energy. Making ATP Generating heat Causing movement (the bacterial flagellum rotates as the gradient is used up) Transports ions across membranes Involved in photosynthesis… 44  Summary of ATP production… In aerobic conditions 1 molecule of glucose can generate 32 molecules of ATP In anaerobic conditions 1 molecule of glucose will generate 2 molecules of ATP Oxidative phosphorylation MUCH more efficient However… the rate of ATP production in anaerobic glycolysis can be 100 x faster Which is why some muscle fibres are specialised for anaerobic glycolysis 45  In Summary… Glucose breakdown releases electrons… Electrons flow down an energy gradient… That drives proton pumps… Back flow of protons drives ATP production… So in conclusion… “Life is nothing but an electron looking for a place to rest” – Albert Szent-Györgyi 46 Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous). 47 MCQ quiz for Lecture 21: ETC and Oxidative Phosphorylation Answers will be uploaded These quizzes are part of your learning for the Biochemistry module They will aid your on-going studies at the University of Westminster 48 Q1) Which of the following provides evidence that supports the chemiosmotic theory of ATP synthesis? A. Protons can be detected when they are pumped out of the inner mitochondrial membrane. B. Imposition of a pH gradient across the membrane results in ATP synthesis. C. There is evidence of vectorial organisation of the electron transport chain. D. A closed compartment is necessary for ATP synthesis – to maintain the proton gradient. E. All of the above. 49 Which of the following statements is false? A. In the presence of oxygen, the maximum yield of ATP (from one molecule of glucose) is 32 molecules. B. In the absence of oxygen, the maximum yield of ATP (from one molecule of glucose) is 2 molecules. C. The Krebs cycle directly uses oxygen in its reactions. D. ATP synthase is separate from the electron transport chain. E. The majority of ATP produced from glucose catabolism is due to electron transfer down the electron transport chain. 50 Which of the following statements is false? A. The electrons in the Electron Transport Chain directly flow to ATP. B. Haem contain proteins are important in Electron Transport Chain. C. ATP synthase is separate from the Electron Transport Chain. D. The outer membrane of mitochondria is permeable to protons. E. Proteins containing iron and sulphur clusters are important in the Electron Transport Chain 51 The oxygen consumed during cellular respiration is involved directly in which process or event? A. Glycolysis B. Accepting electrons at the end of the electron transport chain C. The citric acid cycle D. The oxidation of pyruvate to acetyl CoA E. The phosphorylation of ADP to form ATP 52 To correctly predict the MAXIMUM amount of ATP which could be produced from a single molecule of glucose it is necessary to know what tissue the glycolysis pathway is occurring in. True or False? 53