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LECTURE PRESENTATIONS For CAMPBELL BIOLOGY, NINTH EDITION Jane B. Reece, Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, Robert B. Jackson Chapter 9 Cellular Respiration and Fermentation Lectures by Erin Barley Kathleen Fitzpatrick © 2011 Pearson Education, Inc. Overview: Life Is Work • living cells require energy from outside sources • some animals obtain energy by eating plants • some animals feed on other organisms that eat plants • energy flows into an ecosystem as sunlight and leaves as heat • photosynthesis generates O2 and organic molecules - used in cellular respiration • cells use chemical energy stored in organic molecules to regenerate ATP - powers work Light energy ECOSYSTEM CO2 H2O Photosynthesis in chloroplasts Organic O 2 molecules Cellular respiration in mitochondria ATP ATP powers most cellular work Heat energy Catabolic Pathways and Production of ATP • catabolism - the breakdown of organic molecules is exergonic – potential energy is stored in chemical bonds – result of the arrangement of electrons in these bonds – breaking of these bonds releases this potential energy • catabolic processes – anaerobic, fermentation & aerobic • Fermentation = partial degradation of sugars that occurs without O2 • Aerobic respiration consumes organic molecules (fuel) and O2 to yield ATP – most prevalent and efficient catabolic process – carried out by eukaryotic cells and most prokaryotic • Anaerobic respiration - similar to aerobic respiration but consumes compounds other than O2 – prokaryotic cells • Cellular respiration includes both aerobic and anaerobic respiration – glycolysis, Kreb’s cycle and electron transport chain – is often used to refer to aerobic respiration • uses carbohydrates, fats, and proteins • most cellular respiration starts with the sugar glucose C6H12O6 + 6 O2 6 CO2 + 6 H2O + Energy (ATP + heat) • breakdown of glucose is exergonic – ΔG = -686 kcal/mol • creates ATP – powers the work of endergonic processes Redox Reactions: Oxidation and Reduction • How do catabolic pathways decompose glucose and yield energy • through the transfer of electrons = oxidationreduction reactions or Redox reactions – relocation of electrons releases stored potential energy • this released energy is ultimately used to synthesize ATP The Principle of Redox • redox reactions = chemical reactions that transfer electrons between reactants • oxidation = a substance loses electrons, or is oxidized • reduction = a substance gains electrons, or is reduced (the amount of positive charge is reduced) becomes oxidized becomes reduced becomes oxidized (loses electron) becomes reduced (gains electron) • the electron donor is called the reducing agent – e.g. Xe- = reduces Y • the electron receptor is called the oxidizing agent – e.g. Y = oxidizes Xe by removing its electrons Reactants becomes oxidized Products Energy becomes reduced Methane (reducing agent) Oxygen (oxidizing agent) Carbon dioxide Water • the problem becomes when you have covalent bonds • i.e. not all redox reactions involve the complete transfer of electrons from one substance to another – but change the degree of electron sharing in covalent bonds • an example is the reaction between methane and O2 – – – – in methane – covalent electrons are shared equally between the C and the Hs of methane and the two Os of O2 - they are equally electronegative BUT - O is a very electronegative element – one of the most potent oxidizing agents when CO2 is formed - electrons are shared less equally between C and O – the O “pulls” on the electrons harder – so the carbon has lost electrons (becomes oxidized) the electrons are also shared less equally in the water – O has gained electrons & become reduced Reactants becomes oxidized Products Energy becomes reduced Methane (reducing agent) Oxygen (oxidizing agent) Carbon dioxide Water • energy must be added to pull an electron away from an atom • the more electronegative an atom is – the more energy will be required for it to become oxidized – like rolling a ball uphill – methane is not very electronegative – little energy is required for it to become oxidized • an electron loses potential energy as it shifts from a less electronegative atom to a more electronegative atom – just as a ball loses potential energy when it rolls downhill – it releases this loss of potential energy as chemical energy - used for work • an electron loses potential energy as it shifts from a less electronegative atom to a more electronegative atom • a redox reaction that moves electrons closer to oxygen – releases chemical energy – e.g. form CO2 from methane – electrons move from the Carbon (when in methane) closer to the Oxygen of CO2 = ENERGY Oxidation of Organic Fuel Molecules During Cellular Respiration • During cellular respiration, the fuel (such as glucose) is oxidized, and O2 is reduced becomes oxidized becomes reduced • the carbon atoms of glucose are not very electronegative • lose their potential energy when they combine with O2 to form CO2 – move away from C and closer to O (more electronegative) • in general – molecules with carbons are good sources of potential energy – electrons will shift toward more electronegative atoms like oxygen Oxidation of Organic Fuel Molecules During Cellular Respiration becomes oxidized becomes reduced • in respiration – the oxidation of glucose (loss of electrons) transfers electrons to a lower energy state (lower G) – as electrons move from a less electronegative compound (sugar) to a more electronegative compound (CO2) – this liberates energy – used to make ATP • only the activation energy barrier prevents the transfer of electrons to the lower energy state • body temperature is not high enough to reach this activation energy barrier – done by enzymes • main sources of ATP in cells – carbohydrates and fats Stepwise Energy Harvest via NAD+ and the Electron Transport Chain • so oxidizing glucose produces more electronegative compounds and releases energy • if you release this energy all at once – not very efficient • in cellular respiration - glucose and other organic molecules are broken down to these electronegative compounds in a series of steps – at key steps – electrons are stripped from glucose and eventually transferred to O2 – in typical oxidation reactions - electrons that are transferred travel with a proton (H+) NAD+ • BUT electrons are not transferred directly – first transferred to NAD+ - functions as a coenzyme • NAD = nicotinamine adenine dinucleotide • as an electron acceptor- NAD+ functions as an oxidizing agent during cellular respiration • each NADH (the reduced form of NAD+) represents stored energy that is tapped to synthesize ATP NAD NADH Dehydrogenase Reduction of NAD (from food) Nicotinamide (oxidized form) Oxidation of NADH Nicotinamide (reduced form) • How does NAD+ trap electrons? • transfer is dependent upon a kind of enzyme called a Dehydrogenase • dehydrogenases remove a pair of hydrogen atoms and a pair of electrons • ACTUALLY – dehydrogenase removes a hydride ion/H- (two electrons and a proton) and a proton (H+) Figure 9.UN04 NAD NADH Dehydrogenase Reduction of NAD (from food) Oxidation of NADH Nicotinamide (oxidized form) Nicotinamide (reduced form) • one electron gets transferred onto the N+ of NAD N • the second electron gets attached to the C4 carbon directly across from the N • this is REDUCTION and produces a reduced form of NAD (NAD-) • one proton attaches to the nicotinamide ring NADH • the other is released into the surroundings Dehydrogenase • the electrons lose very little of their potential energy when they are transferred to NAD+ • each NADH represents stored energy that can be tapped to make ATP when the electrons “fall” down an energy gradient to oxygen 2H 1/ 2 O2 1/ 2 O2 (from food via NADH) Explosive release of heat and light energy Free energy, G 2 H+ 2 e Free energy, G • cellular respiration uses an Electron Transport Chain to break this fall into several energy releasing steps • NADH shuttles the electrons to the top of the hill • O2 captures these electrons along with H+ to form water H2 1/2 O2 Controlled release of energy for synthesis of ATP ATP ATP ATP 2 e 2 H+ H2O (a) Uncontrolled reaction H2O (b) Cellular respiration • electron transfer from NADH to O2 = -53 kcal/mol • use of several redox reactions to allow electrons to fall from one carrier to another 2H 1/ 2 O2 1/ 2 O2 (from food via NADH) Explosive release of heat and light energy Free energy, G 2 H+ 2 e Free energy, G • each “downhill” carrier is more electronegative than its “uphill” carrier & capable of oxidizing it • electrons eventually fall down the ETC to a far more stable location in an oxygen atom H2 1/2 O2 Controlled release of energy for synthesis of ATP ATP ATP ATP 2 e 2 H+ H2O (a) Uncontrolled reaction H2O (b) Cellular respiration BioFlix: Cellular Respiration © 2011 Pearson Education, Inc. The Stages of Cellular Respiration • Harvesting of energy from glucose has three stages: – Glycolysis –anaerobic process that breaks down glucose into two molecules of pyruvate • cytosol – the citric acid cycle (Kreb’s Cycle) - completes the breakdown of glucose • starting material = acetyl CoA (derived from pyruvate) • matrix of the mitochondria (cytosol in prokaryotes) – Oxidative phosphorylation = ETC and chemiosmosis • generates most of the ATP • powered by redox reactions • inner mitochondrial membrane (plasma membrane in prokaryotes) Electrons carried via NADH and FADH2 Electrons carried via NADH Glycolysis Glucose Pyruvate CYTOSOL Pyruvate oxidation Acetyl CoA Citric acid cycle Oxidative phosphorylation: electron transport and chemiosmosis MITOCHONDRION ATP ATP ATP Substrate-level phosphorylation Substrate-level phosphorylation Oxidative phosphorylation • oxidative phosphorylation accounts for almost 90% of the ATP generated by cellular respiration • a smaller amount of ATP is formed in glycolysis and the citric acid cycle by substrate-level phosphorylation • ALSO = some of the steps of Glycolysis and Citric Acid Cycle are redox reactions involving dehydrogenases removing protons and electrons and creating NADH • for each molecule of glucose degraded to CO2 and water by respiration- the cell makes up to 32 molecules of ATP Enzyme Enzyme ADP P Substrate ATP Product Glycolysis harvests chemical energy by oxidizing glucose to pyruvate Energy Investment Phase • Glycolysis (“splitting of sugar”) breaks down glucose into two molecules of pyruvate • Glycolysis has two major phases – Energy investment phase – Energy payoff phase • Glycolysis occurs whether or not O2 is present Glucose 2 ADP 2 P 2 ATP used Energy Payoff Phase 4 ADP 4 P 2 NAD+ 4 e 4 H+ 4 ATP formed 2 NADH 2 H+ 2 Pyruvate 2 H2O Net Glucose 4 ATP formed 2 ATP used 2 NAD+ 4 e 4 H+ 2 Pyruvate 2 H2O 2 ATP 2 NADH 2 H+ Figure 9.9-1 Glycolysis: Energy Investment Phase Glucose ATP Glucose 6-phosphate ADP Hexokinase 1 1. Hexokinase – transfers a phosphate group from ATP to glucose glucose-6-phosphate - makes the glucose it more chemically reactive Figure 9.9-2 Glycolysis: Energy Investment Phase Glucose ATP Glucose 6-phosphate Fructose 6-phosphate ADP Hexokinase 1 Phosphoglucoisomerase 2 2. Phosphoglucoisomerase – enzyme that catalyzes the rearrangement of G6P into fructose-6-phosphate Figure 9.9-3 Glycolysis: Energy Investment Phase Glucose ATP Glucose 6-phosphate Fructose 6-phosphate ATP Fructose 1,6-bisphosphate ADP ADP Hexokinase 1 Phosphoglucoisomerase Phosphofructokinase 2 3 3. Phosphofructokinase – transfers a phosphate group from ATP to the opposite end of F6P fructose-1,6-bisphosphate -phosphofructokinase is the rate-limiting enzyme for the glycolytic portion of cellular respiration Figure 9.9-3 Glycolysis: Energy Investment Phase Glucose ATP Glucose 6-phosphate Fructose 6-phosphate ATP Fructose 1,6-bisphosphate ADP ADP Hexokinase 1 Phosphoglucoisomerase Phosphofructokinase 2 3 -phosphofructokinase catalyzes an irreversible step in glycolysis -same is true for hexokinase and pyruvate kinase -its activity is regulated by the reversible binding of an allosteric effector -inhibited by high levels of ATP – acts to lower its affinity for fructose-6-phosphate -ATP binds to the enzyme at a regulatory site – changes the shape of the enzyme’s catalytic site -reduced binding to F6P results -enzyme can also can be inhibited through a drop in pH Figure 9.9-4 Glycolysis: Energy Investment Phase Glucose ATP Glucose 6-phosphate Fructose 6-phosphate ATP ADP ADP Hexokinase 1 Fructose 1,6-bisphosphate Phosphoglucoisomerase Phosphofructokinase 2 3 Aldolase Dihydroxyacetone phosphate 4 Glyceraldehyde 3-phosphate Isomerase 5 4. Aldolase – cleaves the 6 carbon F-1,6-BP into two 3-Carbon sugars dihydroxyacetone phosphate and glyceraldehyde-3phosphate - are isomers of each other -DHAP is a aldose, G3P is a ketose -if this could reach equilibrium – 96% would be DHAP -BUT G3P is used as soon as it is formed – never reaches Eq. To step 6 Figure 9.9-4 Glycolysis: Energy Investment Phase Glucose ATP Glucose 6-phosphate Fructose 6-phosphate ATP ADP ADP Hexokinase 1 Fructose 1,6-bisphosphate Phosphoglucoisomerase Phosphofructokinase 2 3 Aldolase Dihydroxyacetone phosphate 4 Glyceraldehyde 3-phosphate Isomerase 5 To step 6 5. Triose phosphate Isomerase – converts DAHP G3P immediately after G3P is used -G3P forms the main glycolytic pathway so the isomerase funnels DHAP into this pathway by converting it into G3P -the following reactions actually run twice!!! (two molecules of G3P are produced) Figure 9.9-5 Glycolysis: Energy Payoff Phase 2 NADH 2 NAD Triose phosphate dehydrogenase 6 + 2 H 2Pi 6. Triose phosphate Dehydrogenase -also known as Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) -first step: removes 2 electrons and 2 protons from 1,3-Bisphosphoglycerate G3P and transfers them to NAD+ to make NADH -so G3P is oxidized and NAD+ is reduced -transfer of electrons to a more electronegative compound (NAD+) releases energy -second step: energy released by this redox reaction is used for phosphorylation of G3P 1,3bisphosphoglycerate Figure 9.9-6 Glycolysis: Energy Payoff Phase 2 ATP 2 NADH 2 NAD Triose phosphate dehydrogenase 6 + 2 H 2 ADP 2 Phosphoglycerokinase 2Pi 1,3-Bisphosphoglycerate 7 3-Phosphoglycerate 7. Phosphoglycerokinase: – 1,3-BPG is unstable and has a high energy potential -allows for the transfer of the phosphate group from C1 to ADP ATP is made via Substrate level Phosphorylation -1,3-BPG is oxidized 3-phosphoglycerate (when the phosphate group is bound to 1,3-BPG - one electron is pulled closer to the O of the sugar – O is more electronegative than P – when the P group is released, it takes it’s electron & the sugar loses it – i.e. oxidized) Figure 9.9-7 Glycolysis: Energy Payoff Phase 2 ATP 2 NADH 2 NAD Triose phosphate dehydrogenase 6 + 2 H 2 ADP 2 2 Phosphoglyceromutase Phosphoglycerokinase 2Pi 1,3-Bisphosphoglycerate 7 3-Phosphoglycerate 8 2-Phosphoglycerate 8. Phosphoglyceromutase: transfer of the remaining phosphate group from C3 to C2 2-phosphoglycerate Figure 9.9-8 Glycolysis: Energy Payoff Phase 2 ATP 2 H2O 2 NADH 2 NAD Triose phosphate dehydrogenase 6 + 2 H 2 ADP 2 2 1,3-Bisphosphoglycerate 7 Enolase Phosphoglyceromutase Phosphoglycerokinase 2Pi 2 9 3-Phosphoglycerate 8 2-Phosphoglycerate Phosphoenolpyruvate (PEP) 9. Enolase: extracts a water molecule from the sugar and forms a double bond between C2 and C3 phosphoenolpyruvate (PEP) Figure 9.9-9 Glycolysis: Energy Payoff Phase 2 ATP 2 ATP 2 H2O 2 NADH 2 NAD Triose phosphate dehydrogenase 6 + 2 H 2 ADP 2 2 1,3-Bisphosphoglycerate 7 Enolase Phosphoglyceromutase Phosphoglycerokinase 2Pi 9 3-Phosphoglycerate 8 2 ADP 2 2-Phosphoglycerate Pyruvate kinase Phosphoenolpyruvate (PEP) 10 Pyruvate 10. Pyrvuate kinase: PEP is unstable and has a high energy potential -allows the transfer of the remaining P group to ADP for the second round of Substrate Level Phosphorylation ATP is made -this final step of glycolysis produces Pyruvate Glycolysis Energy Tally • 2 NADH from glycolysis = 4 ATP from ETC • 4 ATP made through substrate phosphorylation • 2 ATP consumed After pyruvate is oxidized, the citric acid cycle completes the energy-yielding oxidation of organic molecules • glycolysis releases less than a quarter of the chemical energy in glucose • most energy is still contained in pyruvate • oxidation of pyruvate releases the remaining energy • in eukaryotes and in the presence of O2- pyruvate enters the mitochondrion - the oxidation of glucose is completed • takes place in the cytosol in prokaryotes Transition Phase: Oxidation of Pyruvate to Acetyl CoA • before the citric acid cycle can begin- pyruvate must be converted to acetyl Coenzyme A (acetyl CoA) – links glycolysis to the citric acid cycle • this transition step is carried out by a multienzyme complex in the matrix that catalyzes three reactions = pyruvate dehydrogenase complex • requires a transport pump to bring pyruvate into the matrix of the mitochondria – powered by the H+ gradient of the electron transport chain 1. removal of the carboxyl group as a molecule of CO2 2 carbon compound 2. oxidation into acetate & transfer of electrons to NAD+ 3. coenzyme A attached via its sulfur atom acetyl coA MITOCHONDRION CYTOSOL CO2 Coenzyme A 3 1 2 Pyruvate Transport protein NAD NADH + H Acetyl CoA Transition Phase Energy Tally • 2 NADH from pyruvate oxidation = 6 ATP from ETC • • • • • 1. ethanol 2. lactate 3. acetyl coA 4. PEP glucose 5. amino acids • the production of lactate or ethanol is a reduction reaction and regenerates NAD+ this sustains glycolysis by replenshing NAD+ • The Citric Acid Cycle Pyruvate • citric acid cycle- also called the Krebs cycle • completes the break down of pyruvate to CO2 CO2 NAD CoA NADH + H Acetyl CoA CoA CoA – two more CO2 produced per pyruvate – 6 total (including 2 from transition) • generates 1 GTP/ATP, 3 NADH, and 1 FADH2 per turn • turns once for every pyruvate made • 2 pyruvates are made for each glucose Citric acid cycle 2 CO2 3 NAD FADH2 3 NADH FAD + 3 H ADP + P i ATP The Citric Acid Cycle • The citric acid cycle has eight steps, each catalyzed by a specific enzyme • two carbons (red) enter in the reduced form as an acetyl group • two different carbons (blue) leave as the oxidized form of CO2 • The acetyl group of acetyl CoA joins the cycle by combining with oxaloacetate, forming citrate • The next seven steps decompose the citrate back to oxaloacetate, making the process a cycle • The NADH and FADH2 produced by the cycle relay electrons extracted from food to the electron transport chain Acetyl CoA CoA-SH 1 Oxaloacetate Citrate Citric acid cycle 1. Addition of Acetyl coA to oxaloacetate to produce citrate -catalyzed by Citrate synthase or CoA Synthase Acetyl CoA CoA-SH H2O 1 Oxaloacetate 2 Citrate Isocitrate Citric acid cycle 2. Isomerization of citrate to iso-citrate: dehydration reaction, followed by a hydration step creates this isomer of citrate -intermediate step creates cis-Aconitate (unstable) Figure 9.12-3 Acetyl CoA CoA-SH H2O 1 Oxaloacetate 2 Citrate Isocitrate NAD Citric acid cycle 3 NADH + H CO2 -Ketoglutarate 3. Oxidation of isocitrate alpha-ketoglutarate: : 1st of the 4 redox reactions in the citric acid cycle -creates an unstable intermediate that loses a CO2 (oxidation) -coupled to the reduction of NAD+ to NADH - catalyzed by isocitrate dehydrogenase Acetyl CoA CoA-SH H2O 1 Oxaloacetate 2 4. Oxidation of -ketoglutarate succinyl coA: 2nd of the 4 redox reactions in the citric acid cycle -loss of another CO2 in the oxidation reaction -reduction of NAD+ to NADH -CoA is added through an unstable bond – ΔG is -8kcal/mol -catalyzed by -ketoglutarate dehydrogenase -3 enzyme complex -reactions similar to the creation of acetyl coA Citrate Isocitrate NAD Citric acid cycle NADH 3 + H CO2 CoA-SH -Ketoglutarate 4 NAD NADH Succinyl CoA + H CO2 Acetyl CoA CoA-SH 5. Conversion to succinate: the energy released by the cleavage of the CoA powers the phosphorylation of GDP GTP -the GTP can be used to generate another ATP through phosphate transfer -catalyzed by Succinyl CoA synthase (similar enzyme as that added CoA to oxaloacetate) H2O 1 Oxaloacetate 2 Citrate Isocitrate NAD Citric acid cycle NADH 3 + H CO2 CoA-SH -Ketoglutarate 4 CoA-SH 5 NAD Succinate GTP GDP CO2 NADH Pi Succinyl CoA + H ADP ATP -in other cells – ATP is made at this step -bacteria, plants & some animal cells Acetyl CoA CoA-SH 6 8: Oxidation of succinate to oxaloacetate: regeneration of oxaloacetate is done in three steps 6. Oxidation to Fumarate – reduction of a FAD FADH2 -FAD is used because the free energy change is not enough to reduce NAD+ 7. Hydration to Malate – addition of water rearranges fumarate into malate 8. Oxidation to oxaloacetate – coupled to reduction of another NAD+ NADH NADH + H H2O 1 NAD 8 Oxaloacetate 2 Malate Citrate Isocitrate NAD Citric acid cycle 7 H2O Fumarate NADH 3 + H CO2 CoA-SH -Ketoglutarate 4 6 CoA-SH 5 FADH2 NAD FAD Succinate GTP GDP ADP ATP Pi Succinyl CoA NADH + H CO2 Acetyl CoA CoA-SH NADH CITRIC ACID CYCLE TALLY -for each acetyl coA entering the cycle + H H2O 1 NAD 8 Oxaloacetate 2 Malate 1.3 NAD+ reduced to NADH (6 total = 18 ATP) 2.1 FAD reduced to FADH2 (2 total = 4 ATP) 3.production of 1 GTP (2 total = 2 ATP in some cells) 4.generation of 2 CO2 (4 total) Citrate Isocitrate NAD Citric acid cycle 7 H2O Fumarate NADH 3 + H CO2 CoA-SH -Ketoglutarate 4 6 CoA-SH 5 FADH2 NAD FAD Succinate GTP GDP ADP ATP Pi Succinyl CoA NADH + H CO2 Cellular Respiration = Theoretical Energy • Glucose contains 686 kcal/mol • ATP contains 7.3 kcal/mol • 36 ATP harvested from each molecule of glucose • 2 from glycolysis – substrate level phosphorylation • 2 from Kreb’s cycle – converted from GTP (not done by all cells) • 32 from the ETC & NADH/FADH2 • 2 NADH – glycolysis • 8 NADH – transition + Kreb’s • 2 FADH2 – Kreb’s • note: NADH in the cytosol is only worth 2ATP; NADH in the mitochondria are worth 3 ATP • 36 moles of ATP x 7.3 kcal/mol = 263 kcal trapped as ATP • efficiency : 263/720 = 36% efficiency in converting the potential energy of glucose into chemical energy During oxidative phosphorylation, chemiosmosis couples electron transport to ATP synthesis • following glycolysis and the citric acid cycle - NADH and FADH2 account for most of the energy extracted from food • these two electron carriers donate their electrons to the electron transport chain - powers ATP synthesis via oxidative phosphorylation • donation of electrons to O2 powers the phosphorylation of ADP ATP • enzyme of the electron transport chain is in the inner mitochodrial membrane (cristae) • most of the chain’s components are multiprotein complexes associated with carriers – carriers alternate reduced and oxidized states as they accept and donate electrons • electrons drop in free energy as they go down the chain and are finally passed to O2, forming H2O – the release free energy is broken up into smaller steps through the development of separate protein complexes • the “fall” is spontaneous as the uphill complex has a higher G value vs. the downhill • as the electrons fall they reduce the next downhill complex • eventually O2 is reduced 50 2 e NAD FADH2 2 e Free energy (G) relative to O2 (kcal/mol) • the electron transport chain generates no ATP directly • ATP is generated as a result in the drop of free energy as the electrons “fall” from one protein complex to the next NADH 40 FMN FeS FeS II Q III Cyt b 30 Multiprotein complexes FAD I FeS Cyt c1 IV Cyt c Cyt a 20 10 0 Cyt a3 2 e (originally from NADH or FADH2) 2 H + 1/2 O2 H2O 50 2 e NAD FADH2 2 e Free energy (G) relative to O2 (kcal/mol) • the ETC couples electron transfer to the generation of a proton-motive force for ATP synthesis NADH 40 FMN FeS FeS II Q III Cyt b 30 Multiprotein complexes FAD I FeS Cyt c1 IV Cyt c Cyt a 20 10 0 Cyt a3 2 e (originally from NADH or FADH2) 2 H + 1/2 O2 H2O – flavoprotein • Complex II – Succinate dehydrogenase – adds additional electrons to the ETC from succinate via FADH2 • Complex III – Cytochrome bc1 – also know as cytochrome c reductase • Complex IV – Cytochrome c oxidase • complexes I, II and III have ironsulfur groups (heme groups) that are critical to their function NADH 50 2 e NAD FADH2 2 e Free energy (G) relative to O2 (kcal/mol) • four protein complexes coupled to specific prosthetic groups: • Complex I – NADH dehydrogenase 40 FMN FeS FeS II Q III Cyt b 30 Multiprotein complexes FAD I FeS Cyt c1 IV Cyt c Cyt a 20 10 0 Cyt a3 2 e (originally from NADH or FADH2) 2 H + 1/2 O2 H2O NADH 50 2 e NAD FADH2 2 e Free energy (G) relative to O2 (kcal/mol) • Complex I – NADH dehydrogenase – flavoprotein because one of its prosthetic groups is flavin mononucleotide (FMN) – 4H+ pumped into intermembrane space – 1. 2 electrons transferred to FMN FMNH2 (reduction in one step) – 2. Oxidation of FMNH2 (in 2 steps) QH2 (ubiquinol) • the two electrons moved via a FeS group Q (ubiquinone) • step A: 1st electron converts Q semiquinone • step B: 2nd electron converts semiquinone into ubiquinol (QH2) • QH2 is lipid soluble and diffuses through the membrane to Complex III 40 FMN FeS FeS II Q III Cyt b 30 Multiprotein complexes FAD I FeS Cyt c1 IV Cyt c Cyt a Cyt a3 20 2 e 10 (originally from NADH or FADH2) 2 H + 1/2 O2 0 QH2 carries 2 electrons H2O • Complex III – Cytochrome bc1 – transfers its electrons via Q-Cycle • 4H+ pumped as a result – QH2 binds to the Qo site on the complex – two electrons take two pathways: – path 1. one electron move to Fe-S group and then to a c1 site • electron then reduces the cytochrome c1 carrier and then a cytochrome c – path 2. 2nd electron moves to a cytochrome bL site a bH site –> the Qi site of the complex • a molecule of Q has bound the Qi site – accepts the electron from bH becomes the unstable semiquinone/Q- (not pictured) • ANOTHER CYCLE IS NEEDED – another QH2 binds the Qi site – 1st electron reduces the 2nd cyto c and the 2nd electron travels via bL/bH to reduce semiquinone to QH2 • this QH2 can cycle back to deliver its electrons to Qo – continuous cycle • so 2 cytochrome c’s are reduced and transferred to Complex IV This complex can “leak” electrons to O2 and create superoxide radicals (water-soluble & found in the intermembrane space) THIS PROTEIN COMPLEX NEEDS 2 MOLECULES OF QH2 TO RUN (4 electrons) -moves 4H+ NADH 50 2 e NAD FADH2 2 e Free energy (G) relative to O2 (kcal/mol) • Complex IV – Cytochrome c Oxidase – 4 molecules of cyto c deliver their electrons to this complex – 4 electrons are transferred to cyto a group cyto a3 group O2 to create two molecules of water – 2 H+ are pumped by this complex – water production: – 2e- + 2H+ + ½ O2 H20 – 4e- + 4H+ + O2 2H20 – this complex can be inhibited by cyanide 40 FMN FeS FeS II Q III Cyt b 30 Multiprotein complexes FAD I FeS Cyt c1 IV Cyt c Cyt a 20 10 0 Cyt a3 2 e (originally from NADH or FADH2) 2 H + 1/2 O2 H2O NADH 50 2 e NAD FADH2 2 e Free energy (G) relative to O2 (kcal/mol) • Complex II –Succinate Dehydrogenase – transfers the electrons produced through the reduction of FAD FADH2 – 2 electrons moved to Q to create QH2 40 FMN FeS FeS II Q III Cyt b 30 Multiprotein complexes FAD I FeS Cyt c1 IV Cyt c Cyt a 20 10 0 Cyt a3 2 e (originally from NADH or FADH2) 2 H + 1/2 O2 H2O Chemiosmosis: The Energy-Coupling Mechanism • Electron transfer in the electron transport chain causes proteins to pump H+ from the mitochondrial matrix to the intermembrane space • H+ then moves back across the membrane, passing through the proton, ATP synthase • ATP synthase uses the exergonic flow of H+ to drive phosphorylation of ATP • This is an example of chemiosmosis, the use of energy in a H+ gradient to drive cellular work • The energy stored in a H+ gradient across a membrane couples the redox reactions of the electron transport chain to ATP synthesis • The H+ gradient is referred to as a proton-motive force, emphasizing its capacity to do work H H H Protein complex of electron carriers Q I IV III II FADH2 NADH H Cyt c FAD 2 H + 1/2O2 NAD ATP synthase H2O ADP P i (carrying electrons from food) ATP H 1 Electron transport chain Oxidative phosphorylation 2 Chemiosmosis ATP-synthase – comprised of a: 1.stator – two “½ channels” for H+ binding 2.rotor – multiple H+ binding sites 3.internal rod 4.catalytic knob INTERMEMBRANE SPACE H Stator Rotor 1. H+ ion flowing down its gradient enters the 1st half-channel in the stator 2. H+ ions enter binding sites within the rotor -binding of the H+ ions changes the rotors Internal rod shape so it rotates 3. each H+ ion makes one complete turn Catalytic knob before leaving the rotor and passing through the second half-channel within the stator 4. spinning of the rotor, rotates the rod and then the ADP knob below it + 5. rotation of the knob activates catalytic sites that Pi add a P group to ADP ATP MITOCHONDRIAL MATRIX http://www.youtube.com/watch?v=3y1dO4nNaKY An Accounting of ATP Production by Cellular Respiration • During cellular respiration, most energy flows in this sequence: glucose NADH electron transport chain proton-motive force ATP • About 34% of the energy in a glucose molecule is transferred to ATP during cellular respiration, making about 32 ATP Electron shuttles span membrane 2 NADH Glycolysis 2 Pyruvate Glucose 2 NADH Pyruvate oxidation 2 Acetyl CoA 2 ATP Maximum per glucose: CYTOSOL MITOCHONDRION 2 NADH or 2 FADH2 6 NADH 2 FADH2 Citric acid cycle Oxidative phosphorylation: electron transport and chemiosmosis 2 ATP about 26 or 28 ATP About 30 or 32 ATP An Accounting of ATP Production by Cellular Respiration • There are several reasons why the number of ATP is not known exactly – 1. the ratio of NADH molecules to ATP is not a whole number – phosphorylation and redox are not directly coupled to each other – 2. NADH made in the cytosol cannot enter the mitochondria • must use a “shuttle” to take these electrons into the matrix • number of ATP made depends on whether FAD or NAD+ is used as that shuttle – 3. use of the proton-motive force can be used for other mitochondrial work • powers the uptake of pyruvate from the cytosol • if this motive force was used exclusively for oxidative phosphorylation = 30 to 32 ATP made Electron shuttles span membrane 2 NADH Glycolysis 2 Pyruvate Glucose 2 NADH Pyruvate oxidation 2 Acetyl CoA 2 ATP Maximum per glucose: CYTOSOL MITOCHONDRION 2 NADH or 2 FADH2 6 NADH 2 FADH2 Citric acid cycle Oxidative phosphorylation: electron transport and chemiosmosis 2 ATP about 26 or 28 ATP About 30 or 32 ATP Fermentation and anaerobic respiration enable cells to produce ATP without the use of oxygen • Most cellular respiration requires O2 to produce ATP • Without O2, the electron transport chain will cease to operate • In that case, glycolysis couples with fermentation or anaerobic respiration to produce ATP • Anaerobic respiration uses an electron transport chain with a final electron acceptor other than O2, for example sulfate • Fermentation uses substrate-level phosphorylation instead of an electron transport chain to generate ATP Animation: Fermentation Overview Right-click slide / select “Play” © 2011 Pearson Education, Inc. • Fermentation consists of glycolysis plus reactions that regenerate NAD+, which can be reused by glycolysis • Two common types are alcohol fermentation and lactic acid fermentation • in alcohol fermentation - pyruvate is converted to ethanol in two steps, with the first releasing CO2 – acetylaldehyde ethanol is catalyzed by Alcohol Dehydrogenase 1 – Alcohol fermentation by yeast is used in brewing, winemaking, and baking – humans can take the ethanol and convert it back into a sugar using Alcohol Dehydrogenase Acetylaldehyde 2 ADP 2 P Glucose i 2 ADP 2 P 2 ATP Glycolysis Glucose 2 ATP i Glycolysis 2 Pyruvate 2 NAD 2 Ethanol (a) Alcohol fermentation 2 NADH 2 H 2 NAD 2 CO2 2 Acetaldehyde 2 NADH 2 H 2 Lactate (b) Lactic acid fermentation 2 Pyruvate • in lactic acid fermentation - pyruvate is reduced to NADH, forming lactate as an end product, with no release of CO2 • Lactic acid fermentation by some fungi and bacteria is used to make cheese and yogurt • Human muscle cells use lactic acid fermentation to generate ATP when O2 is scarce 2 ADP 2 P Glucose i 2 ADP 2 P 2 ATP Glycolysis Glucose 2 ATP i Glycolysis 2 Pyruvate 2 NAD 2 Ethanol (a) Alcohol fermentation 2 NADH 2 H 2 NAD 2 CO2 2 Acetaldehyde 2 NADH 2 H 2 Lactate (b) Lactic acid fermentation 2 Pyruvate Comparing Fermentation with Anaerobic and Aerobic Respiration • all three pathways use glycolysis (net ATP = 2) to oxidize glucose and harvest chemical energy of food • in all three - NAD+ is the oxidizing agent that accepts electrons during glycolysis (i.e. that gets reduced) • BUT these processes have different final electron acceptors: – aerobic respiration – O2 – anaerobic respiration – other atoms – fermentation - pyruvate or acetaldehyde • Cellular respiration produces 32 ATP per glucose molecule; fermentation produces 2 ATP per glucose molecule • Obligate anaerobes carry out fermentation or anaerobic respiration and cannot survive in the presence of O2 • Yeast and many bacteria are facultative anaerobes, meaning that they can survive using either fermentation or cellular respiration • In a facultative anaerobe, pyruvate is a fork in the metabolic road that leads to two alternative catabolic routes Glucose CYTOSOL Glycolysis Pyruvate No O2 present: Fermentation Ethanol, lactate, or other products O2 present: Aerobic cellular respiration MITOCHONDRION Acetyl CoA Citric acid cycle Glycolysis and the citric acid cycle connect to many other metabolic pathways Proteins Amino acids Carbohydrates Sugars Fats GlycerolFatty acids Glycolysis Glucose Glyceraldehyde 3- P NH3 Pyruvate Acetyl CoA Citric acid cycle Oxidative phosphorylation Biosynthesis (Anabolic Pathways) • the body uses small molecules to build other substances • these small molecules may come directly from food, from glycolysis, or from the citric acid cycle • about 10 of the 20 amino acids can be made by converting substrates from the citric acid cycle – rest are called essential amino acids • glucose can be made from pyruvate – gluconeogenesis • fatty acids can be made from acetyl coA Regulation of Cellular Respiration via Feedback Mechanisms Glucose • feedback inhibition is the most common mechanism for control • if ATP concentration begins to drop, respiration speeds up • control of catabolism is based mainly on regulating the activity of enzymes at strategic points in the catabolic pathway • key point of regulation: – phosphofructokinase and ATP/AMP + citrate – hexokinase – pyruvate kinase Inhibits AMP Glycolysis Fructose 6-phosphate Stimulates Phosphofructokinase Fructose 1,6-bisphosphate Inhibits Pyruvate ATP Citrate Acetyl CoA Citric acid cycle Oxidative phosphorylation