Ch 07 - TCA Cycle & ETC PDF

Chapter 7 The Common Catabolic Pathway: Citric Acid Cycle, the Electron Transport Chain, and ATP Biosynthesis Biochemistry First Edition John Tansey Lecture PowerPoints Tanea Reed Chapter 7 Outline 7.1 The citric acid cycle 7.2 The electron transport chain 7.3 ATP biosynthesis Copyright © 2019 John...

Chapter 7 The Common Catabolic Pathway: Citric Acid Cycle, the Electron Transport Chain, and ATP Biosynthesis Biochemistry First Edition John Tansey Lecture PowerPoints Tanea Reed Chapter 7 Outline 7.1 The citric acid cycle 7.2 The electron transport chain 7.3 ATP biosynthesis Copyright © 2019 John Wiley & Sons, Inc. Where have all the carbons (C) gone? Where in a cell do these processes occur? What about the electrons (e-)? …and can we follow the energy (DG)? Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved. Learning Objectives § Describe how acetyl-CoA is oxidized and other molecules are transformed in the citric acid cycle. § Illustrate how electron transport results in protons (H+) getting pumped out of the mitochondrial matrix, and the formation of water. § Describe how the cell uses the electrochemical gradient produced in electron transport to synthesize ATP. Copyright © 2019 John Wiley & Sons, Inc. Section 7.1 Learning Objective § Describe how acetyl-CoA is oxidized and other molecules are transformed in the citric acid cycle. Copyright © 2019 John Wiley & Sons, Inc. The Citric Acid Cycle § Also known as the Krebs cycle and tricarboxylic acid (TCA) cycle § Eight reactions that serve as a central metabolic hub § Generates the following from each acetyl-CoA equivalent: 2 CO2 [carbons] 1 GTP or ATP [energy] 3 NADH/H+ (~2.5 ATP per NADH) [electrons] 1 FADH2 (~1.5 ATP per FADH2) [electrons] Copyright © 2019 John Wiley & Sons, Inc. The Citric Acid Cycle Figure 7.2 Citric acid cycle. Copyright © 2019 John Wiley & Sons, Inc. Metabolon Defined § A metabolon is a group of enzymes performing reactions with a common function § Enzymes can be localized in an organelle or part of an organelle § Associated with glycolysis, glycogenolysis, fatty acid biosynthesis, and the electron transport chain § Substrate chanelling may occur Copyright © 2019 John Wiley & Sons, Inc. Substrate Channeling Defined § Substrate channeling is the diversion of the product of one enzymatic reaction directly into a subsequent reaction, to increase reaction rate and efficiency. Helps keep “small” substrates on the right track! Common for one- or two-carbon compounds Often requires biological tethers Co-enzyme A is an example (e.g. in acetyl-CoA) Copyright © 2019 John Wiley & Sons, Inc. Biological tethers allow flexibility: useful for substrate channeling in multi-enzyme complexes Acetyl-CoA § Important intermediate in several pathways § Product of pyruvate oxidation (PDH complex) § Product of fatty acid catabolism § Chemical bonds are energetically rich. § Carries 2C acetyl group Copyright © 2019 John Wiley & Sons, Inc. TCA Cycle § What to know for each step? § Enzyme name(!) and whether it catalyzes a reversible reaction § Substrate (usually the product of the previous rxn) Number of carbons! (exact structure will help, but is optional) § Product § Overall mechanism (is oxidation/reduction occurring?) § Energetics – is it considered favourable? § Extras – cofactors, coenzymes, etc. Copyright © 2019 John Wiley & Sons, Inc. 12 Citrate Synthase § Condensation of acetyl CoA with oxaloacetate § Produces citrate § Irreversible Copyright © 2019 John Wiley & Sons, Inc. Aconitase § Isomerization of citrate to isocitrate § Reversible reaction § Produces isocitrate Copyright © 2019 John Wiley & Sons, Inc. Two-step Reaction: Aconitase Isomerization by dehydration/rehydration Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved. Isocitrate Dehydrogenase § Oxidation of isocitrate to α-ketoglutarate § NAD+ is reduced to NADH/H+.. § Produces a-ketoglutarate (5C) + CO2 § Irreversible Copyright © 2019 John Wiley & Sons, Inc. α-Ketoglutarate Dehydrogenase § α-ketoglutarate is oxidized to succinyl-CoA (4C). § Rate-determining step – irreversible § NAD+ is reduced to NADH/H+. Copyright © 2019 John Wiley & Sons, Inc. α-Ketoglutarate Dehydrogenase Complex § Multienzyme complex § Three different subunits (analogous to PDH complex): E1—TPP decarboxylase E2—dihydrolipoyl transferase E3—dihidyrolipoyl dehydrogenase Copyright © 2019 John Wiley & Sons, Inc. This map is important! Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved. TCA so far: one synthesis one rearrangement two reductive oxidations (dehydrogenases) loss of two carbons generation of two NADH molecules …and finally: We made it to HERE Copyright © 2019 John Wiley & Sons, Inc. Origin of C-atoms in CO2 COOH H2C COOH C COOH HC COOH H2C COOH C H COOH H2C HO Citrate HO Isocitrate H2C COOH H2C CH2 O C COOH a-ketoglutarate COOH CH2 O C SCoA Succinyl-CoA Carbons shown in RED are incoming C from Acetyl-CoA Both carbon atoms lost as CO2 derived from oxaloacetate Succinyl CoA Synthetase § Substrate-level phosphorylation Nucleotide is generated directly by phosphorylating a nucleoside § Reversible reaction § Produces succinate and ATP or GTP Copyright © 2019 John Wiley & Sons, Inc. Succinate Dehydrogenase § Oxidation of succinate – reversible reaction § FAD is reduced to FADH2. § Produces fumarate, a trans-dioic acid Copyright © 2019 John Wiley & Sons, Inc. Succinate Dehydrogenase § Bound to mitochondrial inner membrane Part of Complex II in the electron-transport chain (ETC) § Reduction of the alkane to alkene requires FADH2 Reduction potential of NAD+ is too low § FAD is covalently bound (this is unusual) § Near equilibrium/reversible Product concentration kept low to pull forward Fumarase § Hydration reaction intraconverts fumarate and malate. § Reversible reaction § Energy is neither consumed nor produced. Copyright © 2019 John Wiley & Sons, Inc. Malate Dehydrogenase § Oxidation reaction § Reversible reaction § NAD+ is reduced to NADH/H+. Copyright © 2019 John Wiley & Sons, Inc. A Schematic View of the Citric Acid Cycle Figure 7.3 Schematic view of the citric acid cycle. Copyright © 2019 John Wiley & Sons, Inc. Electrons are Funneled into ATP Synthesis Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved. Energetics of the Citric Acid Cycle Copyright © 2019 John Wiley & Sons, Inc. Regulation of the Citric Acid Cycle § Based on substrate availability Increased levels of substrates generally increase flux through the pathway § NADH inhibits all three dehydrogenases. § Allosteric regulators: ATP Ca2+ Succinyl CoA Copyright © 2019 John Wiley & Sons, Inc. Regulation of the Citric Acid Cycle Figure 7.4 Regulation of the citric acid cycle. Copyright © 2019 John Wiley & Sons, Inc. Regulation of Citric Acid Cycle Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved. Anaplerotic Reactions § Anaplerotic reactions replenish levels of citric acid cycle intermediates through a number of mechanisms. Figure 7.5 Central role of the citric acid cycle in metabolism. Copyright © 2019 John Wiley & Sons, Inc. Break Time! § Review the overall pathways of carbohydrate catabolism from glucose to carbon dioxide § What are the fates of the carbon atoms? § What are the fates of the electrons? Next time we’ll introduce my favourite topic: Electron Transport & Oxidative Phosphorylation! J Copyright © 2019 John Wiley & Sons, Inc. Section 7.2 Learning Objective § Illustrate how electron transport results in protons (H+) getting pumped out of the mitochondrial matrix, and the formation of water. Copyright © 2019 John Wiley & Sons, Inc. Reducing Equivalents Why “reducing equivalents”? Think “redox”: what happens to glucose? What does reduction mean in central metabolism? “Adding” electrons to ETC, but not to build molecules NADH is key e- carrier Figure 7.11 Reducing equivalents come from multiple metabolic pathways. Copyright © 2019 John Wiley & Sons, Inc. Electron Transport Chain § Occurs in the matrix and inner mitochondrial membrane Figure 7.12 Overview of the electron transport chain. Copyright © 2019 John Wiley & Sons, Inc. Electron Carriers in the Electron Transport Chain NAD & FAD: organic carriers Quinones: lipid carriers Hemes & iron-sulfur clusters: biometallic carriers Figure 7.13 Electron carriers in the electron transport chain. Copyright © 2019 John Wiley & Sons, Inc. Mitochondrial Shuttles § Some compounds cannot cross the mitochondrial inner membrane (but glycolysis happens in cytosol…) § Two important shuttles that transport metabolites from the mitochondrial matrix to the cytosol Glycerophosphate shuttle Aspartate-malate shuttle § Move NADH equivalents across inner mitochondrial membrane (that’s a way to get electrons into ETC) Copyright © 2019 John Wiley & Sons, Inc. Mitochondrial Shuttles: dehydrogenases Figure 7.14 Mitochondrial shuttles. Copyright © 2019 John Wiley & Sons, Inc. Mitochondrial Shuttles: malate/aspartate Copyright © 2019 John Wiley & Sons, Inc. Mitochondrial Electron Transport Chain § What to know for each step? § Name of complex (!) and whether it translocates protons § Electron donor (often from previous reactions/complex) Compound/structure Location § Electron acceptor Compound/structure Location § Overall mechanism—location & pathway of electrons § Extras – cofactors, coenzymes, etc. Copyright © 2019 John Wiley & Sons, Inc. 42 Complex I § NADH dehydrogenase § Site of NADH oxidation § Pumps 4H+ out of the mitochondrial matrix. § Electrons are transferred to ubiquinone (Q). § QàQH2 Copyright © 2019 John Wiley & Sons, Inc. Complex I § NADH dehydrogenase § Site of NADH oxidation § Pumps 4H+ out of the mitochondrial matrix. § Electrons are transferred to ubiquinone (Q). § QàQH2 Copyright © 2019 John Wiley & Sons, Inc. Electron Transport in Complex I Figure 7.16 Redox states of ubiquinone and FAD. Copyright © 2019 John Wiley & Sons, Inc. Complex II § Succinate dehydrogenase (yes, that one!) § Generates FADH2 and reduced ubiquinone (QH2)! § No protons are pumped out of the matrix. Figure 7.17A Complex II. Copyright © 2019 John Wiley & Sons, Inc. Complex III § Ubiquinone/cytochrome c reductase § Electrons transferred to cytochrome c (one at a time!) § Pumps 4H+ out of the mitochondrial matrix (per QH2) Figure 7.19A Complex III. Copyright © 2019 John Wiley & Sons, Inc. Q Pool Defined § Combination of oxidized and reduced forms of ubiquinone found in the mitochondrial membrane Copyright © 2019 John Wiley & Sons, Inc. Cytochrome c § A soluble electron carrier § Receives electrons from Complex III § Heme protein that carries one electron in the heme group (Tip: cytochromes are heme-containing iron proteins) Figure 7.21 Cytochrome c. Copyright © 2019 John Wiley & Sons, Inc. Q Cycle and Electron Transport § Big Problem: ETC are leaky! QH2 is a twoelectron carrier Cyt c is a singleelectron carrier § How to transfer e- without generating radicals?! Figure 7.20 Q cycle and electron transport. Copyright © 2019 John Wiley & Sons, Inc. The Q Cycle Part 1 Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved. The Q Cycle Parts 1 & 2 Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved. Complex IV § Cytochrome c oxidase § Takes 4e− from cytochrome c § Pumps 2H+ out of the mitochondrial matrix § Reduces O2 to H2O § Uses four different electron carriers (Fe and Cu) Figure 7.22A Complex IV. Copyright © 2019 John Wiley & Sons, Inc. Complex IV § Cytochrome c oxidase § Takes 4e− from cytochrome c § Pumps 2H+ out of the mitochondrial matrix § Reduces O2 to H2O § Uses four different electron carriers (Fe and Cu) § O2 is the terminal electron acceptor Figure 7.22A Complex IV. Copyright © 2019 John Wiley & Sons, Inc. Respirasome Defined § Respirasome is an aggregated supercomplex containing Complexes I, III, and IV. § Assists with substrate channeling Figure 7.24 Respirasome. Copyright © 2019 John Wiley & Sons, Inc. Poisons That Inhibit the Electron Transport Chain Drug or poison Rotenone (insecticide) Complex Carrier bound I Fe-S II Ubiqinone binding site Antimycin A1 III Cyt bH in the Qn site Cyanide (CN–) IV Heme cyt a3 Amytal (amobarbital, barbituates) Demerol (meperidine) Carboxin (fungicide) 2-thionyltrifluoroacetone Azide (N3–) Carbon monoxide (CO) 2,4-dinitrophenol (DNP) ATP synthase Dicumarol FCCP Copyright © 2019 John Wiley & Sons, Inc. Uncoupling agent Structures of Poisons to the Electron Transport Chain Figure 7.25 Poisons of electron transport. Copyright © 2019 John Wiley & Sons, Inc. Uncouplers and the Electron Transport Chain § Uncouplers interfere with electron transport. § Heat is generated instead of ATP. § How does that work? Copyright © 2019 John Wiley & Sons, Inc. Section 7.3 Learning Objective § Describe how the cell uses the electrochemical gradient produced in electron transport to synthesize ATP. Copyright © 2019 John Wiley & Sons, Inc. ATP Synthase § Also known as F0/F1 ATPase § Contains multiple subunits in both complexes § Enzyme mainly responsible for ATP production § Multimeric enzyme that uses electrochemical energy from the proton gradient to produce ATP from ADP and Pi Copyright © 2019 John Wiley & Sons, Inc. Structure of ATP Synthase Figure 7.26A Reactions of ATP synthase. Copyright © 2019 John Wiley & Sons, Inc. Mechanism of ATP Synthase § Protons move between a and b subunits of F0 complex. § Ring of c subunit rotates. § γ and ε subunits to also move through the hexamer of α and β subunits. § Conformational change occurs. § ATP is formed by binding of ADP and Pi. Copyright © 2019 John Wiley & Sons, Inc. ATP Synthase Rotation Empty ES [TS] EP? Rotational catalysis! Figure 7.26C Reactions of ATP synthase. Copyright © 2019 John Wiley & Sons, Inc. Empty! ATP Synthase ADP + Pi in ATP out! Spinning shaft forces conformational changes in F1: open à loose à tight Repeat! Protons moving through F0 turn gamma subunit (shaft) Powered by PMF from ETC Evidence of ATP Synthase Rotation Figure 7.27 Rotation of the ATP synthase can be demonstrated in the laboratory. Copyright © 2019 John Wiley & Sons, Inc. Proton Motive Force Defined § Proton motive force is the electrochemical potential derived from the uneven distribution of electrons across the inner mitochondrial matrix. § Separated into a chemical and electric potential § ΔG = −2.303 RT ΔpH + nℱ ΔΨm § Bottom line: PMF is a double-whammy Copyright © 2019 John Wiley & Sons, Inc. Chemical Potential Defined § Chemical potential is the higher concentration of protons on the outside of the membrane than on the inside. § If gradient is related to [H+] it can be expressed as ΔpH Copyright © 2019 John Wiley & Sons, Inc. Electric Potential Defined § Electric potential is the added positive charge that accumulates on the outside of the membrane. § Measured in V or mV § Voltage drop across the membrane Copyright © 2019 John Wiley & Sons, Inc. Gibbs Free Energy Correlation to Membrane Potential § Membrane potential (ΔΨm ) = Ψin-Ψout § Ψin = voltage inside the membrane § Ψout = voltage outside the membrane § ΔG = nℱΔΨm § ℱ (Faraday’s constant) = 96.48 kJ/V * mol Copyright © 2019 John Wiley & Sons, Inc. ATP:ADP Translocase Defined § ATP:ADP translocase is an enzyme that transports ATP from the mitochondria in exchange for ADP Transmembrane domain is an integral membrane protein spanning six α helices Conformational change occurs Driven by [ATP]/[ADP] § Phosphate translocase Replaces PO4- “lost” as ATP Driven by PMF Copyright © 2019 John Wiley & Sons, Inc. ATPases § Use ATP hydrolysis to perform their proscribed functions in reverse § Five different types F-type A-type V-type P-type E-type Copyright © 2019 John Wiley & Sons, Inc. ATPase Categories ATPase Feature Inhibitor F-type (F0/F1 ATPase) Have isolated factor involved in ADP phosphorylation Oligomycin Dicyclohexylcarbodiimide (DCC) Aurovertin B A-type Found in Archea Similar structure to F0/F1 ATPase V-type Found in vacuoles Use energy of ATP hydrolysis to pump H+ or Na+ into vacuoles Involved in endocytosis, protein trafficking, active transport of metabolite and neurotransmitter release Bafilomycin Concanamycin Apicularen Lobatamide P-type Pumps that use ATP hydrolysis to move ions from one side of phospholipid bylayer to another Single transmembrane protein Omeprazole (Prilosec) Lansoprazole (Prevacid) E-type Extracellular ATPases Hydrolyze extracellular ATP and ADP or other nucleotide triphosphates Involved in platelet aggregation, transplant rejection, and parasite survival Copyright © 2019 John Wiley & Sons, Inc. Copyright Copyright © 2019 John Wiley & Sons, Inc. All rights reserved. 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