Chapter 06 *Lecture Outline

Chapter 06 *Lecture Outline
*See separate FlexArt PowerPoint slides for all figures and tables pre-inserted into PowerPoint without notes and animations. 1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1

A Glimpse of History Biologists had noticed that in vats of grape juice, alcohol and CO2 are produced while yeast cells increase in number But idea not widely accepted, mocked by some In 1850s, Louis Pasteur set out to prove Simplified setup: clear solution of sugar, ammonia, mineral salts, trace elements Added a few yeast cells—as they grew, sugar decreased, alcohol level increased Strongly supported idea, but Pasteur failed to extract something from inside the cells that would convert sugar In 1897, Eduard Buchner, a German chemist awarded Nobel Prize in 1907 showed that crushed yeast cells could convert sugar to ethanol and CO2;

Microbial Metabolism All cells need to accomplish two fundamental tasks Synthesize new parts Cell walls, membranes, ribosomes, nucleic acids Harvest energy to power reactions Sum total of these is called metabolism Human implications Used to make biofuels Used to produce food Important in laboratory Invaluable models for study Unique pathways potential drug targets

6.1. Principles of Metabolism
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Can separate metabolism into two parts Catabolism Processes that degrade compounds to release energy Energy captured to make ATP Anabolism Biosynthetic processes Assemble subunits of macromolecules Use ATP to drive reactions Processes intimately linked CATABOLISM ANABOLISM Energy source (glucose) Cell structures (cell wall, membrane, ribosomes, surface structures) Energy Macromolecules (proteins, nucleic acids, polysaccharides, lipids) Energy Subunits (amino acids, nucleotides, sugars, fatty acids) Energy Precursor metabolites Waste products Nutrients (acids, carbon dioxide) (source of nitrogen, sulfur, etc.) Catabolic processes harvest the energy released during the breakdown of compounds and use it to make ATP. The processes also produce precursor metabolites used in biosynthesis. Anabolic processes (biosynthesis) synthesize and assemble subunits of macromolecules that make up the cell structures. The processes use the ATP and precursor metabolites produced in catabolism.

Harvesting Energy Energy is the capacity to do work
Two types of energy Potential: stored energy (e.g., chemical bonds, rock on hill, water behind dam) Kinetic: energy of movement (e.g., moving water) Energy in universe cannot be created or destroyed, but it can be converted between forms This is the 1st Law of Thermodynamics

Harvesting Energy Photosynthetic organisms harvest energy in sunlight
Power synthesis of organic compounds from CO2 Convert kinetic energy of photons to potential energy of chemical bonds Chemoorganotrophs obtain energy from organic compounds Depend on activities of photosynthetic organisms Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Radiant energy (sunlight) Photosynthetic organisms harvest the energy of sunlight and use it to power the synthesis of organic compounds from CO2. This converts radiant energy to chemical energy. Chemical energy (organic compounds) Chemoorganotrophs degrade organic compounds, harvesting chemical energy. (top): © Photodisc Vol. Series 74, photo by Robert Glusie; (bottom): © Digital Vision/PunchStock

Harvesting Energy Free energy is energy available to do work
E.g., energy released when chemical bond is broken Compare free energy of reactants, products Exergonic reactions: reactants have more free energy Energy is released in reaction Endergonic reactions: products have more free energy Reaction requires input of energy Change in free energy is same regardless of number of steps involved (e.g., converting glucose to CO2 + H2O) Cells use multiple steps when degrading compounds Energy released from exergonic reactions powers endergonic reactions

Components of Metabolic Pathways
Series of chemical reactions that convert starting compound end product May be linear, branched, cyclical Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Starting compound Intermediatea Intermediateb End product (a) Linear metabolic pathway Intermediateb1 End product1 Starting compound Intermediatea (b) Branched metabolic pathway Intermediateb2 End product2 Starting compound Intermediated End product Intermediatea Intermediatec Intermediateb (c) Cyclical metabolic pathway

Role of Enzymes Biological catalysts: accelerate conversion of substrate into product by lowering activation energy Highly specific: one at each step Reactions would occur without, but extremely slowly Activation energy without an enzyme Activation energy with an enzyme Energy of reactants Relative energy Energy of products Progress of reaction (a) Enzyme a Enzyme b Enzyme c Starting compound Intermediatea Intermediateb End product (b)

Role of ATP Adenosine triphospate (ATP) is energy currency Composed of ribose, adenine, three phosphate groups Adenosine diphospate (ADP) acceptor of free energy Cells produce ATP by adding Pi to ADP using energy Release energy from ATP to yield ADP and Pi Three processes to generate ATP Substrate-level phosphorylation Exergonic reaction powers Oxidative phosphorylation Proton motive force drives Photophosphorylation Sunlight used to create proton motive force to drive Unstable (high-energy) bonds P ~ P ~ P ATP Pi Pi Energy used The energy comes from catabolic reactions. Energy released The energy drives anabolic reactions. P ~ P ADP

Role of the Chemical Energy Source and the Terminal Electron Acceptor Some atoms, molecules more electronegative than others Greater affinity for electrons Energy released when electrons move from low affinity molecule to high affinity molecule (E.g., glucose to O2) Terminal electron acceptors Energy sources Energy release Organic carbon compounds Organic carbon compounds H2 CO2 H2S S0 SO4 FeOOH Fe2+ Relative tendency to give up electrons Relative tendency to give up electrons NH4+ NO2– ( to form NH4+) NO3– ( to form NH4+) Mn2+ MnO2 NO3– ( to form NH2) O2 (a) Energy is released when electrons are moved from an energy source with a low affinity for electrons to a terminal electron acceptor with a higher affinity.

Role of the Chemical Energy Source and the Terminal Electron Acceptor (continued…) More energy released when difference in electronegativity is greater Electron donor: Energy source Acceptor: Terminal electron acceptor Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Glucose as an energy source Terminal electron acceptors Inorganic energy sources Terminal electron acceptors Glucose H2 H2S CO2 Pyruvate Relative tendency to give up electrons Relative tendency to give up electrons Fe2+ Relative tendency to give up electrons NO3– (to form NH4–) O2 O2 (b) Three examples of chemoorganotrophic metabolism (c) Three examples of chemolithotrophic metabolism

Prokaryotes remarkably diverse in using energy sources and terminal electron acceptors Organic, inorganic compounds used as energy source O2, other molecules used as terminal electron acceptor Electrons removed through series of oxidation-reduction reactions or redox reactions Substance that loses electrons is oxidized Substance that gains electrons is reduced Electron-proton pair, or hydrogen, actually moves Dehydrogenation = oxidation Hydrogenation = reduction Transfer of electrons e– e– Compound X + Compound Y Compound X (oxidized) + Compound Y (reduced) X loses electron(s). Y gains electron(s). X is oxidized by the reaction. X is the reducing agent. Y is reduced by the reaction. Y is the oxidizing agent.

Role of Electron Carriers Energy harvested in stepwise process Electrons transferred to electron carriers, which represent reducing power (easily transfer electrons to molecules) Raise energy level of recipient molecule NAD+/NADH, NADP+/NADPH, and FAD/FADH2

Precursor Metabolites
Precursor metabolites are intermediates of catabolism that can be used in anabolism Serve as carbon skeletons for building macromolecules E.g., pyruvate can be converted into amino acids alanine, leucine, or valine

Precursor Metabolites
Recall that E. coli can grow in glucose-salts medium Contains just glucose, inorganic salts Glucose is energy source Glucose is starting point for all cellular components Includes proteins, lipids, carbohydrates, nucleic acids Some glucose molecules completely oxidized for energy; others used in biosynthesis Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Glucose molecules To: Lipid synthesis To: Amino acid synthesis To: Carbohydrate synthesis To: Nucleic acid synthesis CO2 molecules + energy

Overview of Catabolism
Three central metabolic pathways Oxidize glucose to CO2 Catabolic, but precursor metabolites and reducing power can be diverted for use in biosynthesis Termed amphibolic to reflect dual role Glycolysis Splits glucose (6C) to two pyruvates (3C) Generates modest ATP, reducing power, precursors Pentose phosphate pathway Primary role is production precursor metabolites, NADPH Tricarboxylic acid cycle Oxidizes pyruvates from glycolysis Generates reducing power, precursor metabolites, ATP

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. GLUCOSE Central metabolic pathways Glycolysis Pentose phosphate pathway Tricarboxylic acid cycle Key outcomes ATP Reducing power Precursor metabolites Electron Transport Chain 2 Pentose phosphate pathway Starts the oxidation of glucose 1 Glycolysis Oxidizes glucose to pyruvate Yields ~ ~ + Reducing power ATP by substrate-level phosphorylation Yields Reducing power Biosynthesis 5 Fermentation Reduces pyruvate or a derivative Pyruvate Pyruvate Acids, alcohols, and gases 3a Transition step CO2 CO2 Yields Reducing power Acetyl- CoA Acetyl- CoA X 2 CO2 CO2 3b TCA cycle Incorporates an acetyl group and releases CO2 (TCA cycles twice) Yields ~ ~ + Reducing power ATP by substrate-level phosphorylation 4 Respiration Uses the electron transport chain to convert reducing power to proton motive force Yields ~ ~ ATP by oxidative phosphorylation

Respiration transfers electrons from glucose to electron transport chain Electron transport chain generates proton motive force Harvested to make ATP via oxidative phosphorylation Aerobic respiration O2 is terminal electron acceptor Anaerobic respiration Molecule other than O2 as terminal electron acceptor Also use modified version of TCA cycle i.e. “Builds a Battery

Fermentation If cells cannot respire, will run out of carriers available to accept electrons Glycolysis will stop Fermentation uses pyruvate or derivative as terminal electron acceptor to regenerate NAD+ Glycolysis can continue

6.2. Enzymes Enzymes are biological catalysts
Name reflects function; ends in -ase Has active site to which substrate binds weakly Causes enzyme shape to change slightly Existing substrate bonds destabilized, new ones form Enzymes are highly specific Enzyme not used up

6.2. Enzymes Enzymes are biological catalysts Substrate
Enzyme-substrate complex formed Products released Enzyme Active site Enzyme unchanged (a) Substrate Substrate Enzyme Enzyme (b) (c) (b, c): From Voet: Biochemistry, 1/e, , 1990, John Wiley & Sons

6.2. Enzymes Cofactors assist some enzymes
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Cofactors assist some enzymes Cofactors can assist different enzymes; fewer types needed Include magnesium, zinc, copper, other trace elements Coenzymes are organic cofactors Include electron carriers FAD, NAD+, NADP+ Enzyme Cofactor Substrate

6.2. Enzymes Environmental Factors Influencing Enzyme Activity
Enzymes have narrow range of optimal conditions Temperature, pH, salt concentration 10°C increase doubles speed of enzymatic reaction up until maximum Proteins denature at higher temperatures Low salt, neutral pH usually optimal Optimum temperature Optimum pH Enzyme activity Enzyme activity 1 2 3 4 5 6 7 8 9 10 11 12 13 Temperature Acidic Basic (a) (b)

6.2. Enzymes Allosteric Regulation
Enzyme activity controlled by binding to allosteric site Distorts enzyme shape, prevents or enhances binding Regulatory molecule is usually end product Allows feedback inhibition Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Enzyme Enzyme Allosteric inhibitor Substrate Allosteric site Active site (b) (a) Allosteric inhibitor Enzyme a Enzyme b Enzyme c Starting compound Intermediatea Intermediateb End product (c)

6.2. Enzymes Enzyme Inhibition
Site to which inhibitor binds determines type Competitive inhibitor binds to active site of enzyme Chemical structure usually similar to substrate Concentration dependent; blocks substrate Example is sulfa drugs blocking folic acid synthesis Structural differences PABA (substrate) H H HO O N C O S O Sulfa (inhibitor) N N H H H H PABA Sulfanilamide Enzyme (a) (b)

6.2. Enzymes Enzyme Inhibition (continued…)
Non-competitive inhibitor binds to a different site Allosteric inhibitors are one example; action is reversible Some non-competitive inhibitors are not reversible E.g., mercury oxidizes the S—H groups of amino acid cysteine, converts to cystine Cystine cannot form important disulfide bond (S—S) Enzyme changes shape, becomes nonfunctional

6.3. The Central Metabolic Pathways
ATP Reducing power: NADH, FADH2, NADPH Precursor metabolites Glucose molecules can have different fates Can be completely oxidized to CO2 for maximum ATP Can be siphoned off as precursor metabolite for use in biosynthesis

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Glycolysis Converts 1 glucose to 2 pyruvates; yields net 2 ATP, 2 NADH Investment phase: 2 phosphate groups added Glucose split to two 3-carbon molecules Pay-off phase: 3-carbon molecules converted to pyruvate Generates 4 ATP, NADH total Nets 2 ATP GLUCOSE 2 Starts the oxidation of glucose Pentose phosphate pathway 1 Oxidizes glucose to pyruvate Glycolysis Yields phosphorylation by substrate-level ATP P ~ P ~ P + Reducing power Yields power Reducing Biosynthesis 5 Reduces pyruvate or a derivative Fermentation 3a Transition step Acids, alcohols, and gases Glucose Yields Reducing power CO2 CO2 Pyruvate Pyruvate x2 CO2 ATP ~ ~ 1 ATP is expended to add a phosphate group. 3b group and releases CO2 Incorporates an acetyl (TCA cycles twice) TCA cycle CO2 ADP ~ Yields by substrate-level phosphorylation ATP P ~ P ~ P + Reducing power 4 power to proton motive force Uses the electron transport Chain to convert reducing Respiration Yields phosphorylation by oxidative ATP P ~ P ~ P Glucose 6-phosphate 2 A chemical rearrangement occurs. Fructose 6-phosphate ATP ~ ~ 3 ATP is expended to add a phosphate group. ADP ~ Fructose 1,6-bisphosphate 4 The 6-carbon molecule is split into two 3-carbon molecules. Dihydroxyacetone phosphate 5 A chemical rearrangement of one of the molecules occurs. Glyceraldehyde 3-phosphate NAD+ NAD+ 6 NADH + H+ The addition of a phosphate group is coupled to a redox reaction, generating NADH and a high-energy phosphate bond. NADH + H+ 1,3-bisphosphoglycerate ~ ~ ADP ~ ~ 7 ATP is produced by substrate-level phosphorylation. ATP ~ ~ ~ ~ 3-phosphoglycerate 8 A chemical rearrangement occurs. 2-phosphoglycerate H2O H2O 9 Water is removed, causing the phosphate bond to become high-energy. Phospho- enolpyruvate ADP ~ ~ 10 ATP is produced by substrate-level phosphorylation. ATP ~ ~ ~ ~ Pyruvate

Pentose Phosphate Pathway Also breaks down glucose Important in biosynthesis of precursor metabolites Ribose 5-phosphate, erythrose 4-phosphate Also generates reducing power: NADPH Yields vary depending upon alternative taken

Transition Step CO2 is removed from pyruvate Electrons reduce NAD+ to NADH + H+ 2-carbon acetyl group joined to coenzyme A to form acetyl-CoA Takes place in mitochondria in eukaryotes GLUCOSE 2 Starts the oxidation of glucose Pentose phosphate pathway 1 Oxidizes glucose to pyruvate Glycolysis Yields phosphorylation by substrate-level ATP ~ ~ + Reducing power Pyruvate Yields CO2 Reducing power Transition step: CO2 is removed, a redox reaction generates NADH, and coenzyme A is added. Biosynthesis 5 Reduces pyruvate or a derivative Fermentation NAD+ Pyruvate Pyruvate 3a Yields Transition step Acids, alcohols, and gases CoA Reducing power CO2 CO2 Acetyl- CoA Acetyl- CoA NADH + H+ x 2 CO2 CoA 3b group and releases CO2 Incorporates an acetyl (TCA cycles twice) TCA cycle CO2 Acetyl-CoA Yields Reducing power 1 The acetyl group is transferred to oxaloacetate to start a new round of the cycle. phosphorylation by substrate-level ATP ~ ~ + 4 power to proton motive force chain to convert reducing Uses the electron transport Respiration CoA Yields phosphorylation by oxidative ATP ~ ~ NADH + H+ Oxaloacetate 2 A chemical rearrangement occurs. 8 A redox reaction generates NADH. Citrate NAD+ Isocitrate NAD+ 3 A redox reaction generates NADH and CO2 is removed. Malate 7 Water is added. H2O NADH + H+ CO2 Fumarate -ketoglutarate NAD+ 4 A redox reaction generates NADH, CO2 is removed, and coenzyme A is added. FADH2 CoA 6 A redox reaction generates FADH2- NADH + H+ CoA FAD CO2 Succinate Succinyl-CoA 5 The energy released during CoA removal is harvested to produce ATP. CoA ~ ~ ~ + Pi ATP ADP

Tricarboxylic Acid (TCA) Cycle (Krebs) Completes oxidation of glucose Produces 2 CO2 2 ATP 6 NADH 2 FADH2 Precursor metabolites GLUCOSE 2 Starts the oxidation of glucose Pentose phosphate pathway 1 Oxidizes glucose to pyruvate Glycolysis Yields phosphorylation by substrate-level ATP ~ ~ + Reducing power Pyruvate Yields CO2 Reducing power Transition step: CO2 is removed, a redox reaction generates NADH, and coenzyme A is added. Biosynthesis 5 Reduces pyruvate or a derivative Fermentation NAD+ Pyruvate Pyruvate 3a Yields Transition step Acids, alcohols, and gases CoA Reducing power CO2 CO2 Acetyl- CoA Acetyl- CoA NADH + H+ x 2 CO2 CoA 3b group and releases CO2 Incorporates an acetyl (TCA cycles twice) TCA cycle CO2 Acetyl-CoA Yields Reducing power 1 The acetyl group is transferred to oxaloacetate to start a new round of the cycle. phosphorylation by substrate-level ATP ~ ~ + 4 power to proton motive force chain to convert reducing Uses the electron transport Respiration CoA Yields phosphorylation by oxidative ATP ~ ~ NADH + H+ Oxaloacetate 2 A chemical rearrangement occurs. 8 A redox reaction generates NADH. Citrate NAD+ Isocitrate NAD+ 3 A redox reaction generates NADH and CO2 is removed. Malate 7 Water is added. H2O NADH + H+ CO2 Fumarate -ketoglutarate NAD+ 4 A redox reaction generates NADH, CO2 is removed, and coenzyme A is added. FADH2 CoA 6 A redox reaction generates FADH2- NADH + H+ CoA FAD CO2 Succinate Succinyl-CoA 5 The energy released during CoA removal is harvested to produce ATP. CoA ~ ~ ~ + Pi ATP ADP

6.4. Respiration Uses reducing power (NADH, FADH2) generated by glycolysis, transition step, and TCA cycle to synthesize ATP Electron transport chain generates proton motive force Drives synthesis of ATP by ATP synthase Process proposed by British scientist Peter Mitchell in 1961 Initially widely dismissed Mitchell conducted years of self-funded research Received a Nobel Prize in 1978 Now called chemiosmotic theory

The Electron Transport Chain—Generating Proton Motive Force
Electron transport chain is membrane-embedded electron carriers Pass electrons sequentially, eject protons in process Prokaryotes: in cytoplasmic membrane Eukaryotes: in inner mitochondrial membrane Energy gradually released Release coupled to ejection of protons Creates electrochemical gradient (“Battery”) Used to synthesize ATP Prokaryotes can also power transporters, flagella Electrons from the energy source 2 e– Energy released is used to generate a proton motive force. High energy Low energy Electrons donated to the terminal electron acceptor. 1/2 O2 2 H+ H2O

Components of an Electron Transport Chain Most carriers grouped into large protein complexes Serve as proton pumps Three general groups are notable Quinones Lipid-soluble molecules Move freely, can transfer electrons between complexes Cytochromes Contain heme, molecule with iron atom at center Several types Flavoproteins Proteins to which a flavin is attached FAD, other flavins synthesized from riboflavin

General Mechanisms of Proton Ejection Some carriers accept only hydrogen atoms (proton-electron pairs), others only electrons Spatial arrangement in membrane shuttles protons to outside of membrane When hydrogen carrier accepts electron from electron carrier, it picks up proton from inside cell or mitochondrial matrix When hydrogen carrier passes electrons to electron carrier, protons released to outside of cell or intermembrane space of mitochondria Net effect is movement of protons across membrane Establishes concentration gradient Driven by energy released during electron transfer

Electron Transport Chain of Mitochondria Complex I (NADH dehydrogenase complex) Accepts electrons from NADH, transfers to ubiquinone Pumps 4 protons Complex II (succinate dehydrogenase complex) Accepts electrons from TCA cycle via FADH2, “downstream” of those carried by NADH Transfers electrons to ubiquinone Complex III (cytochrome bc1 complex) Accepts electrons from ubiquinone from Complex I or II 4 protons pumped; electrons transferred to cytochrome c Complex IV (cytochrome c oxidase complex) Accepts electrons from cytochrome c, pumps 2 protons Terminal oxidoreductase, meaning transfers electrons to terminal electron acceptor (O2)

The Electron Transport Chain of Mitochondria
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. GLUCOSE 2 Pentose phosphate Starts the oxidation of glucose pathway 1 Oxidizes glucose to pyruvate Glycolysis Yields phosphorylation by substrate-level ATP P ~ P ~ P + Reducing power Yields Reducing power Eukaryotic cell Biosynthesis 5 Reduces pyruvate Fermentation or a derivative Pyruvate Pyruvate Acids, alcohols, and gases 3a Transition step Yields CO2 CO2 Reducing power Acetyl- CoA Acetyl- CoA x 2 CO2 CO2 3b group and releases CO2 Incorporates an acetyl TCA cycle (TCA cycles twice) Inner mitochondrial membrane Yields phosphorylation by substrate-level ATP Reducing power 4 power to proton motive force Uses the electron transport Respiration chain to convert reducing Yields phosphorylation by oxidative ATP P P P Electron Transport Chain Use of Proton Motive Force Proton motive force is used to drive: ATP synthase (ATP synthesis) Complex I Complex III Complex IV 4 H+ 4 H+ 2 H+ 10 H+ Intermembrane space Ubiquinone Cytochrome c Path of electrons 2 e– Mitochondrial matrix 2 H+ 1/2 O2 Complex II NADH Terminal electron acceptor + H2O NAD+ H+ 3 ATP + 3 Pi 3 ADP

Electron Transport Chain of Prokaryotes Tremendous variation: even single species can have several alternate carriers E. coli serves as example of versatility of prokaryotes Aerobic respiration in E. coli Can use 2 different NADH dehydrogenases Proton pump equivalent to complex I of mitochondria Can produce several alternatives to optimally use different energy sources, including H2 Lack equivalents of complex III or cytochrome c Quinones shuttle electrons directly to ubiquinol oxidase, a terminal oxidoreductase Two versions for high or low O2 concentrations

Electron Transport Chain of Prokaryotes (cont…) Anaerobic respiration in E. coli Harvests less energy than aerobic respiration Lower electron affinities of terminal electron acceptors Some components different Can synthesize terminal oxidoreductase that uses nitrate as terminal electron acceptor Produces nitrite E. coli converts to less toxic ammonia Sulfate-reducers use sulfate (SO42–) as terminal electron acceptor Produce hydrogen sulfide as end product

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Prokaryotic cell Cytoplasmic membrane Electron Transport Chain Uses of Proton Motive Force ATP synthase (ATP synthesis) Active transport (one mechanism) Rotation of a flagella NADH dehydrogenase Ubiquinol oxidase v e f o r c e r i v e : H+ (0 or 4) H+ (2 or 4) 10 H+ H+ H+ Proton motive force is used to drive: Outside of cytoplasmic membrane Transported molecule Ubiquinone Path of electrons 2 e– – Cytoplasm 2 H+ 1/2 O2 Succinate dehydrogenase NADH Terminal electron acceptor + NAD+ H2O H+ 3 ATP + 3 Pi 3 ADP

ATP Synthase—Harvesting the Proton Motive Force to Synthesize ATP Energy required to establish gradient Released when gradient is eased ATP synthase allows protons to flow down gradient in controlled manner Uses Proton energy to add phosphate group to ADP 1 ATP formed from entry of ~3 protons Calculating yields Based on experiments on rat mitochondria: ~2.5 ATP made per electron pair from NADH ~1.5 ATP made per electron pair from FADH2

Calculating theoretical maximum yields In prokaryotes: Glycolysis: 2 NADH 6 ATP Transition step: 2 NADH  6 ATP TCA Cycle: 6 NADH  18 ATP; 2 FADH2  4 ATP Total maximum oxidative phosphorylation yield = 34 ATP Slightly less in eukaryotic cells NADH from glycolysis in cytoplasm transported across mitochondrial membrane to enter electron transport chain Requires ~1 ATP per NADH generated

ATP Yield of Aerobic Respiration in Prokaryotes Substrate-level phosphorylation: 2 ATP (from glycolysis; net gain) 2 ATP (from the TCA cycle) 4 ATP (total) Oxidative phosphorylation: 6 ATP (from reducing power gained in glycolysis) 6 ATP (from reducing power gained in transition step) 22 ATP (from reducing power gained in TCA cycle) 34 (total) Total ATP gain (theoretical maximum) = 38

ATP Yield of Aerobic Respiration in Prokaryotes
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. GLUCOSE 2 Starts the oxidation of glucose Pentose phosphate pathway 1 Oxidizes glucose to pyruvate Glycolysis Yields phosphorylation by substrate-level ATP Reducing power GLUCOSE Glycolysis Oxidizes glucose to pyruvate Yields power Reducing Biosynthesis 5 Reduces pyruvate or a derivative Fermentation ~ ~ Pyruvate Pyruvate 3a Transition step Acids, alcohols, and gases Yield 2 ATP Reducing power CO2 CO2 Acetyl- CoA CoA Acetyl- net gain = 0 x 2 CO2 ~ ~ CO2 3b group and releases CO2 (TCA cycles twice) Incorporates an acetyl TCA cycle 2 ATP Yields phosphorylation by substrate-level ATP power Reducing 4 power to proton motive force chain to convert reducing Uses the electron transport Respiration Yields phosphorylation by oxidative ATP 2 NADH ~ ~ Oxidative phosphorylation 6 ATP ~ ~ Substrate-level phosphorylation 2 ATP Pyruvate Pyruvate 2 NADH ~ ~ Oxidative phosphorylation 6 ATP Acetyl- CoA Acetyl- CoA 6 NADH ~ ~ Oxidative phosphorylation x 2 CO2 18 ATP 2 FADH2 ~ ~ Oxidative phosphorylation CO2 4 ATP TCA cycle Incorporates an acetyl group and releases CO2 (TCA cycles twice) ~ ~ Substrate-level phosphorylation 2 ATP

6.5. Fermentation Fermentation used when respiration not an option
E. coli is facultative anaerobe Aerobic respiration, anaerobic respiration, and fermentation Streptococcus pneumoniae lacks electron transport chain Fermentation only option ATP-generating reactions are only those of glycolysis Additional steps consume excess reducing power Regenerate NAD+ GLUCOSE 2 Starts the oxidation of glucose Pentose phosphate pathway 1 Oxidizes glucose to pyruvate Glycolysis Yields phosphorylation by substrate-level ATP P ~ P ~ P + power Reducing Yields power Reducing Biosynthesis 5 Reduces pyruvate or a derivative Fermentation Pyruvate Pyruvate 3a Transition step Acids, alcohols, and gases Yields Reducing power CO2 CO2 Acetyl- CoA CoA Acetyl- x 2 CO2 3b CO2 group and releases CO2 Incorporates an acetyl (TCA cycles twice) TCA cycle Yields phosphorylation by substrate-level ATP P ~ P ~ P + power Reducing 4 power to proton motive force chain to convert reducing Uses the electron transport Respiration Yields phosphorylation by oxidative ATP P ~ P ~ P NADH + H+ NAD+ O O OH O H3C C C O– H3C C C O– H Pyruvate Lactate (a) Lactic acid fermentation CO2 NADH + H+ NAD+ O O O OH H3C C C O– H3C C H H3C C H H Pyruvate Acetaldehyde Ethanol (b) Ethanol fermentation

6.5. Fermentation Fermentation end products varied; helpful in identification, commercially useful Ethanol Butyric acid Propionic acid 2,3-Butanediol Mixed acids Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Pyruvate Fermentation pathway Lactic acid Ethanol Butyric acid Propionic acid Mixed acids 2,3-Butanediol Microorganisms Streptococcus Lactobacillus Saccharomyces Clostridium Propionibacterium E. coli Enterobacter End products Lactic acid Ethanol Butyric acid Butanol Acetone Isopropanol Propionic acid Acetic acid Acetic acid Lactic acid Succinic acid Ethanol CO2 CO2 CO2 CO2 CO2 H2 H2 H2 (yogurt, dairy, pickle), b (wine, beer), (acetone): © Brian Moeskau/McGraw- Hill; (cheese): © Photodisc/McGraw-Hill; (Voges-Proskauer Test), (Methyl-Red Test): © The McGraw-Hill Companies, Inc./Auburn University Photographic Services

6.6. Catabolism of Organic Compounds Other than Glucose
Microbes can use variety of compounds Excrete hydrolytic enzymes; transport subunits into cell Degrade further to appropriate precursor metabolites Polysaccharides and disaccharides Amylases digest starch; cellulases digest cellulose Disaccharides hydrolyzed by specific disaccharidases Lipids Fats hydrolyzed by lipases; glycerol converted to dihydroxyacetone phosphate, enters glycolysis Fatty acids degraded by β-oxidation to enter TCA cycle Proteins Hydrolyzed by proteases; amino group deaminated Carbon skeletons converted into precursor molecules

6.6. Catabolism of Organic Compounds Other than Glucose
Microbes can use variety of compounds (cont…) Convert to precursor metabolites Enter appropriate metabolic pathways POLYSACCHARIDES Starch Cellulose DISACCHARIDES Lactose Maltose Sucrose LIPIDS (fats) PROTEINS lipases proteases amylases cellulases disaccharidases glycerol Amino acids + deamination monosaccharides (simple sugars) GLUCOSE fatty acids NH3 Pentose phosphate pathway Glycolysis Applies to both branches In glycolysis ß-oxidation removes 2-carbon units. Pyruvate Pyruvate Acetyl- CoA Acetyl- CoA X 2 TCA cycle

6.7. Chemolithotrophs Prokaryotes unique in ability to use reduced inorganic compounds as sources of energy E.g., hydrogen sulfide (H2S), ammonia (NH3) Produced by anaerobic respiration from inorganic molecules (sulfate, nitrate) serving as terminal electron acceptors Important example of nutrient cycling Four general groups

6.8. Photosynthesis Photosynthesis
Plants, algae, several groups of bacteria General reaction is where X indicates element such as oxygen or sulfur Can be considered in two distinct stages Light reactions (light-dependent reactions) Capture energy and convert it to ATP Light-independent reactions (dark reactions) Use ATP to synthesize organic compounds Involves carbon fixation Light Energy 6 CO H2X C6H12O X + 6 H2O

6.8. Photosynthesis Photosynthesis (continued…)
Many variations found in approaches Oxygenic and anoxygenic

6.8. Photosynthesis Capturing Radiant Energy
Colors observed are those of wavelength reflected Pigments are located in photosystems within membranes Chlorophylls (plants, algae, cyanobacteria) Bacteriochlorophylls (anoxygenic bacteria) Absorb different wavelengths than chlorophylls Accessory pigments absorb at additional wavelengths Carotenoids (many photosynthetic prokaryotes and eukaryotes) Phycobilins (cyanobacteria, red algae) Antennae pigments form complex Funnel energy to reaction-center pigment

6.8. Photosynthesis Reaction-center pigments
Donate excited electrons to electron transport chain Chlorophyll a (plants, algae, cyanobacteria) Bacteriochlorophylls (anoxygenic bacteria) Cyanobacteria: photosystems in membranes of stacked structures inside cell termed thylakoids Plants, algae: thylakoids in stroma of chloroplast Endosymbiotic theory explains Purple bacteria: in cytoplasmic membrane, extensive infoldings Green bacteria: specialized chlorosomes attached to cytoplasmic membrane Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Photosystem Electron transport chain Radiant energy Reaction-center chlorophyll e– Chlorophyll molecule Photosynthetic membrane

Converting Radiant Energy into Chemical Energy
Light-dependent reactions in cyanobacteria and photosynthetic eukaryotes Two distinct photosystems (I and II) Cyclic photophosphorylation Photosystem I alone produces ATP Reaction-center chlorophyll is terminal electron acceptor Non-cyclic photophosphorylation Used when cells need both ATP and reducing power Electrons from photosystem II drive photophosphorylation Are then donated to photosystem I Photosystem II replenishes electrons by splitting water Generates oxygen (process is oxygenic) Electrons from photosystem I reduce NADP+ to NADPH

Excited chlorophyll Electron carrier Proton gradient formed for ATP synthesis e– Excited chlorophyll NADP reductase Electron carrier H+ e– NADPH e– NADP+ Reaction- center chlorophyll P r o t o n p u m p Electron carrier Reaction- center chlorophyll Energy of electrons e- Radiant energy Water-splitting enzyme Radiant energy 2 Z H2O e– 4 H + O2 Photosystem II Proton pump Photosystem I NADP reductase

Light-dependent reactions in anoxygenic photosynthetic bacteria Each has single photosystem Cannot use water as electron donor, so anoxygenic Use electron donors such as hydrogen gas (H2), hydrogen sulfide (H2S), organic compounds Purple bacteria: photosystem similar to photosystem II Energy of electrons insufficient to reduce NAD+ Instead expend ATP to use reversed electron transport Green bacteria: photosystem similar to photosystem I Electrons can generate proton motive force or reduce NAD+

6.9. Carbon Fixation Chemolithoautotrophs, photoautotrophs use CO2 to synthesize organic compounds: carbon fixation In photosynthetic organisms: light-independent reactions Consumes lots of ATP, reducing power Reverse process of oxidizing compounds to CO2 liberates a lot of energy! Calvin cycle most commonly used Three essential stages Incorporation of CO2 into organic compounds Reduction of resulting molecule Regeneration of starting compound Six “turns” of cycle: net gain of one fructose-6-phosphate Consumes 18 ATP, 12 NADPH per fructose molecule

ribulose 1,5-bisphosphate 1,3-bisphosphoglycerate
6.9. Carbon Fixation Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 6 CO2 1 1 Carbon dioxide is added to ribulose 1,5-bisphosphate to start a new round of the cycle. 12 molecules 3-phosphoglycerate 6 molecules ribulose 1,5-bisphosphate ~ ~ 12 ATP STAGE 1 ~ 12 ADP 12 molecules 1,3-bisphosphoglycerate ~ STAGE 3 6 ADP STAGE 2 ~ ~ 12 NADPH + H+ 6 ATP 6 molecules ribulose 5-phosphate 12 molecules glyceraldehyde 3-phosphate 12 NADP+ Series of complex reactions 12 Pi 3 3 Ribulose 1,5-bisphosphate is regenerated so that the cycle Can continue. 2 2 ATP and NADPH are used to reduce the product of stage1, producing glyceraldehyde 3-phosphate, which can be used in biosynthesis. 1 molecule fructose 6-phosphate Cell components

6.10. Anabolic Pathways—Synthesizing Subunits from Precursor Molecules
Prokaryotes remarkably similar in biosynthesis Synthesize subunits using central metabolic pathways If enzymes lacking, end product must be supplied Fastidious bacteria require many growth factors Lipid synthesis requires fatty acids, glycerol Fatty acids: 2-carbon units added to acetyl group from acetyl-CoA Glycerol: dihydroxyacetone phosphate from glycolysis Nucleotide synthesis DNA, RNA initially synthesized as ribonucleotides Purines: atoms added to ribose 5-phosphate to form ring Pyrimidines: ring made, then attached to ribose 5-phosphate Can be converted to other nucleobases of same type

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Pentose phosphate pathway Ribose 5-phosphate Erythrose 5-phosphate Nucleotides amino acids (histidine) Amino acids (phenylalanine, tryptophan, tyrosine) Lipids (glycerol component) (cysteine, glycine, serine) tryptophan, tyrosine) (aspartate, asparagine, isoleucine, lysine, methionine, threonine) TCA cycle (arginine, glutamate, glutamine, proline) (fatty acids) (alanine, leucine, valine) Peptidoglycan Lipopolysaccharide (polysaccharide) Glucose 6-phosphate Fructose 6-phosphate Dihydroxyacetone phosphate 3-phosphoglycerate Phosphoenolpyruvate Pyruvate Acetyl-CoA Oxaloacetate - ketoglutarate Glycolysis X 2

Amino Acid Synthesis Synthesis of glutamate provides mechanism for incorporation of nitrogen into organic material Ammonium (NH4+) commonly used via glutamate synthesis Transamination can generate other amino acids NH2 α-ketoglutarate NH3 (ammonia) Glutamate is synthesized by adding ammonia to the precursor metabolite α-ketoglutarate. Aspartate NH2 Oxaloacetate Glutamate The amino group (NH2) of glutamate can be transferred to other carbon compounds to produce other amino acids.

Amino Acid Synthesis (continued…) Aromatic amino acids: carefully regulated branch points Tryptophan is feedback inhibitor of enzyme that directs branch to its synthesis Pathway instead leads to tyrosine, phenylalanine Tyrosine, phenylalanine likewise inhibit first enzyme of branch leading to their synthesis The three amino acids also regulate formation of original 7-carbon compound (three different enzymes catalyze) From glycolysis Phenylalanine Compound a Branch point II 3-C 7-C compound Branch point I + Tyrosine 4-C Compound b Tryptophan From pentose phosphate pathway

Chapter 06 *Lecture Outline

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Chapter 06 *Lecture Outline

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