Cellular Respiration Lesson 20 Handout Fall 2024 PDF

Biosc 0155 Fall 2024 11/19/2024 Lesson 20 Handout Cellular Respiration Vocabulary you should know: glycolysis citric acid...

Biosc 0155 Fall 2024 11/19/2024 Lesson 20 Handout Cellular Respiration Vocabulary you should know: glycolysis citric acid cycle (CAC) cellular respiration guanosine triphosphate (GTP) homeostasis inner mitochondrial membrane NAD+/NADH mitochondrial matrix FAD/FADH2 intermembrane space (IMS) substrate-level phosphorylation electron transport chain (ETC) pyruvate ubiquinone (Q) cellular respiration cytochrome c (cytc) homeostasis proton-motive force mitochondrial matrix chemiosmosis coenzyme A (CoA) ATP synthase acetyl CoA oxidative phosphorylation pyruvate aerobic respiration pyruvate dehydrogenase anaerobic respiration Learning Objectives: Students should be able to: Describe the inputs and outputs of glycolysis. Explain the difference between the energy investment and energy payoff stages of glycolysis. Recognize substrate-level phosphorylation reactions. Know the three enzymes that catalyze irreversible steps in glycolysis. Explain how phosphofructokinase is regulated. Recognize the structures of glucose, glucose-6-phosphate, and pyruvate. Explain the four components of cellular respiration. Describe pyruvate oxidation and the citric acid cycle, including the inputs and outputs of each process. Analyze biological redox reactions to identify the oxidizing and reducing agents. Know the cellular location of glycolysis, and pyruvate processing, the citric acid cycle, and electron transport. Describe the regulation of pyruvate oxidation and CAC. Describe the components of the respiratory chain and how a proton gradient is established. Know the cellular location of electron transport. Explain how the action of ATP synthase is dependent on a proton motive force. © 2024 Laura S. Zapanta, Ph.D. 1 Biosc 0155 Fall 2024 11/19/2024 Describe two experiments that provided evidence for chemiosmosis. Know the cellular location of electron transport and oxidative phosphorylation. Compare and contrast aerobic respiration and anaerobic respiration. Recommended End of Chapter Questions: Chapter 9:1-16 © 2024 Laura S. Zapanta, Ph.D. 2  Biosc 0155 Fall 2024 11/19/2024 Cellular Respiration Honors Foundations of Biology 1 Lesson 20 – November 19, 2024 1 1 Glucose metabolism is central to all metabolism. All organisms use glucose to build complex carbohydrates, precursors to fats, and other compounds. Cells recover glucose by breaking down these molecules. Glucose is also the primary energy source of cells. 2 2 © 2024 Laura S. Zapanta, Ph.D. 3  Biosc 0155 Fall 2024 11/19/2024 Glucose is used to make ATP through cellular respiration or fermentation. 3 3 In the presence of oxygen, cellular respiration oxidizes glucose to make ATP. Carbon atoms of glucose are oxidized to form carbon dioxide. Oxygen atoms in oxygen are reduced to form water. The resulting change in free energy is used to synthesize ATP. C6H12O6 + 6 O2 ® 6 CO2 + 6 H2O + energy 4 4 © 2024 Laura S. Zapanta, Ph.D. 4  Biosc 0155 Fall 2024 11/19/2024 Cellular respiration has four steps. 5 5 Lesson 20.1 – Glycolysis 6 © 2024 Laura S. Zapanta, Ph.D. 5  Biosc 0155 Fall 2024 11/19/2024 In glycolysis, glucose is broken down into two molecules of pyruvate. The potential energy released is used to phosphorylate ADP to ATP. 7 7 Glycolysis is a metabolic pathway consisting of 10 steps. All glycolysis reactions occur in the cytosol of eukaryotic cells. 8 8 © 2024 Laura S. Zapanta, Ph.D. 6  Biosc 0155 Fall 2024 11/19/2024 Glycolysis has two phases: an energy investment phase and an energy payoff phase. 9 The first part of glycolysis is an energy investment phase. The cell uses (spends) ATP to make higher energy molecules. 10 10 © 2024 Laura S. Zapanta, Ph.D. 7  Biosc 0155 Fall 2024 11/19/2024 The second part of glycolysis is the energy payoff phase. “Investment” repaid with interest in the form of ATP and NADH 11 11 Products of Glycolysis From one molecule of glucose: 2 pyruvate 2 ATP 2 NADH 12 12 © 2024 Laura S. Zapanta, Ph.D. 8  Biosc 0155 Fall 2024 11/19/2024 Two types of reactions occur repeatedly in glycolysis. Redox Substrate-level Reactions Phosphorylations 13 13 REDOX REVIEW Which substrate is oxidized in the reaction catalyzed by glyceraldeyde 3-phosphate dehydrogenase? Reduced? 14 14 © 2024 Laura S. Zapanta, Ph.D. 9  Biosc 0155 Fall 2024 11/19/2024 In glycolysis, ATP is produced through substrate- level phosphorylation. This is the enzyme-catalyzed transfer of a phosphate group from a phosphorylated substrate to ADP to form ATP. 15 15 SUBSTRATE-LEVELPHOSPHORYLATION Which steps of glycolysis are substrate-level phosphorylations? Which molecules donate phosphate to ADP? 16 16 © 2024 Laura S. Zapanta, Ph.D. 10  Biosc 0155 Fall 2024 11/19/2024 Chemical logic of pathway (part 1) 17 Chemical logic of pathway (part 2) 18 © 2024 Laura S. Zapanta, Ph.D. 11  Biosc 0155 Fall 2024 11/19/2024 Three steps in glycolysis are irreversible. Step 1 – catalyzed by hexokinase Step 3 – catalyzed by phosphofructokinase Step 10 – catalyzed by pyruvate kinase DG = –18.8 kJ/mol DG = –26.8 kJ/mol DG = –33.9 kJ/mol 19 Hexokinase uses glucose and ATP to make glucose-6-phosphate. Because glucose-6-phosphate cannot exit the cell through glucose transporters, this “traps” glucose in the cell. 20 © 2024 Laura S. Zapanta, Ph.D. 12  Biosc 0155 Fall 2024 11/19/2024 Glucose-6-phosphate is an inhibitor of hexokinase. Product inhibition Binds to a regulatory site away from active site 21 An important site of regulation in glycolysis is the step catalyzed by the enzyme phosphofructokinase. High levels of ATP inhibit the enzyme through feedback inhibition. 22 22 © 2024 Laura S. Zapanta, Ph.D. 13  Biosc 0155 Fall 2024 11/19/2024 Phosphofructokinase has two binding sites for ATP: the active site and a regulatory site. 23 23 PFK activity decreases when ATP concentration increases. At low [ATP], the catalytic sites (active sites) are occupied by ATP and the regulatory sites are not. At high [ATP], regulatory sites become occupied too, lowering affinity for fructose-6-phosphate. 24 24 © 2024 Laura S. Zapanta, Ph.D. 14  Biosc 0155 Fall 2024 11/19/2024 PFK INHIBITION PFK catalyzes step 3 in glycolysis. If it is inhibited by high levels of ATP, which intermediate(s) will increase in concentration? Decrease in concentration? 25 25 Pyruvate kinase catalyzes the last step in glycolysis producing ATP and pyruvate. ATP is also an allosteric inhibitor of this enzyme. 26 © 2024 Laura S. Zapanta, Ph.D. 15  Biosc 0155 Fall 2024 11/19/2024 Lesson 20.2 – Pyruvate Processing 27 The pyruvate formed in glycolysis can be broken down in two different ways depending on oxygen availability. 28 © 2024 Laura S. Zapanta, Ph.D. 16  Biosc 0155 Fall 2024 11/19/2024 The remaining steps of cellular respiration occur in the mitochondria. 29 29 Pyruvate from glycolysis is transported from the cytosol into mitochondria. Bender and Martinou (2016) BBA Mol Cell Res 1863:2436-2442. 30 30 © 2024 Laura S. Zapanta, Ph.D. 17  Biosc 0155 Fall 2024 11/19/2024 Pyruvate oxidation is catalyzed by the pyruvate dehydrogenase enzyme complex in the mitochondrial matrix. 31 31 Products of Pyruvate Processing From one molecule of pyruvate: 1 acetyl CoA 1 NADH 1 CO2 32 32 © 2024 Laura S. Zapanta, Ph.D. 18  Biosc 0155 Fall 2024 11/19/2024 Pyruvate oxidation is under both positive and negative control. Pyruvate dehydrogenase is inhibited by High levels of ATP High levels of acetyl CoA and NADH Pyruvate dehydrogenase is stimulated by High levels of AMP High levels of coenzyme A (CoA) and NAD+ 33 33 Lesson 20.3 – The Citric Acid Cycle 34 © 2024 Laura S. Zapanta, Ph.D. 19  Biosc 0155 Fall 2024 11/19/2024 The acetyl CoA enters the citric acid cycle (CAC). Occurs in the mitochondrial matrix 35 This pathway is a cycle because the starting molecule (oxaloacetate) is regenerated in the last step. 36 36 © 2024 Laura S. Zapanta, Ph.D. 20  Biosc 0155 Fall 2024 11/19/2024 Acetyl CoA is oxidized to two molecules of CO2. The CAC completes glucose oxidation. 37 37 Products of Citric Acid Cycle From one molecule of acetyl CoA (1 turn of the cycle): 3 NADH 1 FADH2 1 GTP 2 CO2 38 38 © 2024 Laura S. Zapanta, Ph.D. 21  Biosc 0155 Fall 2024 11/19/2024 The citric acid cycle can be turned off at multiple points by feedback inhibition. The cycle slows down when ATP and NADH are plentiful. 39 39 Cellular Respiration: Glycolysis through CAC C6H12O6 + 10 NAD+ + 2 FAD + 4 ADP + 4 Pi + 6 H2O 6 CO2 + 10 NADH + 2 FADH2 + 4 ATP + 10 H+ 2 CO2 – pyruvate processing 4 CO2 – CAC 2 FADH2 – CAC 2 ATP – glycolysis 2 ATP – CAC 2 NADH – glycolysis 2 NADH – pyruvate processing 6 NADH – CAC 40 40 © 2024 Laura S. Zapanta, Ph.D. 22  Biosc 0155 Fall 2024 11/19/2024 Most of glucose’s original energy is contained in the electrons transferred to NADH and FADH2. 41 41 NADH and FADH2 carry two electrons each. http://what-when-how.com/wp-content/uploads/2011/05/tmp462_thumb_thumb2.jpg 42 42 © 2024 Laura S. Zapanta, Ph.D. 23  Biosc 0155 Fall 2024 11/19/2024 Lesson 20.4 – The Electron Transport Chain 43 During the fourth step of cellular respiration, the high potential energy of the electrons carried by NADH and FADH2 is gradually decreased as they move through a series of redox reactions. 44 44 © 2024 Laura S. Zapanta, Ph.D. 24  Biosc 0155 Fall 2024 11/19/2024 The proteins involved in these reactions make up a respiratory chain, also called the electron transport chain (ETC). The proteins are embedded within the inner mitochondria membranes. The respiratory proteins contain chemical groups that facilitate redox reactions. Figure 10.1 McKee & McKee Biochemistry: The Molecular Basis of Life, 4/e©2009 45 45 Mobile electron carriers shuttle electrons between the proteins in the ETC. Ubiquinone (Coenzyme Q) Cytochrome C Tymoczko Biochemistry A Short Course ©2010 Figure 19.9 http://upload.wikimedia.org/wikipedia/commons/thumb/0/07 /Cytochrome_c.png/600px-Cytochrome_c.png 46 46 © 2024 Laura S. Zapanta, Ph.D. 25  Biosc 0155 Fall 2024 11/19/2024 O2 is the final electron acceptor. The transfer of electrons along with protons to oxygen forms water. 47 47 The energy released as electrons move through the ETC is used to move protons across the mitochondrial inner membrane into the intermembrane space. 48 48 © 2024 Laura S. Zapanta, Ph.D. 26  Biosc 0155 Fall 2024 11/19/2024 Electrons from NADH enter the respiratory chain at Complex I. 2 electrons passed from NADH to Q Provides enough energy to pump 4 H+ from matrix to IMS 49 49 Electrons from FADH2 enter the respiratory chain at Complex II. 2 electrons passed from NADH to Q No protons moved with Complex II 50 50 © 2024 Laura S. Zapanta, Ph.D. 27  Biosc 0155 Fall 2024 11/19/2024 Q delivers electrons to Complex III Electrons are passed to cytochrome c (Cyt c) Provides enough energy to move 4 H+ into IMS 51 Cyt c delivers electrons to Complex IV. Electrons reduce O2 to H2O Provides enough energy to pump 2H+ into IMS 52 © 2024 Laura S. Zapanta, Ph.D. 28  Biosc 0155 Fall 2024 11/19/2024 Reactions of the Respiratory Chain Summary 53 53 All eukaryotes and many prokaryotes use oxygen as the final electron acceptor of electron transport chains in aerobic respiration. Oxygen is the most effective electron acceptor because it is highly electronegative. A large difference between the potential energy of NADH and O2 electrons. Allows the generation of a large proton-motive force for ATP production. 54 54 © 2024 Laura S. Zapanta, Ph.D. 29  Biosc 0155 Fall 2024 11/19/2024 Some prokaryotes use other electron acceptors in anaerobic respiration. 55 55 Energy released by electron transfer is used to move protons into the IMS, creating a proton gradient. This proton gradient will be used to make ATP. 56 56 © 2024 Laura S. Zapanta, Ph.D. 30  Biosc 0155 Fall 2024 11/19/2024 Lesson 20.5 – Oxidative Phosphorylation ATP Synthase 57 Oxidative Phosphorylation Process in which ATP is formed as a result of the transfer of electrons from NADH or FADH2 to O2 Occurs in mitochondria Major source of ATP (aerobic organisms) 58 58 © 2024 Laura S. Zapanta, Ph.D. 31  Biosc 0155 Fall 2024 11/19/2024 How is ATP made in the mitochondria? Throughout the 1950’s scientists studying respiratory chain components assumed one of the complexes must make ATP through substrate level phosphorylation. In 1960, Efraim Racker discovered ATP synthase in mitochondrial membranes. 59 ATP synthase is an enzyme complex in the inner mitochondrial membrane that is made of two components: A membrane-bound, proton- transporting base (F0 unit) An ATPase “knob” (F1 unit) 60 60 © 2024 Laura S. Zapanta, Ph.D. 32  Biosc 0155 Fall 2024 11/19/2024 Peter Mitchell’s chemiosmosis hypothesis proposed a link between the electron transport chain and ATP synthase. The real job of the ETC is to move protons across the inner membrane of mitochondria from the matrix to the intermembrane space. After a proton gradient is established an enzyme in the inner membrane, like ATP synthase, synthesizes ATP from ADP and Pi. 61 61 Experimental Evidence for the Chemiosmotic Mechanism 62 62 © 2024 Laura S. Zapanta, Ph.D. 33  Biosc 0155 Fall 2024 11/19/2024 TABLE OF THE DAY Textbook Fig. 9.14 Data in Depth Exercise: André Jagendorf and Ernest Uribe at Johns Hopkins University performed the experiment outlined in Figure 9.9 to establish that an H+ concentration gradient ultimately drives the synthesis of ATP in chloroplasts. Thylakoids were preincubated in a solution at pH 3.8 and then quickly transferred to an ATP synthase reaction mixture containing ADP and inorganic phosphate (Pi) at pH 8. ATP formation was measured in two ways. The first used the enzyme luciferase, which catalyzes the formation of a luminescent molecule if ATP is present. The second used molybdate to measure phosphorylation directly. The data are displayed in the table. 63 Additional Evidence for Chemiosmosis 64 © 2024 Laura S. Zapanta, Ph.D. 34  Biosc 0155 Fall 2024 11/19/2024 ATP synthase uses the proton gradient established by the respiratory chain to make ATP. ATP synthesis catalyzed by ATP synthase is called oxidative phosphorylation. Proton gradient provides the energy. 65 65 ATP synthase acts as a molecular motor. Protons flowing through the F0 unit spin the rotor. The rotor spins the F1 unit. As the F1 unit spins, its subunits change shape and catalyze the phosphorylation of ADP to ATP. 66 66 © 2024 Laura S. Zapanta, Ph.D. 35  Biosc 0155 Fall 2024 11/19/2024 67 Most of the ATP obtained from glucose oxidation occurs via oxidative phosphorylation. About 32 ATP produced from 1 molecule of glucose in cellular respiration: 2 ATP from glycolysis 2 ATP from citric acid cycle 28 from oxidative phosphorylation (ATP synthase) 68 68 © 2024 Laura S. Zapanta, Ph.D. 36  Biosc 0155 Fall 2024 11/19/2024 ATP PRODUCTION It has been determined that each molecule of NADH leads to the production of ~2.5 ATP, and each molecule of FADH2 produces ~1.5 ATP. Why is less ATP made with FADH2? HINT: How is ATP synthase “powered”? 69 69 The electron transport and oxidative phosphorylation are tightly coupled. Disruption of either process will affect ATP synthesis. 70 © 2024 Laura S. Zapanta, Ph.D. 37  Biosc 0155 Fall 2024 11/19/2024 Three classes of inhibitors decrease rates of mitochondrial ATP synthesis. Inhibitors of ETC Uncouplers (allow protons to diffuse through inner membrane) Inhibitors of ATP synthase or ATP/ADP translocase 71 71 GRAPH OF THE DAY Zhang, J et al. (2022) Molecular Medicine Reports, 25, 91. https://doi.org/10.3892/mmr.2022.12607 72 © 2024 Laura S. Zapanta, Ph.D. 38  Biosc 0155 Fall 2024 11/19/2024 Uncoupling via UCP1 facilitates thermogenesis. 73 Lesson 20.6 - Fermentation 74 © 2024 Laura S. Zapanta, Ph.D. 39  Biosc 0155 Fall 2024 11/19/2024 When O2 is present the NAD+ required for cellular respiration is regenerated by the ETC. 75 Glycolysis can produce ATP in the absence of oxygen. NAD+ is needed for glycolysis to occur. 76 76 © 2024 Laura S. Zapanta, Ph.D. 40  Biosc 0155 Fall 2024 11/19/2024 Fermentation is the metabolic pathway that regenerates NAD+ from NADH in the absence of oxygen. 77 77 In lactic acid fermentation, pyruvate accepts electrons from NADH, and lactate and NAD+ are produced. 78 78 © 2024 Laura S. Zapanta, Ph.D. 41  Biosc 0155 Fall 2024 11/19/2024 In alcohol fermentation, pyruvate is converted to acetaldehyde and CO2, and acetaldehyde accepts electrons from NADH to produce ethanol and NAD+. 79 79 Fermentation is extremely inefficient compared to cellular respiration. Organisms never use fermentation if an appropriate electron acceptor is available for cellular respiration. 80 80 © 2024 Laura S. Zapanta, Ph.D. 42