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Transcript
210 MCB 3020, Spring 2005 Chapter 5: Nutrition and Metabolism I 211 The Generation of Energy: I. Metabolism (metabolic reactions) II. Nutrients III. Energy IV. Review of free energy V. Enzymes VI. Energy generation: oxidation and reduction reactions I. Metabolism (metabolic reactions) 212 • all of the biochemical reactions in a cell • includes catabolic (degradative) and anabolic (biosynthetic) reactions 213 1. Catabolism • the breakdown of complex molecules into simpler compounds with the release of energy 2. Anabolism • the biosynthesis of complex molecules from simpler compounds with the input of energy B. Catabolic reactions generate ATP. ATP is 214 used for biosynthesis and cell maintenance. energy source Catabolism waste products ATP, reductant small molecules Anabolism macromolecules (polymers) 215 C. ATP is called the energy currency of the cell. • catabolic reactions release energy and store it as ATP. • anabolic (biosynthetic) reactions require energy in the form of ATP. II. Nutrition A. Nutrients chemicals taken up from environment and used for cellular reactions 1. macronutrients 2. micronutrients 3. growth factors 216 1. Macronutrients: chemicals taken up and required in relatively large amounts C + K H Mg2+ O + Na N 2+ Ca P Fe2+/Fe3+ S 217 218 Where do macronutrients occur in cells? C H O N P S Fe many organic molecules amino acids, nucleic acids, cell walls, etc. nucleic acids, phospholipids cysteine, methionine, vitamins like CoA Electron transport proteins 2. Micronutrients: inorganic required in small amounts 219 chemicals • also called trace elements • usually metals in metabolic enzymes • examples Co (the metal center of vitamin B12) Cu (found in electron transport proteins) Se (found in selenocysteine) Ni, Zn, Mn, V, W 220 3. Growth factors: organic chemicals required in small amounts by some (but not all) cells a. Examples: vitamins, like B1, B6, B12, biotin some amino acids purines, pyrimidines b. Many vitamins are precursors of coenzymes used in metabolism. Vitamin B2 (riboflavin) niacin (nicotinic acid) B12 folate 221 Coenzyme FAD, FMN NAD, NADP cobalamin tetrahydrofolate Coenzymes are molecules that work together with enzymes to catalyze chemical reactions. 222 B. Cells can be grown in laboratory cultures. Two classes of culture media 1. Chemically defined medium exact chemical composition is known; contains precise amounts of pure chemicals added to distilled water 2. Complex (undefined) medium exact chemical composition is not known; contains digests of milk proteins, yeast, soybeans, etc. that have growth factors Different organisms can have vastly different nutritional requirements. 223 Escherichia coli can grow on a simple defined medium. It can synthesize most of the organic molecules required for biosynthesis. Leuconostoc mesenteroides needs added amino acids, purines, pyrimidines, and vitamins for growth because it cannot synthesize these molecules by itself. Laboratory growth medium for E. coli Glucose K2HPO4 (NH4)2SO4 H2O KH2PO4 MgSO4 224 CaCl2 minerals Growth medium for L. mesenteroides Glucose, H2O, K2HPO4, KH2PO4, NH4Cl, MgSO4, Na acetate, alanine arginine asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, adenine, guanine, uracil, xanthine biotin, folate, nicotinic acid, pyridoxal, pyridoxamine pyridoxine, riboflavin, thiamine, pantothenate, para-aminobenzoic acid, trace elements (don't memorize) III. Energy Why do cells need energy? Where do organisms get energy? How do cells use energy sources? 225 A. Why do cells need energy? • growth and biosynthesis • motility • nutrient uptake • reproduction • maintenance, etc. nutrients polysaccharides 226 B. Where do organisms get energy? Chemotrophs chemicals 227 Phototrophs light Chemoorganotrophs Chemolithotrophs organic chemicals (eg. sugars) inorganic chemicals (eg. H2, NH3, H2S) C. How do chemotrophs derive energy228 from energy sources? Organisms capture energy that is released when an organic or inorganic chemical is oxidized. Remember: oxidation is the loss of electrons glucose + 6 O2 6 CO2 + 6 H2O G°’ = - 686 kcal/mol D. Units of energy 229 kcal (kilocalorie) • a unit of energy • amount of heat energy required to raise the temperature of 1 kg of water 1°C • 1 kcal = 4.184 kilojoules (kJ) • 1 kcal = 1 “nutritional” calorie IV. Review of free energy (G) • energy that is available to do useful work Review from General Chemistry: G = H - T S 230 change in entropy change in enthalpy (total energy) change in free energy 231 standard For biological reactions, the conditions for measuring the change in free energy (G°’ ) are • 25°C • pH 7 • reactants and products initially present at 1 M concentration A. The G°’ can tell us about the 232 direction a reaction tends to occur. A+B Free Energy C+D A+B G°’ is negative C+D Progress of reaction If G°’ is (-) products have lower free energy than substrates 233 1. If G°’ is negative • free energy is released • the reaction is exergonic • the reaction tends to occur in the direction written Examples: H2 + 1/2 O2 H2O glucose + 6 O2 6 CO2 + 6 H2O ATP + H2O ADP + PO4- - 57 kcal/mol - 686 kcal/mol - 7.3 kcal/mol 2. If G°’ is positive • energy input is usually required • the reaction is endergonic • the reaction does not tend to occur in the direction written Free Energy C+D A+B G°’ is positive Progress of reaction 234 If G°’ is (+) products have higher free energy than substrates B. Coupled reactions 235 Exergonic reactions (-G°’) can be used to "drive" endergonic reactions (+G°’) to make the overall "coupled" reaction favorable. Reaction 1 A B Go’ = +20 kJ/mole D Go’ = -30 kJ/mole Reaction 2 C Reactions 1 and 2 coupled A+C B+D Go’ = -10 kJ/mole 236 C. Equilibrium A+B C+D • equilibrium occurs when the rates of the forward and reverse reactions are equal • usually at equilibrium, the concentrations of the products and reactants are not equal C. Equilibrium (contd.) • if the G°’ is large and negative, equilibrium lies towards product; very little of the reactants remain A+B C+D 237 D. “Activation energy” is required to break bonds. 238 H2 + 1/2 O2 H2O G°’ = - 57 kcal/mol If H2 and O2 are mixed without a catalyst, no detectable amount of water is formed in our lifetime. Why? Because before water is formed, chemical bonds have to be broken. 239 Activation energy: energy required to bring molecules to the reactive state Free Energy H2 + 1/2 O2 Activation energy H2O Progress of reaction 240 E. Catalysts chemicals that increase the reaction rate by lowering the activation energy Free Energy H2 + 1/2 O2 G Activation energy of catalyzed reaction H2O Progress of reaction Properties of catalysts 241 • increase the rate of the reaction, • but DO NOT change the G, • DO NOT change the equilibrium • many reactions in living organisms are catalyzed by biological molecules called enzymes V. Enzymes • biological catalysts • most enzymes are proteins, a few are nucleic acids (ribozymes or catalytic RNAs) • most enzymes catalyze specific reactions or sets of reactions 242 A. Enzyme catalysis Enzyme (E): usually a protein Substrates (S): reactants, S starting materials Products (P): ending materials 243 Substrate(s) first combine with the enzyme to form an enzyme-substrate (E-S) complex. 244 B. Typical enzymatic reaction sequence: S E E E E E + S E-S E-P E + P Enzyme-substrate complex At end of reaction, the enzyme returns to its original form C. Important notes on enzymes 245 • Enzymes DO NOT alter the equilibrium of the reaction. • Enzymes can catalyze exergonic and endergonic reactions. • Substrates bind at the enzyme active site. • Many enzymes contain nonprotein components: coenzymes (loosely bound) or prosthetic groups (tightly bound). Important notes on enzymes (contd.) 246 • Enzymes tend to be sensitive to pH and temperature. • Enzymes are often named after the substrate or the reaction catalyzed, plus the ending “-ase” (eg. cellulase breaks down cellulose, ATP synthase makes ATP). D. Sometimes enzymes change shape247 when substrates bind (“induced fit”) glucose + hexokinase (a protein used in glycolysis) Active site 248 E. Metabolic reactions are catalyzed by enzymes. CH OH 2 glucose 12 enzymes HO OH O OH OH ethanol + CO2 glucose fermentation (anaerobic) Respiration of glucose (aerobic) glucose + 6 O2 ~36 [ADP + Pi] ~30 enzymes ~36 ATP 6 CO2 + 6 H2O 249 250 VI. Energy generation: A. Oxidation and reduction reactions For chemotrophs, utilization of a chemical energy source involves oxidation and reduction reactions (redox reactions). Oxidation and reduction reactions “LEO says GER” Loss of Electrons = Oxidation H2 2 H+ + 2 eGlucose (C6H12O6) 12 H+ + 12 e- + 6 CO2 Gain of Electrons = Reduction 1/2 O2 + 2 H+ + 2 e- H2O 251 B. Complete redox reactions can be divided 252 into oxidative and reductive half reactions. Oxidative half-reaction: H 2 2 H + + 2 e- Reductive half-reaction: 1/2 O2 + 2 H+ + 2 e- H2O Complete reaction: H2 + 1/2 O2 H2O e- donor e- acceptor H2 and H+ are called a redox couple. 253 C. Because electrons do not typically exist alone in solution, complete redox reactions need an electron donor (eg. H2) and an electron acceptor (eg. O2) glucose + 6 O2 primary terminal electron donor e- acceptor 6 CO2 + 6 H2O D. Energy is released when an energy source is oxidized. H2 + 1/2 O2 H2O glucose + 6 O2 6 CO2 + 6 H2O 254 G°’ - 57 kcal/mol - 686 kcal/mol Oxidative half-reaction H2 2 H+ + 2 e- 255 E. Cells oxidize energy sources and harness the energy released to make ATP. H2 1/2 O2 H2 Explosive release of energy as heat can't be harnessed to do work 2 H+ H2 O H2 + 1/2 O2 H2O 2 e- Hydrogen atoms separated into protons & electrons Some released energy is harnessed to make ATP Electron transport system 2 e2 H+ 1/2 O2 H2 O G°’ = - 57 kcal/mol Study Objectives 256 1. Understand metabolism, catabolism, anabolism, and the role of ATP in metabolism. 2. Know the differences between macronutrients, micronutrients, and growth factors. Know where they occur in biological molecules and the examples presented in class. 3. Contrast defined and complex media. Know one reason why nutritional requirements differ among organisms. 4. Give examples of energy-requiring processes in the cell. 5. Define chemotrophs, phototrophs, chemoorganotrophs, chemolithotrophs. (eg. chemotrophs are organisms that use chemicals as an energy source.) Given an energy source (eg. NH3), be able to identify the type of catabolism being used (eg. chemolithotrophy). 6. Understand the terms kcal and free energy. What predictions can be made from the Go' value of a reaction. What is reaction coupling and how can it be used by the cell? 257 7. Understand equilibrium, activation energy, catalysts and their properties. Understand the effect of catalysts on equilibrium. Can catalysts make a nonspontaneous reaction spontaneous? 8. Understand enzymes and all the properties presented in class. What is the function of enzymes in the cell? 9. Define oxidation, reduction, half reactions, redox couples, electron donor, electron acceptor. 10. Describe how cells derive energy from an energy source. What are the roles of the primary electron donor and the terminal electron acceptor? Energy generation and glycolysis I. Oxidation of the energy source II. Reduction of NAD+ III. Making ATP through substrate level phosphorylation IV. Glycolysis V. Reoxidation of NADH 258 I. Oxidation of the energy source: 259 A. Energy released when an energy source is oxidized can be conserved in the form of high energy chemical bonds. oxidation waste glucose products ADP + Pi ATP chemicals with high energy bonds 260 B. Electrons are transferred during catabolism. glucose [carbon] energy source primary electron donor electrons one or more intermediate electron carriers, e.g. NAD+ terminal electron acceptor (the last molecule to accept electrons), e.g. O2 261 C. Redox terminology 1. Oxidation is the loss of electrons Glucose (C6H12O6) 12 H+ + 12 e- + 6 CO2 Compounds become oxidized after losing electrons. An oxidant is a compound that accepts electrons. It can oxidize other compounds. TB 262 2. Reduction is the gain of electrons Compounds become reduced after gaining electrons. A reductant is a compound that donates electrons. It can reduce other compounds. TB D. Redox reactions 263 electron donor A(red) + B(ox) A(ox) + B(red) electron acceptor e.g. glucose + 6 O2 6 CO2 + 6 H2O TB 1. Redox couples are substances interconverted by redox reactions A(red) + B(ox) 264 A(ox) + B(red) A(ox)/A(red) is a redox couple (CO2/ glucose) B(ox)/B(red) is a redox couple (O2/ H2O) Note: the oxidized substance is written to the left. Two redox couples are needed for a redox reaction.TB Example: pyruvate and lactate are a redox couple. pyruvate/lactate half-reaction (hypothetical) pyruvate + 2H+ + 2e- lactate pyruvate can be reduced to lactate lactate can be oxidized to pyruvate o E' (reduction potential) = -0.19 volts 265 2. Redox couples have associated standard reduction potentials (Eo'). 266 (Eo') is a measure of the tendency of a redox couple to donate electrons in a redox reaction. Eo' values can be summarized in a "table of reduction potentials." In this table, the REDUCED substance of the redox couple is written on the right. TB 3. Partial table of reduction potentials 267 Oxidized form / Reduced form Reduction potential Eo' (Volts) CO2 / glucose (C6H12O2) 2 H+ / H 2 NAD+ / NADH pyruvate / lactate fumarate / succinate NO3- / NO2- (- 0.43) (- 0.42) (- 0.32) (- 0.19) (+ 0.03) (+ 0.42) O2 / H2O (+ 0.82) a. In a table of reduction potentials, the reduced compound of redox couple with a more negative Eo' 268 can give electrons to the oxidized compound of a redox couple lower in the table b. Example Two redox couples 269 NAD+/NADH Eo’ = –0.32 V pyruvate/lactate Eo’ = -0.19 V In a redox reaction, NADH can donate electrons to pyruvate. NADH + pyruvate NAD+ + lactate 4. Eo' is the change in standard reduction potential. 270 Reduction potential Eo' (Volts) CO2 / glucose (C6H12O2) (- 0.43) Eo' O2 / H2O (+ 0.82) 5. A large Eo' corresponds to a large Go'.271 Go' = -nF Eo' (don't memorize equation) Reduction potential Eo' (Volts) small Eo' CO2 / glucose (- 0.43) = -0.24 V pyruvate / lactate (- 0.19) not much energy O2 / H2O (+ 0.82) large Eo' = -1.25 V (lots of energy) 6. Electrons can be transferred to intermediate272 electron carriers in a series of redox reactions. (glucose) (H2O) A(red) A(ox) B(ox) B(red) C(red) C(ox) (CO2) (O2) A(red) = primary electron donor (energy source) B = intermediate electron carrier TB C (ox) = terminal electron acceptor II. NAD+ is an intermediate electron carrier. A(red) NAD+ C(red) 273 A(ox) NADH C(ox) "A" and "C" can be many numerous compounds many of which are catabolic intermediates. TB A. NAD+ and NADP+ 274 1. NAD+ nicotinamide adenine dinucleotide carries 2 electrons and a proton; usually involved in catabolic rxns 2. NADP+ similar to NAD+ with an extra PO4-; usually involved in biosynthesis B. The NAD+/NADH couple (Eo' = –0.32V)275 + NAD NADH + H NH2 adenine O OH HO O P-P O N+ HO OH 2e– + 2H+ + H H H O NH2 N + H+ R TB (look at but don't memorize structures) C. NAD+ must be recycled 276 NAD+ is made by cells in limited amounts. The reduction of NAD+ to NADH depletes NAD+. NAD+ must be regenerated by the oxidation of NADH to NAD+. TB III. Making ATP by substrate level phosphorylation (SLP) 277 A. Substrate Level Phosphorylation: *ATP synthesis driven by a high-energy compound, NOT the proton motive force (PMF). Example PEP + ADP pyruvate + ATP TB Ex. of Substrate level phosphorylation278 COO- P ~ P OCH2 O R CO~ P CH2 PEP + ADP COO- pyruvate + ATP C=O CH3 P ~ P ~ P OCH2 O R B. High energy compounds 279 Compounds that can release large amounts of energy when they react. Catabolism conserves energy in the form of high energy compounds which can be used to perform cellular work. TB 280 High energy compounds Go' of hydrolysis (kJ / mol) phosphoenolpyruvate 1,3-bisphosphoglycerate acetyl phosphate succinyl CoA, acetyl CoA ATP ADP -52 -52 -45 -32 -32 1. ATP is the most important high energy compound in cells. ATP + H2O o G ' 281 ADP + Pi = - 32 kJ / mol 2. ADP ADP + H2O AMP + Pi o G ' = – 32 kJ/mol TB 3. Phosphoenolpyruvate (PEP) PEP + H2O o G ' COOCO~PO3 CH2 282 pyruvate + Pi = - 52 kJ / mol COOC=O + PO43CH3 TB 4. 1,3-bisphosphoglycerate (BPG) 283 BPG + H2O 3-phosphoglycerate + Pi o G ' = – 52 kJ/mol The hydrolysis of the above high energy compounds is coupled to energy-consuming cellular reactions to drive them forward. TB SLP and glycolysis 284 During glycolysis, the hydrolysis of the high-energy compounds PEP or 1,3-bisphosphoglycerate (BPG) is "coupled" to ATP synthesis. This is an example of SLP. PEP + ADP pyruvate + ATP TB AMP and glucose-6-phosphate are examples of compounds with low energy bonds. o G ' 285 of hydrolysis -14 kJ / mol IV. Glycolysis 286 A. Overall reaction of glycolysis: Glucose 2 pyruvate + 2 NADH + 2 ATP • one pathway of making energy from glucose • glucose is partially oxidized to pyruvate • NAD+ is the intermediate electron carrier that accepts the electrons • ATP is made by substrate level phosphorylation (SLP) • glycolysis occurs in the cytoplasm B. Important steps in glycolysis glucose (C6) energy input 287 ATP ATP hexose splitting redox 2 NADH step ATP 2 ATP synthesis 2 ATP by SLP 2 pyruvate (C3) C. Individual steps of glycolysis (1) glucose hexokinase (1) 288 ATP* ADP glucose-6-phosphate energy input* TB (1) glucose-6-phosphate (1) fructose-6- phosphate 289 TB (1) fructose-6- phosphate 290 ATP* ADP (1) fructose-1,6- bisphosphate energy input* TB (1) fructose-1,6- bisphosphate (C6 molecule) 291 splitting reaction dihydroxyacetone phosphate (C3 molecule) glyceraldehyde 3- phosphate (C3 molecule) TB (2) glyceraldehyde-3- phosphate Pi (2) + NAD (2) NADH + (2) (2) 292 + H 1,3-bisphosphoglycerate Redox reaction TB (2) 1,3 bisphosphoglycerate (BPG) 293 2 ADP (2) 2 ATP 3-phosphoglycerate substrate level phosphorylation TB (2) 2-phosphoglycerate (2) phosphoenolpyruvate 294 TB (2) phosphoenolpyruvate 295 2 ADP 2 ATP (2) pyruvate substrate level phosphorylation TB V. Reoxidation of NADH to NAD+ Important: in the cell NAD+ is limited, so NADH must be reoxidized 296 A. The reoxidation of electron carriers297 All organisms on earth that have been studied use one or more of 3 general methods to reoxidize electron carriers 1. Fermentation 2. Aerobic respiration 3. Anaerobic respiration Organisms that use all three methods usually prefer aerobic respiration. TB Glycolysis 298 glucose 2 pyruvate 2 NAD+ 2 NADH + H+ re-oxidation 1. Fermentation reactions 2. Aerobic respiration 3. Anaerobic respiration TB 1. Fermentation 299 • catabolic process in which NADH is re-oxidized using a compound derived from the growth substrate • ATP synthesis is by substrate level phosphorylation (SLP) only. • Generally used when O2 is not available TB a. Fermentation producing ethanol 300 2 NAD+ 2 NADH + H+ glucose 2 pyruvate 2 CO2 2 CH3CH2OH 2 Ethanol + 2NAD 2 Acetaldehyde 2NADH + + H TB b. Fermentation producing lactate 301 COO2 NAD+ 2 NADH + C=O glucose 2 pyruvate CH + 3 2NADH + H H+ + 2NAD COOHC-OH 2 lactate CH3 TB 302 Fermentation does not use electron transport chains for the reoxidation of electron carriers. Cytoplasmic enzymes catalyze the reoxidation of NADH. Many different fermentations are known. Some of the products of fermentation are valuable. TB Study objectives 303 1. Describe how cells derive energy from an energy source. What are the roles of the primary electron donor and the terminal electron acceptor? 2. Understand redox reactions and the terminology used to talk about them. 3. Understand redox couples. 4. Be able to use the table of standard reduction potentials to predict the direction of a redox reaction. 5. Understand the relationship between Eo' and Go'. (Basically, a large Eo' corresponds to a large Go' ) You will NOT be asked to do a calculation. 6. Understand how NAD functions in cells. 7. Compare and contrast NAD and NADP. 8. Understand high energy compounds. Know the examples of high energy and presented in class. Know that GTP is a high energy compound. 9. Describe substrate level phosphorylation. Understand the difference between substrate level phosphorylation and oxidative phosphorylation. 10. Understand the process of glycolysis. Know the overall reaction. Memorize all the steps. Know which steps involve energy input, hexose splitting, redox reactions, substrate level phosphorylation, ATP synthesis. 304 11. What are the 3 general methods microbes use to reoxidize reduced electron carriers formed during catabolic processes? 12. Why must reduced electron carriers be reoxidixed? 13. Understand fermentation and its purpose. Memorize the examples and reactions presented in class. Respiration and the TCA cycle: I. Aerobic respiration of glucose II. TCA cycle III. Electron carriers IV. Electron transport system V. Oxidative phosphorylation 305 Reoxidation of NADH Growth substrates 306 Oxidized products Oxidized electron carriers Reduced electron carriers re-oxidation 1. Fermentation 2. Aerobic respiration 3. Anaerobic respiration TB I. Aerobic respiration of glucose 307 one way to get more energy out of glucose than by fermentation glucose + 6 O2 6 CO2 + 6 H2O Fermentation: ~2 ATP / glucose Respiration: ~36 to 38 ATP / glucose 308 A. Respiration 1. Oxidation of an organic energy source in the presence of an external terminal electron acceptor "external" terminal electron acceptor glucose + 6 O2 6 CO2 + 6 H2O organic energy source 1. terminal electron acceptor: the last molecule to receive the electrons during catabolism 309 2. "external" terminal electron acceptor: terminal electron acceptor that is NOT derived from the energy source 310 B. Aerobic respiration 1. Terminal electron acceptor is O2 Anaerobic respiration "external" terminal electron acceptor is NOT O2 eg. NO3- (nitrate), Fe3+, SO4-, CO2, CO32-, succinate or another organic molecule 311 B. Aerobic respiration (continued) 2. Reoxidation of reduced electron carriers with O2 occurs via intermediate electron carriers arranged as electron transport chains (respiratory chains). 3. ATP synthesis occurs mainly by oxidative phosphorylation 312 C. Aerobic respiration of glucose complete oxidation of glucose to CO2 higher energy yield than fermentation C6H12O6 6 O2 6 CO2 + 6 H2 O Respiration: 36 to 38 ATP glucose 2 C3H6O3 lactic acid Fermentation: 2 ATP 313 D. Oxidative phosphorylation (electron transport phosphorylation) ATP synthesis at the expense of a proton gradient (proton motive force) produced across a membrane by an electron transport system H+ ATP H+ H+ H+ H+ H+ H+ + + H H H+ ADP + Pi Cytoplasmic membrane in prokaryotes Inner mitochondrial membrane in eukaryotes E. Overview: aerobic glucose respiration 314 glucose membrane NADH acCoA pyruvate NADH TCA FADH2 GTP NADH NADH NADH e- outside 2 H+ NAD+ proton motive force 2 H+ H+ 315 II. Glucose respiration to CO2 and the TCA cycle glucose 1 NADH pyruvate Glucose respiration CO2 2 acCoA TCA CO2 3 NADH 4 NAD+ CO2 1. Glycolysis 2. Conversion of pyruvate (3C) to acetyl CoA (2C) 3. Oxidation of acetyl CoA in TCA cycle 4. Reoxidation the intermediate electron acceptors 5. ATP synthesis by oxidative phosphorylation 316 A. Conversion of pyruvate to acetyl CoA • pyruvate oxidation produces NADH • decarboxylation makes CO2 glucose glycolysis 2 pyruvate (3C) 2 CoA + + 2 NAD 2 NADH 2 acetyl CoA (2C) 2 CO2 317 B. Oxidation of acetyl CoA in the TCA cycle (tricarboxylic acid cycle) acetyl CoA also called the citric acid cycle NADH NADH TCA CO2 FADH2 GTP CO2 NADH • two carbons are oxidized to CO2 per acetyl CoA • 3 NADHs and 1 FADH2 are made per acetyl CoA • one GTP is made by substrate level phosphorylation 1. Acetyl CoA (C2) condenses with 318 oxaloacetate (C4) to form citrate (C6). O CH3C~SCoA acetyl CoA (2C) COOO=CH oxaloacetate CH2 COO- citrate (6C) (4C) (5C) CH2COOHOCH2COOCH2COO- 2. Redox reactions, decarboxylations, SLP319 CoA + NAD+ NADH pyruvate acetyl CoA (2C) CO2 (3C) citrate oxaloacetate NADH NAD+ (6C) (4C) FADH2 NAD+ (5C) NADH FAD *SLP CoA GTP GDP + Pi NAD+ NADH CO2 CO2 3. The TCA Cycle 320 acetyl CoA (2C) oxaloacetate NADH NAD+ FADH2 malate fumarate FAD SLP citrate aconitate (6C) (4C) succinate CoA GTP GDP + Pi (5C) isocitrate NAD+ NADH CO2 -ketoglutarate succinyl CoA NAD+ NADH CO2 4. In the TCA cycle, there are 4 redox 321 reactions (3 NADH and 1 FADH2) and two decarboxylations. Four oxidative steps in the TCA cycle isocitrate -ketoglutarate -ketoglutarate succinyl CoA succinate fumarate (FADH2) malate oxaloacetate 2 decarboxylations (CO2 removed) d. There is one substrate level phosphorylation in the TCA cycle. succinyl CoA succinate GTP is made and is easily converted to ATP 322 e. Sum of reactions for NADH pyruvate oxidation CO acCoA pyruvate and TCA cycle 323 2 pyruvate 3 CO2 NADH 4 NADH must be 1 FADH2 reoxidized 1 GTP by SLP TCA FADH2 GTP NADH CO2 NADH CO2 15 ATP equivalents per pyruvate III. Reoxidation of NADH and FADH2 with O2 occurs via intermediate electron carriers arranged as electron transport chains in the membrane. 324 e– 2H 2H NAD+ NADH + H+ e– Q e– 1/2 O2 + e– 2H+ H2O TB A. Intermediate electron carriers 325 1. NADH dehydrogenases Protein complexes that accept protons and electrons from NADH. 2H NAD+ NADH + H+ [2H] = 2 protons + 2 electrons TB 2. Flavoproteins 326 Proteins with FAD or FMN (flavin adenine dinucleotide or flavin mononucleotide) as a prosthetic group. Flavoproteins carry protons and electrons. 2H TB FMN and FAD (isoalloxazine ring) H3C H3C N N 327 O NH N Oxidized form O R Flavin couples FMN / FMNH2 FAD / FADH2 FMN and FAD are functionally equivalent, but have a different R-group TB Iron-sulfur center S E-cys-S Fe E-cys-S 328 S-cys-E Fe S S-cys-E 2Fe2S E-cys-S = the sulfur of a cys residue of the protein is bonded to the iron TB 329 Iron-sulfur center E-cys-S S Fe Fe S Fe S S Fe S-cys-E S-cys-E E-cys-S 4Fe4S TB 4. Quinones Small molecules (nonprotein) 330 Quinones carry both protons and electrons. Quinones can diffuse within the membrane. TB 331 Quinone OH O O CH3O CH3O R CH3O R HO CH3O Q QH2 Oxidized Reduced TB Diffusion of quinones within the cell membrane. 332 cytoplasm TB 5. Cytochromes 333 Proteins that contain the heme prosthetic group. Cytochromes carry electrons only. cytochrome c e– e– e– cytochrome cytochrome bc1 aa3 TB 334 Cytochrome heme protein Fe Fe3+/Fe2+ The iron carries the electrons TB B. Electron Transport Chains A series of electron carriers arranged within a membrane. Many different electron transport chains are known and they all function similarly. 335 • electron transport chains can oxidize intermediate electron carriers like NADH and FADH2 and create proton gradients (PMF) TB 1. Electron transport chain of E. coli. flavoprotein quinone 336 cytochrome c Q NADH dehydrogenase iron-sulfur protein cytochrome cytochrome bc1 aa3 cytoplasm TB a. In aerobic respiration, the electron transport337 chain is used to reoxidize NADH with O2. 2H = 2 protons and 2 electrons e– 2H 2H 2e– Q e– e– NAD NADH + H+ cytoplasm 1/2 O2 + 2H+ H2O TB b. Oxidation via electron transport allows338 proton pumping. A proton gradient (PMF) is formed across the membrane. 2H+ 2H 2H NAD NADH + H+ 2H+ Q 2e– e– e– e– 2H+ + 222H cytoplasm 1/2 O2 + 2H+ H2O TB 339 2. Proton motive force (PMF) an energized state of the membrane created by a proton gradient + + + + + -- - - - + -+ + - - - - + + + + + + + + + + + + - - - - - - - H+ OH- H+ + H - - - - - - - + + + + + + H+ H+ H+ H+ + + H H H+ PMF about -20 kJ/mol In prokaryotes, H+ are pumped out of the cell. The outside becomes slightly acidic and positively charged relative to the inside. 340 3. Results of the electron transport chain a. intermediate electron carriers (e.g. NADH and FADH2) are reoxidized b. electrons are ultimately transferred to O2, making water c. proton motive force (PMF) is created, which can be used for ATP synthesis 341 V. Oxidative phosphorylation (electron transport phosphorylation) ATP synthesis driven by PMF The F1F0 ATPase synthesizes ATP using the PMF. A. Chemiosmosis (Peter Mitchell, 1961): Use of an ion gradient (like PMF) to drive ATP synthesis TB B. ATP synthesis using PMF 342 Energy is released when the H+ gradient is dissipated. The energy can do work (make ATP, rotate flagella, take up nutrients). H+ ATP 2 to 4H+ ADP + Pi H+ H+ H+ H+ + H+ H H+ PMF -20 kJ/mol ATP synthase F1F0 ATPase H+ H+ 343 H+ Fo F1: catalyzes ATP synthesis cytoplasm ADP + Pi H+ + + H H ATP TB C. How many ATPs can be synthesized when NADH and FADH2 are reoxidized through an electron transport chain (respiratory chain)? NADH FADH2 ~ 3 ATP ~ 2 ATP 344 D. Comparison of glucose fermentation 345 and respiration in bacteria glucose fermentation: 2 ATP per glucose glucose respiration (bacteria): 38 ATP per glucose glycolysis: 2 ATP (net) 2 NADH (2 x 3 ATP) 2 pyr 2 acCoA: 2 NADH 2 ATP 6 ATP 6 ATP 2 acCoA (2) x TCA cycles: (2) x 3 NADH (2 x 3 x 3 ATP) 18 ATP (2) x 1 FADH2 (2 x 1 x 2 ATP) 4 ATP (2) x 1 GTP 2 ATP Total: 38 ATP Study objectives for lecture 9 346 1. Understand respiration. Contrast fermentation and respiration. 2. Understand oxidative phosphorylation. Contrast oxidative phosphorylation and substrate level phosphorylation. 3. Describe the overall process of glucose respiration and the five steps presented in class. 4. Memorize and understand the reaction of pyruvate conversion to acetyl CoA. 5. Understand the TCA cycle. Know that the TCA cycles begins with the reaction of acetyl CoA and oxaloacetate to make citrate. How does the TCA cycle help cells produce energy? 6. Memorize ALL the steps in the TCA cycle. Know which steps involve redox reactions, substrate level phosphorylation, decarboxylation. You do not need to memorize the structures. 7. In respiration, glucose is oxidized completely to CO2. How is this done? Where is CO2 released? What happens to the electrons? What is the role of oxygen in respiration? How is energy conserved as ATP? How do cells derive energy from glucose in respiration? In fermentation? 8. What are electron transport chains? What is their role in metabolism? 347 9. Compare and contrast the electron carriers used in electron transport chains. Understand the particular features of each electron carrier. You do not need to memorize the structures. 10. Which electron carrier is nonprotein? 11. Can cytochromes carry protons? 12. Describe how electron transport chains are used to synthesize ATP. Understand how electron transport in the membrane generates proton motive force. Recall that proton motive force can be used to produce ATP. Continued on next slide 348 1. Describe oxidative phosphorylation. Define chemiosmosis. 2. Know the general structure of the F1/F0 ATPase. What is its function? 3. How is the reoxidation of intermediate electron carriers related to ATP synthesis? 4. Which method allows production of more ATP: aerobic respiration or fermentation? 5. Starting with glucose, describe how ATP is made from glucose in fermentation and respiration. 6. Describe how glycolysis, the TCA cycle, electron transport chains, and ATP synthesis are connected in respiration.