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ATP Formation by Electron-Transport Chains Mitochondrial Electron-Transport Components of the Electron-Transport Chain Oxidative Phosphorylation Recycling of Cytoplasmic NADH Photosynthetic Electron-Transport Synthesis of Carbohydrates by the Calvin Cycle 1 Introduction Up to this point, we have dealt with • Oxidation of substrates. • Collection of electrons by cofactors. Energy from the cofactors is recovered using O2 as the final electron acceptor. This is accomplished using a series of carriers in the inner mitochondrial membrane . 2 Mitochondrial electron transport Stage I and II of carbohydrate catabolism converge at the mitochondria. Stage I Stage II citric acid cycle electron-transport oxidative phosphorylation 3 Mitochondrial electron transport • Extensive inner membrane folding in the mitochondria provides a large surface area. • There are many molecular systems on this membrane for production of ATP. • Electron-transport chain components are arranged in packages called respiratory assemblies. • There are also knob-like spheres called F1 particles. 4 Electron-transport and oxidative phosphorylation Electrons obtained from nutrients and metabolic intermediates are transferred to NAD+ and FAD. dehydrogenase AH2 + NAD+ BH2 + FAD A + NADH + H+ dehydrogenase B + FADH2 Since NAD+ and FAD are in limited supply, they must be recycled. 5 Electron-transport and oxidative phosphorylation Recycling is accomplished by oxidation and transfer of electrons to oxygen. ADP + Pi ATP NADH + H+ + 1/2 O2 ADP + Pi FADH2 + 1/2 O2 NAD+ + H2O ATP FAD + H2O NAD+ and FAD are then available for additional oxidative metabolism. The energy released during electron transport is coupled to ATP synthesis. 6 Electron-transport chain Composed of four large protein complexes. • Complex I - NADH-Coenzyme Q reductase • Complex II - Succinate-Coenzyme Q reductase • Complex III - Cytochrome c reductase • Complex IV - Cytochrome c oxidase Many of the components are integral membrane proteins with prosthetic groups to move electrons. 7 Electron-transport chain Two important characteristics of the electron-transport chain • order of electron carriers • quantity of energy produced Electron carriers are arranged in order of increasing electron affinity. This results in the spontaneous flow of electrons from carrier to carrier. 8 Flow of electrons 9 Energy produced The amount of energy can be calculated in terms of Go’ : NADH + H+ + 1/2 O2 NAD+ + H2O Go’ = - 220 kJ/mol FADH2 + 1/2 O2 FAD + H2O Go’ = - 152 kJ/mol Note: ADP + Pi ATP Go’ = +31 kJ/mol 10 Components of the electron transport chain Complex I • Electrons flow from NADH to flavin mononucleotide (FMN) - similar to FAD. • Electrons then flow to a prosthetic group on an iron-sulfur cluster - iron cycles between 3+ and 2+ states. • Complex I terminates at ubiquinone - also called coenzyme Q or CoQ. 11 Components of the electron transport chain 2 H+ Complex I QH2 2 one-electron FMNH2 2 one-electron Fe-S transfers transfers FMN Q 2 H+ NADH H+ NAD + 12 Components of the electron transport chain flavoprotein 13 Iron-sulfur clusters S Cys Fe Cys S Fe Cys S S S Cys Cys Fe S S S Fe S Cys Cys S S Fe S S Fe S Cys protein 14 Components of the electron transport chain CoQ - ubiquinone Highlighted region serves as an anchor to inner mitochondrial membrane. O H3CO CH3 CH3 H3CO (CH2 CH C CH2)10 H O 15 Reduction of CoQ Oxidized form Ubiquinone (CoQ) Reduced form Ubiquinol (CoQH2) O OH H3CO CH3 H3CO CH3 H3CO R H3CO R O O e- + OH H3CO CH3 H3CO R H+ OH intermediate,semiquinone e- + H+ 16 Components of the electron transport chain Complex II • Entry point for both FADH2 and Complex I. • Succinate dehydrogenase From the citric acid cycle. Directs transfer of electrons from succinate to CoQ via FADH2. • Acyl-CoA dehydrogenase From -oxidation of fatty acids. It also transfers electrons to CoQ via FADH2. Both enzymes have iron-sulfur clusters as prosthetic groups and are integral proteins. 17 Components of the electron transport chain All electrons from FADH2 and NADH must pass through CoQ. innermembrane space I Fe-S FMN II FAD NADH NAD + CoQ Fe-S FAD Succinate Fatty acyl CoA matrix 18 Components of the electron transport chain Complex III Electron transfer from ubiquinol to cytochrome c. cytochrome c heme prosthetic group 19 Components of the electron transport chain Protein S H3C CHCH 3 H3C CH3 N N Fe N H3C H3C Structure of cytochrome c heme group. N CH2CH2COO- CH2CH2COO20 Components of the electron transport chain Complex IV • Combination of cytochromes a and a3 cytochrome c oxidase. • Consists of 10 protein subunits, 2 types of prosthetic groups - 2 heme and 2 Cu. • Cytochromes a and a3 are the only species capable of direct transfer of electrons to oxygen. 21 Components of the electron transport chain Complex I CoQ Complex III cyt c1 cyt b NADH cyt c Complex IV (Cu) cyt a/a3 O 2 matrix 22 Oxidative phosphorylation • The electron-transport chain moves electrons from NADH and FADH2 to O2. • The next step is the phosphorylation of ADP to produce ATP. Catalyzed by the inner membrane enzyme ATP synthase. • The steps are coupled - electrons do not flow to oxygen unless ATP is needed. Each NADH produces 3 ATP Each FADH2 produces 2 ATP 23 Coupling of electron-transport with ATP synthesis Chemiosmotic coupling mechanism • Electron-transport causes unidirectional movement of H+ into the innermembrane space. • The results in a H+ gradient being produced. • The gradient then drives the synthesis of ATP. 24 Coupling of electron-transport with ATP synthase Outer mitochondrial membrane H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ Inner mitochondrial membrane H+ H + H+ Electron Transport Chain F1-ATP synthase complex H+ ADP + Pi ATP 25 Components of ATP synthase These are knob-like projections into the matrix side of the inner membrane. Two units • F1 contains the catalytic site for ATP synthesis. • F0 serves as a transmembrane channel for H+ flow. F1-F0 complex serves as the molecular apparatus for coupling H+ movement to ATP synthase. 26 Components of ATP synthase H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ F0 F 1 27 Regulation of oxidative phosphorylation • Electrons do not flow unless ADP is present for phosphorylation • Increased ADP levels cause an increase in catabolic reactions of various enzymes including: glycogen phosphorylase phosphofructokinase citrate synthase 28 Uncoupling of electron-transport and oxidative phosphorylation • In some special cases, the coupling of the two processes can be disrupted. • Large amounts of O2 are consumed but no ATP is produced. • Used by newborn animals and hibernating mammals. • Occurs in ‘brown fat’- dark color due to high levels of mitochondria which contain thermogenin (uncoupling protein). • Thermogenin allows the release of energy as heat instead of ATP. 29 Energy production from glucose Glycolysis 2 ATP 2 NADH 3 ATP/NADH Citric Acid Cycle 2 GTP 1 ATP/GTP 6 NADH 3 ATP/NADH 2 FADH2 2 ATP/FADH2 * 4 ATP in muscle and brain. 36 ATP / glucose 2 ATP 6 ATP* 2 ATP 18 ATP 4 ATP 38 ATP (in heart) 30 Energy production from glucose Mitochondria Glycolysis Glucose 2 Pyruvate 2 NADH 2 Acetyl CoA 2 NADH 6 NADH+ 2 FADH2 2 GTP Oxidative phosphorylation 2 ATP 32-34 ATP 2 ATP 31 Recycling of cytoplasmic NADH Different methods are used to recycle NADH. This accounts for the different energy productions from glucose. Glycerol-3-phosphate shuttle Used by skeletal muscles and the brain Malate-aspartate shuttle Used by the heart and liver 32 Glucose-3-phosphate shuttle NAD + + NAD cytoplasmic cytosolic NADH + H++H+ glycerol-3-phosphate NADH glycerol-3-phosphate dehydrogenase dehydrogenase Glycerol-3-phosphate Dihydroxyacetone phosphate mitochondrial glycerol-3-phosphate dehydrogenase Cytoplasm FAD FADH2 II Q III Mitochondrial matrix 33 Malate-aspartate shuttle Matrix L-aspartate L-aspartate -ketoglutarate Glycolysis cytoplasmic aspartate aminotransferase L-glutamate oxaloacetate cytoplasmic malate dehydrogenase NADH + H+ L-malate + NAD -ketoglutarate mitochondrial aspartate aminotransferase L-glutamate mitochondrial malate dehydrogenase L-malate NAD+ oxaloacetate 3 ATP NADH + H+ 34 Photosynthetic electron transport Heterotrophs Obtain energy by ingestion of other plants and animals. Phototrophs Absorb solar radiation and divert the energy through the electron transport chain. They can produce their own carbohydrates from CO2 and H2O 35 Photosynthetic electron transport Two type of reactions. Light reactions - photo phase Absorb energy using chlorophyll and other pigments. Dark reactions - synthesis phase Carbon metabolism to make carbohydrates. Light is not directly required. 36 Chloroplast The apparatus for light absorption and carbon fixing in eukaryotic photosynthetic cells. Outer membrane Innermembrane space Inner membrane Thylakoid Granum Stroma 37 Chloroplast Stroma Gel-like, unstructured matrix within the inner compartment. It contains the enzymes for the dark reactions. Thylakoids Membranes folded into sacs that are the sites for light receiving pigments, electron carriers and ATP synthesis. They are arranged into stacks called grana. 38 Biomolecules and light Several types of light absorbing pigments are used. Green plants Chlorophylls a and b. Bacteria Bacteriochlorophyll. Accessory pigments Carotenes and phycobilins - absorb light outside the range of chlorophyll. 39 Chlorophyll a H O C saturated bond in bacteriochlorophyll in chlorophyll b CH 2 CH 2 CH3 CH3 C O H 3C in bacteriochlorophyll I N II CH2CH3 III CH3 N Mg H3C CH3 CH 3 CH3 CH 3 O H3C phytol side chain N IV H C O N C H2 C H2 H H H3CO O C O 40 Carotenes -carotene OH HO lutein 41 Phycoerytherin CH3 CH2 in phycocyanin COO - COO - CH 3 H3C O CH N H CH2 CH2 CH2 H3C CH3 H3C N H N H unsaturated bond in phycocyanin CH N H O 42 Photosynthetic light reactions Electrons flow through an electron transport chain from water to an electron acceptor. NADP+ is the acceptor in green plants. 2 H2O + 2 NADP+ light 2 H+ + O2 + 2 NADPH 43 Photosystems h Chl Chl Cat Chl Chl Cat Chl Chl Chl Chl Chl a Reaction Center Cat Cat Chl Chl Chl Chl Cat Two types Each contain one primary acceptor molecule - usually chlorophyll Chl Chl A set of accessory molecules help funnel additional light. Cat 44 Photosystems Photosystem I - P700 • Chlorophyll a and accessory pigments • Absorb in 600-700 nm range Photosystem II - P680 • Chlorophyll a, b and accessory pigments • Absorb light with a maximum at 680 nm All photosynthetic cells have P700. Both are present in O2 evolving organisms higher plants, algae and cyanobacteria. 45 Linkage of photosystems I and II In green plants, the two systems are linked. • Light is absorbed by Photosystem I. • Energy is transmitted to the P700 center and an electron is excited. • Electron is passed via an electron transport chain. • The ‘electron hole’ is filled by another electron transport chain driven by Photosystem II. 46 Photosystem I Reduction potential, V Photosystem I -1.0 P700* A0 A1 -0.5 0.0 Fe-S Complex P700 Ferredoxin-NADP+ reductase light +0.5 Ferredoxin NADP + NADPH + H+ + proton gradient 47 Photosystem II -1.0 Reduction potential, V Photosystem II P680* -0.5 0.0 Q A Watersplitting complex Q B 2HO 2 +0.5 P680 +1.0 O2 + 4 H+ + proton gradient light 48 Linkage of photosystems I and II light 49 Photosystems I and II Net reaction 2 H2O + 2 NADP+ 8 h O2 + 2 NADPH + 2 H+ Eight photons are required to transfer four electrons. 50 Photophosphorylation • Converting light into chemical bonds- very similar to oxidative phosphorylation. • Photoinduced electron transfer from water to NADP+ pumps H+ through thylkaloid membrane - from stromal side to inner compartment. • Protein complexes CF0 and CF1 are the ATP synthases of chloroplasts. 51 Photophosphorylation proton pump within the light-induced electron transport system. H+ H + Low Mg2+ High H+ Lumen thylakoid membrane H+ H+ + High Mg2+ Low H ADP ATP Stroma 52 Photophosphorylation Process is non-cyclic • Starts with H2O and ends with NADPH and O 2. • Products will accumulate as long as there is light. A cyclic process exists for photosystem I. • No H2O is consumed and no NADPH or O2 is produced. • ADP is phosphorylated. 53 Cyclic photophosphorylation P700* A0 A1 Fe-S Complex Cytochrome bf complex Ferredoxin P700 light proton gradient Plastocyanin 54 Synthesis of carbohydrates The Calvin Cycle • The ‘dark’ reactions - fixation of carbon from CO2. • Four stages - fix one carbon at a time. • Six cycles per glucose. Overall reaction for one glucose 6 CO2 + 12 NADPH + 12H+ + 18 ATP + 12 H2O C6H12O6 + 12 NADP+ + 18 ADP + 18 Pi 55 Calvin cycle Stage 1 • Addition of CO2 to an acceptor molecule. • Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) catalyzes the addition of CO2 • The ribulose-1,5-bisphosphate that is produced will immediately cleave into two molecules of 3-phosphoglycerate. 56 Calvin cycle Stage 1 O O O O- P O - O CH2 C H C OH H C OH + CO2 O O P CH 2 O C C C H CH2 O O- H+ O O- P C H2O H O- 2 O OH C O- C OH O O P C H2 O- O- H+ CH 2 O O- O- O P O- 3-phosphoglycerate O- ribulose-1,5bisphosphate -keto acid intermediate 57 Calvin cycle Stage 2 • Phosphorylation of the C1 carboxyl group, producing 1,3-bisphosphoglycerate. • Stromal 1,3-bisphosphoglycerate (1,3-PBG) is reduced to glyceraldehyde-3-phosphate. ATP COOH C OH CH2OPO32- ADP O COPO32H C OH 3-phosphoglycerate 2CH OPO 2 3 kinase NADPH + H+ NADP+ + Pi glyceraldehyde 3-phosphate dehydrogenase H O C H C OH CH2OPO32- 58 Calvin cycle Stage 3 • Carbohydrates are formed from glyceraldehyde-3phosphate. The same gluconeogenesis pathways used earlier are used. glyceraldehyde-3-phosphate isomerase DHAP + glyceraldehyde-3-phosphate fructose-1,6-bisphosphate + H2O fructose-6-phosphate glucose-6-phosphate dihydroxyacetone phosphate aldolase phosphatase isomerase phosphoglucomutase fructose-1,6-bisphosphate fructose-6-phosphate + Pi glucose-6-phosphate glucose-1-phosphate 59 Calvin cycle Stage 4 • Only one of each six cycles results in carbohydrate production. • The other passes through the cycle are used to regenerate the ribulose-1,5-bisphosphate. • The first step is the conversion of glyceraldehyde3-phosphate to dihydroxyacetone phosphate. H H O C H C OH H C H isomerase H C OH H C O H C H O - O P O O O- - O P O O60 3-phosphoglycerate H 2O CO 2 ADP ATP ADP Calvin cycle Glycerate-1,3 bisphosphate Ribulose-1,5bisphosphate NADPH NAD + Pi ATP Dihydroxyacetone phosphate (DHAP) Ribulose-5phosphate X5P F6P E4P X5P S7P R5P Pi Glucose Sucrose, starch, cellulose, etc. Glucose-6phosphate FbisP Glyceraldehyde-3 phosphate (G3P) G3P DHAP G3P DHAP G3P Fructose-6phosphate Pi Fructose-1,6bisphosphate 61 Photorespiration Rubisco can act as an oxygenase by substituting O2 for CO2. CH2OPO32C - O O CHOH CH2OPO32- rubisco + O2 - CHOH O C + C O O CHOH CH2OPO32- CH2OPO32- 62 Photorespiration • This appears to be a counter productive path - oxygen is consumed. • Some plants have adapted this process as an optional pathway for carbon fixation. (sugar cane, corn, sorghum, ...) • This can be described by the Hatch-Slack pathway - C4 pathway 63 Hatch-Slack pathway NADPH + H+ HPO24 oxaloacetate malate dehydrogenase PEP carboxykinase HCO 3 NADP+ malate malic enzyme pyruvate phosphate dikinase phosphoenolpyruvate CO 2 malate AMP + PP i Mesophyll cell pyruvate ATP + H 2PO4- pyruvate NADPH + H+ NADP + + CO2 to Calvin Cycle Bundle sheath cell 64