FIMS Biochemistry – Citric Acid Cycle and ETC - PBC9400 PDF

Certificate in Health Professions Preparation FIMS Biochemistry – PBC9400 Week 9 Citric Acid Cycle and Electron Transport Chain Thomas A. Panavelil, Ph.D., M.S., M.B.A. Learning Objectives: Citric Acid Cycle and Electron Transport Chain 1. Describe the citric acid cycle and where elect...

Certificate in Health Professions Preparation FIMS Biochemistry – PBC9400 Week 9 Citric Acid Cycle and Electron Transport Chain Thomas A. Panavelil, Ph.D., M.S., M.B.A. Learning Objectives: Citric Acid Cycle and Electron Transport Chain 1. Describe the citric acid cycle and where electrons and carbons go. 2. Describe how electrochemical gradients are produced. 3. Explain how ATP is produced during chemiosmosis. 4. Describe the theoretical energy yield for cellular respiration. 5. Identify entry points of fats and proteins in cellular respiration. 6. Describe key intermediates, key regulators, and negative feedback mechanism of cellular respiration Lecturio videos: https://nova.lecturio.com/#/lecture/c/8060/8024/41244 https://nova.lecturio.com/#/lecture/c/8060/8024/41246 2 Respiration Conversion of nutrients into useful energy for the cell Carbohydrates Proteins Fats Sugars Amino acids Fatty acids & glycerol Acetyl-CoA Metabolism Fats Polysaccharides Proteins Fatty acids Monosaccharides Amino acids Acetyl-CoA NAD+ ADP Citric acid Electron Oxidative cycle transport phosphorylation NADH ATP Electron Transport Chain (ETC) and Chemiosmosis (1:57) https://nova.lecturio.com/#/lecture/c/8242/35814 5 Anaplerotic/Cataplerotic Reactions of the Citric Acid Cycle Incoming Outgoing Leu Lys Phe Trp Tyr Acetoacetate Arg Gln Glu His Pro Ala Cys Gly Leu Leu Lys -Ketoglutarate Arg Gln Glu Pro Lys Ser Val Ala Cys Gly Citrate Phe Trp Tyr Ile Trp Leu Thr Trp Ser Acetyl Phosphoenol- Pyruvate Acetyl -CoA Succinyl- Ile Met Val pyruvate -CoA CoA Oxalo- acetate Asn Asp Ile Fumarate Phe Tyr Lys Met Thr Pyruvate pathways Pyruvate Mitochondrial enzyme complex linking dehydrogenase glycolysis and TCA cycle. Differentially complex regulated in fed/fasting states (active in fed state). Reaction: pyruvate + NAD+ + CoA > acetyl CoA + CO2 + NADH The complex contains 3 enzymes (Pyruvate dehydrogenase or Pyruvate decarboxylase,. dihydrolipoyl transacetylase & dihydrolipoyl dehydrogenase) that require 5 cofactors: 1. Pyrophosphate (B1, thiamine; TPP) 2. FAD (B2, riboflavin) 3. NAD (B3, niacin) 4. CoA (B5, pantothcnic acid) 5. Lipoic acid Activated by exercise, which: increase >NAD+/NADH ratio increase ADP increase Ca2+ Metabolic Fates of Pyruvate Acetyl-CoA Oxygen present Lactate Pyruvate Alcohol dehydrogenase decarboxylase dehydrogenase NAD+ NADH H+ CO2 NADH NAD+ O + H+ O H H + H+ H H H OH OH H C C H C C O OH O H O H H Lactate Pyruvate Acetaldehyde Ethanol Oxygen absent Oxygen absent Animals Bacteria/yeast Glycolysis Pyruvate Dehydrogenase Ethanol NAD+ No O2 CO2 NADH Mitochondrial enzyme Pyruvate Acetaldehyde Very large multimeric complex NAD+ O2 Three subunits E1, E2, E3 NAD+ NADH NADH + CO2 Acetyl-CoA Steps in Pyruvate Oxidation Bacteria/yeast Acetaldehyde NAD+ (Mostly in low O2) NADH FAD FADH2 Electron transfer CO2 to lipoamide-S-S Pyruvate TPP- Acetyl- Acetyl-CoA Acetaldehyde Lipoamide E1 E2 E3 Pyruvate dehydrogenase  Pyruvate Dehydrogenase Complex (PDH) Three enzymes and five cofactors make-up the PDH complex. Four cofactors are derived from vitamins. The oxidative decarboxylation mechanism is irreversible. Regulation of PDH represents precise regulated flux through a metabolic step. 12  Making acetyl-CoA Pyruvate may diffuse through large openings in the outer membrane and then get into the matrix via an H+-coupled pyruvate-specific symporter in the inner membrane, the mitochondrial pyruvate carrier (MPC). After reaching the matrix, pyruvate can be oxidized to CO2 by the TCA cycle. Mitochondrial pyruvate is also the major substrate supporting the anabolic processes of gluconeogenesis and lipogenesis. https://taylor.lab.uiowa.edu/ 13 Pyruvate Dehydrogenase Mechanism TPP-Acetaldehyde Acetyl-Lipoamide E1 Pyruvate Electron transfer to lipoamide-S-S E3 Acetyl-CoA  Making acetyl-CoA (cont.) In the mitochondria, an enzyme called pyruvate dehydrogenase snatches CO2 from pyruvate and adds coenzyme A, making acetyl-CoA (activated acetate). In the process, two electrons are also transferred to a nearby NAD+, making NADH. This step links glycolysis to the citric acid cycle, but it’s not considered part of either process. Logical point for regulation that determines the rate of catabolic activity. Coenzyme A has a reactive thiol group (-SH) that is critical to its role as an acyl carrier in many metabolic processes. Acyl groups are covalently linked to the thiol group, forming thioesters. 15 Lipoamide Oxidized/Reduced O HN.. S S O HN... Lipoic acid component oxidized Lysine side chain O HN.. SH SH O HN... Lipoic acid component reduced Lysine side chain Acetyl-CoA Acetyl-CoA NH2 HO N O O N P S O O O N N P CH2 N N H H O OH O OH O HO O OH P HO O Citric Acid Cycle NAD+ NADH + H+ Isocitrate Pyruvate Isocitrate -Ketoglutarate dehydrogenase dehydrogenase NAD+ CoA-SH CO2 + Aconitase -Ketoglutarate NADH + NAD+ NADH + H+ dehydrogenase + H+ + CO2 Citrate CoA-SH Succinyl-CoA Citrate synthase GDP + Pi Acetyl-CoA Succinyl-CoA Pyruvate synthease CoA-SH Oxaloacetate + GTP Malate Succinate NADH + H+ dehydrogenase Succinate dehydrogenase NAD+ FAD HCO3 ADP Malate Fumarase + ATP + Pi Fumarate FADH2 Pyruvate carboxylase H2O  Krebs Cycle (7:08) https://nova.lecturio.com/#/lecture/c/8242/35816 The citric acid cycle is a set of 8 enzymatic reactions that start with acetyl-CoA, and four of the enzymes (half of the total) are dehydrogenases. And in this process, Acetyl-CoA gets converted into carbon dioxide. 19 20 Nobel prize to Hans Adolf Krebs for discovery of the citric acid cycle and to Fritz Albert Lipmann in 1953 for discovery of coenzyme A and its importance in intermediary metabolism. Carl and Gerty Cori studied how the body metabolizes glucose and advanced the understanding of how the body produces and stores energy. Their findings were particularly useful in the development of treatments for diabetes. In 1947 the Coris shared a Nobel Prize for their discoveries. 21 The Overall Picture of the TCA 22 The Overall Picture of the TCA Cycle 23 Citrate Synthase Citrate Oxaloacetate Acetyl-CoA + Oxaloacetate Very Citrate negative synthase + ° + Citrate + CoA-SH H Acetyl-CoA CoA-SH Aconitase H Citrate 3 Aconitase HO Isocitrate H Isocitrate Dehydrogenase Isocitrate + NAD+ Isocitrate dehydro- genase -ketoglutarate + NADH + CO2 + NAD+ + NADH + CO2 First oxidative decarboxylation α-keto-glutarate Dehydrogenase -ketoglutarate + NAD+ + CoA-SH α-keto- glutarate dehydro- genase Succinyl-CoA + NADH + CO2 + NAD+ + CoA-SH + NADH + CO2 Second oxidative decarboxylation Succinyl-CoA Synthetase Symmetrical product Succinyl-CoA + GDP + Pi Succinyl- CoA synthetase Succinate+ GTP + CoA-SH + GDP + Pi + GTP + CoA-SH Only substrate level phosphorylation in cycle Succinate Dehydrogenase Succinate + FAD Succinate Dehydro- genase Fumarate + FADH2 + FAD + FADH2 Third oxidation of cycle Succinate Dehydrogenase Enzyme is embedded in inner mitochondrial membrane Only enzyme of citric acid cycle not found in matrix Succinate Dehydrogenase Enzyme is embedded in inner mitochondrial membrane Only enzyme of citric acid cycle not found in matrix Electron Movement through the Succinate Dehydrogenase Complex Electron movement Intermembrane space SDHC SDHD Q QH2 Q Heme QH2 SDHB Matrix SDHA FAD FADH2 Reaction: Succinate Fumarate Fumarase Fumarate + H2O Fumarase L-Malate + H2O Malate Dehydrogenase ° Reaction pulled by citrate synthase reaction L-Malate + NAD+ Malate dehydro- genase Oxaloacetate + NADH + NAD + + NADH Fourth and final oxidation of cycle Citric Acid Cycle NAD+ NADH + H+ Isocitrate Pyruvate Isocitrate -Ketoglutarate dehydrogenase dehydrogenase NAD+ CoA-SH CO2 + Aconitase -Ketoglutarate NADH + NAD+ NADH + H+ dehydrogenase + H+ + CO2 Citrate CoA-SH Succinyl-CoA Citrate synthase GDP + Pi Acetyl-CoA Succinyl-CoA Pyruvate synthease CoA-SH Oxaloacetate + GTP Malate Succinate NADH + H+ dehydrogenase Succinate dehydrogenase NAD+ FAD HCO3 ADP Malate Fumarase + ATP + Pi Fumarate FADH2 Pyruvate carboxylase H2O Citric Acid Cycle Summary Input 2 carbons (1 Acetyl-CoA) Release 2 CO2 molecules Four oxidations 3 NADH, 1 FADH2, 1 GTP per turn of cycle Each Citric Acid Cycle Intermediate Functions in Other Pathways Citrate Isocitrate α-ketoglutarate Succinyl-CoA glyoxylate cycle, glyoxylate cycle amino acid/nitrogen heme synthesis, allosteric effector, metabolism amino acid shuttle system metabolism Succinate Fumarate Malate Oxaloacetate glyoxylate glyoxylate glyoxylate glyoxylate metabolism, odd metabolism, metabolism, shuttle metabolism, chain fatty acid nucleotide system gluconeogenesis, metabolism metabolism amino acid metabolism Clinical correlation! Adequate intake of these vitamins is essential, because deficiencies can disrupt the TCA and impact overall health as consequence. Thiamine deficiency can lead to a disease called beriberi, in which the central nervous system and then the heart can’t work properly. Likewise, niacin deficiency can cause a disease called pellagra, characterized by 4 “D”s: diarrhea, dermatitis, dementia and, if the deficiency isn’t corrected, it can cause death. 38 Glyoxylate Cycle NAD+ NADH + CO2 H2O Isocitrate Aconitase Isocitrate dehydro- -Ketoglutarate Cis-aconitate Isocitrate NAD + + CoA genase H2O lyase -Ketoglutarate NADH + CO 2 Aconitase dehydrogenase Glyoxylate CoA Citrate Succinyl-CoA Acetyl-CoA Citrate Succinyl-CoA ADP + Pi + H2O synthase Malate synthease Acetyl-CoA synthetase + H2O ATP + CoA Two per turn Oxaloacetate Malate of cycle dehydro- CoA Succinate genase Succinate NADH + H+ dehydro- genase Fumarase FAD NAD+ Malate Fumarate FADH2 H2O Overview of the GlyoxylateCycle Aconitase 1Isocitrate 1 Citrate Isocitrate Excessoxaloacetate is lyase used for gluconeogenesis 1 1 2 Acetyl-CoA 1 1 1Glyoxalate Glucose 2 Oxaloacetate + 1succinate Malate Succinate synthase dehydrogenase 1Fumarate 2 Malate Fumarase Why is glyoxylate cycle? The primary function of the glyoxylate cycle is to allow growth when glucose is unavailable and two-carbon compounds, such as ethanol and acetate, are the only carbon sources. Isocitrate Lyase Isocitrate Succinate + Glyoxylate + Malate Synthase Acetyl-CoA + Glyoxylate L-Malate + GlyoxylateCycle Summary Input 4 carbons (2-Acetyl-CoA) Releases 0 CO2 molecules Produces one extra oxaloacetate Two oxidations 1 NADH, 1 FADH2 1 (extra ) oxaloacetate per turn of cycle Net synthesis of glucose from Acetyl-CoA  Succinate Thiokinase → Kinase adds a –P somewhere… The next step is: succinate thiokinase removes the CoA from succinyl CoA, turning it into a 4-carbon succinate molecule. It also couples a phosphate and GDP molecule to the reaction, making GTP. 45 Citric Acid Cycle Summary Input 2 carbons (1 Acetyl-CoA) Releases 2 CO2 molecules Four oxidations 3 NADH, 1 FADH2, 1 GTP per turn of cycle No net synthesis of glucose from Acetyl-CoA  Control of TCA The control of the citric acid cycle is based on energy level of the cell. Hormones don’t play a role in its regulation. 47 Summary of TCA One acetyl-CoA molecule made 3X NADH, 1X FADH2, 1X GTP and 2X CO2. The CO2s leave the cell and are transported in the blood as bicarbonate thanks to enzymes called carbonic anhydrases. In the ETC, each NADH makes 3 ATPs, and each FADH2 makes 2 ATPs. Our 1 GTP yields the energy equivalent of 1 ATP. And so, we make a total of 12 ATP molecules per acetyl-CoA. And since one glucose molecule splits into 2 pyruvates, each glucose molecule yields 24 ATP in the citric acid cycle. 48 49 Ketone Body Metabolism 2 Acetyl-CoA Thiolase Acetoacetyl-CoA HMG-CoA synthase -hydroxy--methylglutaryl CoA (HMG-CoA) O CoA-SH Acetyl-CoA CoA-SH OH CH3 O O O O H3C S CoA S CoA + HC 3 S CoA H3C S CoA O OH HMG-CoA lyase -hydroxybutarate Non-enzymatic dehydrogenase Acetyl-CoA decarboxylation OH O NADH + H+ NAD+ O O CO2 O O H3C O H3C CH3 D--hydroxybutyrate Acetoacetate Acetone Formation of HMG-CoA H3C OH O O O O + H2O CoA -OOC CoA H3C S CoA H3C S CoA S Acetoacetyl-CoA Acetyl-CoA 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) 2 H+ + 2 NADPH CoA + 2 NADP+ Mitochondria Cytoplasm H3C OH OH CH3 O -OOC + -OOC O H3C S CoA Cholesterol Acetoacetate Acetyl-CoA Mevalonate Cellular Respiration Occurs in 4 Phases The Power Plant of The Cell Mitochondria are considered the power plant of the cell.  Electron Transport Chain (ETC) (1:58) https://nova.lecturio.com/#/lecture/c/8242/35818 53 Learning Objectives After this Lecture You Will Be Able to: Diagram where electrons and carbon go. Describe how a gradient is produced in ETC. Explain how ATP is produced in chemiosmosis.  Electrochemical Gradient (4:05) https://nova.lecturio.com/#/lecture/c/8060/8024/41246 55 Glycolysis Literally Means Splitting Sugar Glycolysis Pyruvate oxidation FADH2 Krebs cycle NADH Electron transport chain & chemiosmosis ATP John E. Hall, Guyton and Hall Textbook of Medical Physiology, 13th Edition, 2016,p. 14, Fig. 2-6, Saunders (Elsevier) Krebs Cycle Consumes the Remaining Carbons of Glucose The Potential Energy of Glucose Is Captured by Electron Carriers CoA Acetyl-CoA Citrate NAD+ NADH CO2 NADH Oxaloacetate 2x For each glucose NAD+ α-ketoglutarate FADH2 NAD+ FAD NADH ATP CO2 ADP + P Succinyl-CoA Krebs Cycle Consumes the Remaining Carbons of Glucose The potential energy of glucose is captured by electron carriers. Krebs Cycle Consumes the Remaining Carbons of Glucose The Potential Energy of2 Glucose Acetyl CoA from each glucose 2(2C) = 4C CoA Acetyl-CoA Citrate NAD+ 4 carbon NADH work bench CO2 Oxaloacetate NADH NAD+ α-ketoglutarate FADH2 NAD+ FAD NADH ATP CO2 The potential energy of glucose is captured by electron carriers. ADP + P Succinyl-CoA 60 Krebs Cycle Consumes the Remaining Carbons of Products of Krebs Glucose The Potential Energy of Glucose CoA 2 CO2 evolve per cycle 2(2C) = 4CO2 per glucose Acetyl-CoA Citrate NAD+ 3 NADH per cycle 2(3 NADH) = 6 NADH CO2 NADH per glucose Oxaloacetate 1FADH2 per cycle = NADH 2 FADH2 per glucose NAD+ α-ketoglutarate FADH2 NAD+ 1ATP per cycle = FAD NADH 2 ATP per glucose ATP CO2 The potential energy of glucose is captured by electron carriers. ADP + P Succinyl-CoA This is Where All the ATP is Made The Electron Transport Chain & Chemiosmosis Glycolysis Formation of an electrochemical gradient Pyruvate oxidation ATP synthase ATP Krebs Lots of ATP cycle Electron transport chain chemiosmosis The ETC & Chemiosmosis Krebs Cycle NADH & FADH2 arrive at the ETC from: Glycolysis Pyruvate oxidation Krebs cycle The ETC & chemiosmosis are paired to form ATP. They occur across the inner mitochondrial membrane. The ETC & Chemiosmosis Krebs Cycle Electron transport chain Proton pumps Intermembrane space Hydrogen gradient ATP synthase The ETC & chemiosmosis are paired to form ATP. They occur across the inner mitochondrial membrane. A Closer Look at the Inner Mitochondrial Membrane The ETCcreates an electrochemical gradient.  NADH electrons from glycolysis enter mitochondria via the malate-aspartate or glycerol-3- Electron transport phosphate shuttle. FADH 2 electrons are transferred to complex II (at a lower energy level than chain and oxidative NADH). The passage of electrons results in the formation of a proton gradient that, coupled to phosphorylation oxidative phosphorylation, drives the production of ATP. ATP Mitochondrial matrix Inner mitochondrial membrane lntermembrane space 2,4-Dinitrophenol A Closer Look at the Inner Mitochondrial Membrane Electrochemical Gradient Intermembrane space Oxygen (½ of O2) The ETCcreates an Mitochondrial matrix electrochemical gradient. Putting it All Together All the pieces of cell respiration come together to form lots of ATP in the final stage. A Closer Look at the Inner Mitochondrial Membrane Oxygen in the Final Electron Acceptor Inner mitochondrial membrane Mitochondrial matrix ATP 2 free 2 electrons ½ of an O2 hydrogen ions exiting ETC molecule A Closer Look at the Inner Mitochondrial Membrane ATP Synthase ATP synthase uses the energy from the gradient to fuel ATP synthesis. ATP A Closer Look at the Inner Mitochondrial Membrane Oxygen in the Final Electron Acceptor Inner mitochondrial membrane ½ O2 Mitochondrial matrix ATP 2 free 2 electrons ½ of an O2 hydrogen ions exiting ETC molecule Putting it All Together Forming Lots of ATP in the Final Stage C6H12O6 + 6 O2 6 CO2 + 6 H2O + energy (heat and ATP) All the pieces of cell respiration come together to form lots of ATP in the final stage. Theoretical Energy Yields for Cell Respiration Most ATP is made during chemiosmosis. Actual yields vary. Theoretical Energy Yields for Cell Respiration Yield Variation Glucose 2 ATP ATP Glycolysis Pyruvate 2 NADH 5 ATP Chemiosmosis Pyruvate oxidation 2 NADH 5 ATP 2 ATP Krebs 6 NADH 1 ATP cycle 5 Chemiosmosis 2 FADH2 3 ATP  ATP Synthase (2:56) https://nova.lecturio.com/#/lecture/c/8242/35822 75 76  Complex V: ATP Synthase matrix Intermembrane space https://www.slideshare.net/DipeshTamrakar2/oxidative-phosphorylation-and-electron-transport-chain?qid=918d3905-b68e-4dcb-9523-1629d6acfbb7&v=&b=&from_search=8 77  Cell respiration (6:00) https://nova.lecturio.com/#/lecture/c/8242/35824 78 Theoretical Energy Yields for Cell Respiration ATP yields / glucose is contentious Most ATP Is Made During Chemiosmosis. Actual Yields Vary Glucose 2 ATP Glycolysis 2.5 ATP per NADH = 28Pyruvate ATP = 30 2 ATP NADH 5 ATP 1.5 ATP per FADH2 2 ATP per glycolysis Pyruvate oxidation 2 NADH ATP Efficency ATP Glucose = 686 kcal/mol Krebs 6 NADH ATP ATP = 7.3kcal/mol cycle 𝑘𝑐𝑎 𝑙 7. 3 (30𝐴𝑇𝑃) 𝑚𝑜𝑙 = 32% 686 𝑘𝑐𝑎𝑙/𝑚𝑜𝑙 2 FADH2 ATP ATPs in Aerobic Respiration  Regulation of Oxidative Phosphorylation https://www.slideshare.net/DipeshTamrakar2/oxidative-phosphorylation-and-electron-transport-chain?qid=918d3905-b68e-4dcb-9523-1629d6acfbb7&v=&b=&from_search=8 81  Many Poisons Inhibit the Respiratory Chain https://www.slideshare.net/DipeshTamrakar2/oxidative-phosphorylation-and-electron-transport-chain?qid=918d3905-b68e-4dcb-9523-1629d6acfbb7&v=&b=&from_search=8 82  Clinical Correlation: Inherited Defects in OxPhos https://www.slideshare.net/DipeshTamrakar2/oxidative-phosphorylation-and-electron-transport-chain?qid=918d3905-b68e-4dcb-9523-1629d6acfbb7&v=&b=&from_search=8 83 Additional ETC References: 1.Ahmad, M, Woiberg, A, Kahwaji, CI (2020). Biochemistry, electron transport chain. StatPearls. Retrieved May 26, 2021 from https://www.ncbi.nlm.nih.gov/books/NBK526105/ 2.Cooper, GM. (2000). The mechanism of oxidative phosphorylation The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates. https://www.ncbi.nlm.nih.gov/books/NBK9885/ 3.Alberts, B, Johnson, A, Lewis, J, et al (2002). Electron-transport chains and their proton pumps. Molecular Biology of the Cell. 4th edition. New York: Garland 4. Science. https://www.ncbi.nlm.nih.gov/books/NBK26904/ 84