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Exam #2 Study Guide

by: Maya Panchal

Exam #2 Study Guide BIOL01203

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Cell Biology Study Guide
Biology 3: Cell Biology
Dr. Demarest
Study Guide
glycolysis, Photosynthesis, Cell Biology, glucose, ATP, pyruvate, plant, cell wall, protein, Stomata, thylakoid, chloroplast
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This 22 page Study Guide was uploaded by Maya Panchal on Wednesday March 30, 2016. The Study Guide belongs to BIOL01203 at Rowan University taught by Dr. Demarest in Fall 2015. Since its upload, it has received 20 views. For similar materials see Biology 3: Cell Biology in Biology at Rowan University.


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Date Created: 03/30/16
BIOLOGY 3 EXAM #2 REVIEW GLYCOLOSIS - Glycolysis (glyco = sugar; lysis = breaking) Goal: break glucose down to form two pyruvates Who: all life on earth performs glyclolysis Where: the cytoplasm Glycolysis produces 4 ATP's and 2 NADH, but uses 2 ATP's in the process for a net of 2 ATP and 2 NADH KREB’S CYCLE- 6 NADH's are generated (3 per Acetyl CoA that enters) 2 FADH 2s generated (1 per Acetyl CoA that enters) 2 ATP are generated (1 per Acetyl CoA that enters) 4 CO 2s are released (2 per Acetyl CoA that enters) Therefore, the total numbers of molecules generated in the oxidation of pyruvate and the Krebs Cycle is: 8 NADH 2 FADH 2 2 ATP 6 CO 2 Electron Transport Phosphorylation (Chemiosmosis) • Goal: to break down NADH and FADH ,2pumping H into the outer compartment of the mitochondria • Where: the mitochondria • In this reaction, the ETS creates a gradient which is used to produce ATP, quite like in the chloroplast • Electron Transport Phosphorylation typically produces 32 ATP's • ATP is generated as H+ moves down its concentration gradient through a special enzyme called ATP synthase GLYCOLYSIS INFORMATION • Glucose Catabolism Yields Much More Energy in the Presence of Oxygen than in Its Absence • Complete oxidation of glucose in the presence of oxygen is called aerobic respiration • Glycolysis is present in all organisms. In most cells, the enzymes for glycolysis are found in the cytosol • In the absence of oxygen glycolysis leads to anaerobic respiration (fermentation) Anaerobic respiration • Even in the absence of oxygen, most organisms can extract limited energy from glucose • Electrons removed during glucose oxidation are returned to an organic molecule later in the same pathway • This is called fermentation Fermentation is a metabolic process whereby electrons released from nutrients are transferred to molecules obtained from the breakdown of those same nutrients Two types of fermentation • In some animals and many bacteria, the end product of fermentation is lactate, and so anaerobic glucose catabolism is called lactate fermentation • In most plant cells and microorganisms such as yeast the process is termed alcoholic fermentation because the end product is ethanol Based on Their Need for Oxygen, Organisms Are Aerobic, Anaerobic, or Facultative • Obligate aerobes have an absolute requirement for oxygen • Obligate anaerobes cannot use oxygen as an electron acceptor; oxygen is toxic to these organisms • Facultative organisms can function under aerobic or anaerobic conditions Glycolysis and Fermentation: ATP Generation Without the Involvement of Oxygen • Anaerobes carry out oxidative reactions without using oxygen as an electron acceptor • Most organisms generate two molecules of ATP for every glucose molecule that is oxidized Glycolysis Generates ATP by Catabolizing Glucose to Pyruvate • Glycolysis (or the glycolytic pathway) is a ten-step reaction sequence that converts one glucose molecule into two molecules of pyruvate • Pyruvate is a three-carbon compound. Both ATP and NADH are produced Important features of the glycolytic pathway: • The initial input of ATP (Gly-1) • The sugar splitting reaction in which glucose is split into two three-carbon molecules • The oxidative event that generates NADH (Gly-6) • The two steps at which the reaction sequence is coupled to ATP generation (Gly-7 and Gly-10) The glycolytic pathway can be divided into three phases 1. Phase I: the preparatory and cleavage steps 2. Phase II: the oxidative sequence, which is the first ATP-generating event 3. Phase III: the second ATP-generating event Phase 2: Oxidation and ATP Generation • The net energy yield of phase one is negative • Two molecules of ATP have been consumed per molecule of glucose • In phase 2, ATP production is linked to an oxidative event, followed by the generation of ATP in phase 3 Gly-6 and Gly-7 The oxidation of glyceraldehyde-3- phosphate to 3-phosphoglycerate is highly exergonic, and drives – the reduction of NAD+ to NADH (Gly-6) – the phosphorylation of ADP with inorganic phosphate, Pi (Gly-7) Important features of Gly-6 and Gly-7 • NAD+ is an electron acceptor • The oxidation is coupled to the formation of a high-energy, doublyphosphorylated intermediate, 1,3- bisphosphoglycerate • ATP is generated by transferring a phosphate group to ADP from a phosphorylated substrate, 1,3- bisphosphoglycerate Summary of Gly-6 and Gly-7 • Gly-6 and Gly-7 can be summarized as - Each reaction involving glyceraldehyde-3- phosphate occurs twice per starting molecule of glucose -The two ATPs invested in the first phase are recovered in the second phase, for no net ATP Phase 3: Pyruvate Formation and ATP Generation • The phosphoester bond of 3-phosphoglycerate is converted to a phosphoenol bond • The phosphate group is moved to the adjacent carbon, forming 2-phosphoglycerate (Gly-8) • Water is removed from the 2-phosphoglycerate by the enzyme enolase (Gly-9) generating the high- energy compound phosphoenolpyruvate (PEP) Rubisco is the most abundant enzyme on the planet. Important for the first step. Phosphoenolpyruvate • Hydrolysis of the phosphoenol bond of PEP is one of the most exergonic known in biological systems • PEP hydrolysis drives ATP synthesis by transferring a phosphate to ADP, catalyzed by the enzyme pyruvate kinase Summary of Glycolysis • The two molecules of ATP formed in the second phosphorylation event (Gly-10) represent the net yield of ATP for the glycolytic pathway • The pathway is highly exergonic Conservation of Glycolysis • The glycolytic pathway is one of the most common and highly conserved metabolic pathways known • Virtually all cells have the ability to convert glucose to pyruvate, extracting energy in the process • The next steps depend on the availability of oxygen The Fate of Pyruvate Depends on Whether Oxygen Is Available • Pyruvate occupies a key position as a branch point in chemotrophic energy metabolism • In the presence of oxygen, pyruvate undergoes further oxidation to acetyl coenzyme A The fate of pyruvate in the absence of oxygen • Under anaerobic conditions no further oxidation of pyruvate occurs • Pyruvate is reduced by accepting the electrons (and protons) that must be removed from NADH • The most common products of pyruvate reduction are lactate or ethanol and CO2 In the Absence of Oxygen, Pyruvate Undergoes Fermentation to Regenerate NAD+ • Fermentation must regenerate NAD+ from NADH so that glycolysis can continue • Cells monitor and stabilize the NAD+/NADH ratio, an indicator of the cell’s redox state (general level of oxidation of cellular components) • Electrons are transferred to pyruvate with two possible outcomes Lactate Fermentation • The anaerobic process that culminates with lactate is called lactate fermentation • Lactate is generated by direct transfer of electrons from NADH to pyruvate by lactate dehydrogenase Gluconeogenesis • Lactate produced in muscles under hypoxic (anaerobic) conditions is transferred to the liver • In the liver, it is converted into glucose by the process of gluconeogenesis • It is the reverse of lactate fermentation but with several differences Fermentation Taps Only a Fraction of the Substrate’s Free Energy but Conserves That Energy Efficiently as ATP • An essential feature of every fermentation process is – no external electron acceptor is involved and no net oxidation occurs • Fermentation gives a modest ATP yield of two ATP per glucose; most of the free energy of the glucose molecule is still present in the lactate or ethanol Free energy of lactate • The two lactate molecules produced from one glucose contain most of the energy present per mole of glucose The Regulation of Glycolysis and Gluconeogenesis • Cells have enzymes for both glycolysis and gluconeogenesis, so the processes must be regulated • S p a t i a l regulation keeps the two processes confined to separate places in the body • There is also temporal regulation in which the two processes take place at different times in one cell Key Enzymes in the Glycolytic and Gluconeogenic Pathways Are Subject to Allosteric Regulation • Allosteric regulation involves the interconversion of an enzyme between two forms, one catalytically active and the other inactive • The enzyme will be active or not depending on whether an allosteric effector is bound to the allosteric site • The effector might be an activator or inhibitor Glycolysis and gluconeogenesis are reciprocally regulated • AMP and acetyl CoA, the two effectors to which both pathways are sensitive, have opposite effects • AMP activates glycolysis and inhibits gluconeogenesis • Acetyl CoA activate gluconeogenesis but inhibits glycolysis Further allosteric regulation of glycolysis and gluconeogenesis • Both pathways are subject to allosteric regulation by compounds involved in respiration • Acetyl CoA and citrate are key intermediates in an aerobic pathway called the tricarboxylic acid cycle • Both have inhibitory effects on glycolysis, decreasing the rate of pyruvate formation Phototrophic Energy Metabolism: Photosynthesis • Most chemotrophs depend on an external source of organic substrates for survival • Photosynthetic organisms produce the chemical energy and organic carbon required by chemotrophs • They use solar energy to reduce CO to2produce carbohydrates, fats, and proteins • Photosynthesis: the conversion of light energy to chemical energy and its subsequent use in synthesis of organic molecules • Phototrophs: organisms that convert solar energy into chemical energy as ATP and reduced coenzymes Types of phototrophs -Photoheterotrophs: organisms that acquire energy from sunlight but depend on organic sources of reduced carbon (heliobacteria, purple non-sulfur bacteria, green non-sulfur bacteria) -Photoautotrophs: organisms that use solar energy to synthesize energy-rich organic molecules using starting materials such as CO a2d H O 2plants, algae, & most photosynthetic bacteria) An Overview of Photosynthesis • Photosynthesis involves two major biochemical processes: - Energy transduction reactions: light energy is captured and converted into chemical energy (ATP (energy) & NADPH (reducing power)) - Carbon assimilation reactions: (carbon fixation reactions) carbohydrates are formed from CO and 2 O 2 (Calvin cycle) The Energy Transduction Reactions Convert Solar Energy to Chemical Energy • Light energy is captured by green pigment molecules called chlorophylls, present in the green leaves of plants and the cells of algae and photosynthetic bacteria • Light absorption by a chlorophyll molecule excites one of its electrons, which is then ejected from the molecule and enters an electron transport system (ETS) Unidirectional proton pumping  The photosynthetic ETS is coupled to unidirectional proton pumping  The electrochemical gradient produced is used to generate ATP through photophosphorylation Photoreduction • Oxygenic phototrophs release oxygen as water is oxidized • Light-dependent generation of NADPH is called photo reduction The Carbon Assimilation Reactions Fix Carbon by Reducing Carbon Dioxide  Most of the energy accumulated by the generation of ATP and NADPH is used for carbon dioxide fixation and reduction  “H 2” is a suitable electron donor, and “A” is the oxidized form of the donor Production of sugars • The intermediates of photosynthesis are used for biosynthesis of a variety of products, including glucose, sucrose, and starch  Sucrose is the major transport carbohydrate in most plants  Starch is the major storage carbohydrate in most plants The Chloroplast Is the Photosynthetic Organelle in Eukaryotic Cells  The most familiar oxygenic phototrophs are the green plants, in which the photosynthetic organelle is the chloroplast  Newly differentiated plant cells have smaller organelles called proplastids, which may develop into any of several types of plastids depending on the function of the cell  Amyloplasts are specialized for storing starch  Chromoplasts give flowers and fruits their distinctive colors Chloroplasts Are Composed of Three Membrane Systems • A chloroplast has both an outer membrane and an inner membrane • These are usually separated by an intermembrane space • The inner membrane encloses the stroma, a gel-like matrix full of enzymes for C, N, and S reduction and assimilation The outer membrane is freely permeable  The outer membrane contains porins  In the inner membrane, transport proteins control the flow of metabolites between the stroma and intermembrane space Thylakoids -Chloroplasts have a third membrane system, called thylakoids -These are flat, saclike structures in the stroma, arranged in stacks called grana -Grana are interconnected by stroma thylakoids -Grana and stroma thylakoids form one single continuous compartment – thylakoid lumen  Protons are pumped across the membrane into the thylakoid lumen during light-driven electron transport  Potential energy that drives ATP synthesis when protons return to the stroma (like water in a dam) Photosynthetic Energy Transduction I: Light Harvesting  The first stage of photosynthetic energy transduction is the capture of solar energy  Light behaves as a stream of particles called photons, each carrying a quantum (indivisible packet) of energy  When a photon is absorbed by a pigment such as chlorophyll, the energy of the photon is transferred to an electron Photoexcitation  The energy transferred from a photon energizes the electron from its ground state in a low-energy orbital, to an excited state in a high-energy orbital  This first step of photosynthesis is called photoexcitation  Different pigments have different absorption spectra, to describe the wavelengths absorbed. Photoexcited electrons are unstable  A photoexcited electron in a pigment molecule is unstable and must either return to the ground state or transfer to a stable high- energy orbital  If it returns to the ground state, the energy is lost as heat or light  The energy can also be transferred to an electron in an adjacent molecule, in a process called resonance energy transfer Transfer of the photoexcited electron  If the excited electron is transferred to another molecule, it is called photochemical reduction  Photochemical reduction is essential for converting light energy into chemical energy Chlorophyll Is Life’s Primary Link to Sunlight • Chlorophyll is found in nearly all photosynthetic organisms • Chlorophyll a and b each have a central porphyrin ring, which absorbs visible light • Their strongly hydrophobic phytol side chains anchor the chlorophylls in the thylakoid membranes Bacteriochlorophyll -Bacteriochlorophyll is a subfamily restricted to anoxygenic phototrophs. It is characterized by a saturated site not found in other chlorophylls. The absorption maxima of bacteriochlorophylls are shifted toward the near-ultraviolet and the far-red regions Accessory Pigments Further Expand Access to Solar Energy • Most photosynthetic organisms also contain accessory pigments, which absorb photons that cannot be captured by chlorophyll  The energy is transferred to a chlorophyll molecule by resonance energy transfer  Two types of accessory pigments are carotenoids and phycobilins Carotenoids  Two carotenoids that are abundant in the thylakoid membranes of most plants and green algae are -carotene and lutein  When not masked by chlorophyll these pigments confer an orange or yellow tint to leaves  They absorb photons from a broad range of the blue region of the spectrum Phycobilins Phycobilins are found only in red algae and cyanobacteria; two common examples are – Phycoerythrin, which allows absorption of light that penetrates the ocean’s surface water – Phycocyanin, which is characteristic of cyanobacteria near the surface of a lake, or on land Light-Gathering Molecules Are Organized into Photosystems and Light-Harvesting Complexes • Functional units, photosystems, contain – Chlorophyll – Accessory proteins –Chlorophyll-binding proteins that stabilize the chlorophyll in a photosystem –Other proteins that bind components of the electron transport system Photosystems and Light- Harvesting Complexes • Most pigments of a photosystem serve as light-gathering antenna pigments • These absorb photons and pass the energy to a neighboring chlorophyll or accessory protein by resonance energy transfer • Some antenna pigments are “wired” together by quantum mechanical probability effects The reaction center • The events that drive electron flow and proton pumping do not begin until the energy reaches the reaction center of a photosystem  Here, two chlorophyll a molecules called the special pair are found These molecules catalyze the conversion of solar energy into chemical energy The light-harvesting complex • Each photosystem is associated with a light-harvesting complex (LHC), which collects light energy and can move in response to changing light conditions • The LHC does not contain a reaction center • Instead it passes the collected energy to a nearby photosystem by resonance energy transfer -Plants and green algae have LHCs composed of 80-250 chlorophyll a and b molecules along with carotenoids and pigment-binding proteins -Together a photosystem and the associated LHCs are referred to as a photosystem complex Oxygenic Phototrophs Have Two Types of Photosystems • In the 1940s Emerson and colleague discovered that two separate photosystems are involved in oxygenic photosynthesis • They found that photosynthesis driven by a combination of wavelengths exceeded the sum of activities with either wavelength alone • This synergistic phenomenon was called the Emerson enhancement effect Photosystems I and II • Photosystem I (PSI) has an absorption maximum of 700nm, whereas photosystem II (PSII) has an absorption maximum of 680 nm • Each electron that passes from water to NADP+ must be photoexcited once for each photosystem • With illumination of 690nm and above, photosynthesis is severely impaired • Each electron is first excited by PSII and then by PSI • The special pair of chlorophyll a molecules in the reaction center of each photosystem are designated P680 for PSII and P700 for PSI • The granal and stromal thylakoid membranes have differing amounts of the two photosystems, which can move to respond to changing light conditions Photosynthetic Energy Transduction II: NADPH Synthesis  The second stage of photosynthesis uses a series of electron carriers to transport electrons from chlorophyll to the coenzyme nicotine adenine dinucleotide phosphate (NADP+)  It forms NADPH when reduced  This is called photoreduction and involves a chloroplast electron transport system (ETS) Similarity to mitochondrial electron transport • The chloroplast electron transport system is similar to that of mitochondria  The complete pathway includes several components  Many of the molecules are similar to those of the mitochondrial ETS— cytochromes, iron-sulfur proteins, etc. G  and E  • Recall that Go (standard free energy) and Eo (standard reduction potential) are opposite in sign • This means that electrons will spontaneously flow toward a compound with a higher reduction potential • Absorption of light by each photosystem boosts electrons to the top of an ETS Electron flow • As electrons flow from PSII to PSI, a portion of their energy is conserved in a proton gradient across the thylakoid membrane • From PSI the electrons flow to ferredoxin and then to NADP+ • NADP+ is the coenzyme mainly used for anabolic pathways, whereas NAD+ is usually involved in catabolic pathways Photosystem II Transfers Electrons from Water to a Plastoquinone • Photosystem II uses electrons from water to reduce a plastoquinone (QB) to plastoquinol (QBH2) • PSII is associated with light-harvesting complex II (LHCII), which contains about 250 chlorophyll and many carotenoid molecules • Energy captured by antenna pigments of PSII or LHCII is funneled to the reaction center • Captured energy in the reaction center lowers the reduction potential of a P680 molecule making it a better electron donor • A photoexcited electron is passed to pheophytin (Ph), a chlorophyll a molecule with two protons in place of the Mg2+ • The charge separation between P680+ and Ph– prevents the electron from returning to its ground state • Solar energy has been harvested and converted into electrochemical potential energy in the form of the charge separation • The electron is passed to QA, a plastoquinone (similar to coenzyme Q) tightly bound to protein D2 • QB receives two electrons from QA and picks up two protons from the stroma to form QBH2 • QBH2 enters a mobile pool of QBH2 inside the photosynthetic membrane, where it passes two electrons and two protons to the cytochrome b6 /f complex • Formation of one mobile plastoquinone molecule depends on two sequential photoreactions • To replace the electron lost to plastoquinone, oxidized P680+ is reduced by an electron from water • PSII includes an oxygen-evolving complex that catalyzes the splitting and oxidation of water, producing O2, electrons, and protons • Two water molecules donate four electrons one at a time to four molecules of P680+ • The protons accumulating in the lumen contribute to an electrochemical proton gradient across the thylakoid membrane, and the O2 diffuses out of the chloroplast • The light-dependent oxidation of water is called water photolysis The Cytochrome b6 /f Complex Transfers Electrons from a Plastoquinone to Plastocyanin • Electrons carried by QBH2 flow through an ETS coupled to unidirectional proton pumping into the lumen • This happens by way of the cytochrome b6 /f complex, which is composed of seven different integral transmembrane proteins including two cytochromes and an iron- sulfur protein The Cytochrome b6 /f complex • QBH2 donates two electrons via cytochrome b6 and the iron-sulfur protein to cytochrome f • Each oxidation of QBH2 releases two protons into the thylakoid lumen Electrons are passed to plastocyanin • Reduced cytochrome f donates electrons to a copper-containing protein called plastocyanin (PC), which is a mobile electron carrier • PC, a peripheral membrane protein on the lumenal side of the thylakoid membrane, carries electrons one at a time to PSI Photosystem I Transfers Electrons from Plastocyanin to Ferredoxin • PSI transfers photoexcited electrons from reduced plastocyanin to the protein ferredoxin, the immediate electron donor to NADP+ • The PSI reaction center includes a chlorophyll a molecule called Ao (instead of pheophytin), as well as phylloquinone and three Fe-Su centers that form an ETS from Ao to ferredoxin Light-harvesting complex I  PSI in plants and green algae is associated with light-harvesting complex I (LHCI), with fewer antennae molecules than LHCII  Energy is funneled to a reaction center with a special pair of chlorophyll a molecules, P700  The energy absorbed by PSI lowers the reduction potential of the P700s so that a photoexcited electron is rapidly passed to Ao Charge separation • The charge separation between P700+ and reduced Ao prevents the electron from returning to the ground state • The electron lost by P700 is replaced by an incoming electron from reduced plastocyanin • From Ao electrons flow exergonically through the ETS to ferredoxin, the final electron acceptor for PSI Ferredoxin • Ferredoxin (Fd) is a mobile iron-sulfur protein found in the stroma Ferredoxin-NADP+ Reductase Catalyzes the Reduction of NADP+ • The final step in photoreduction is the transfer of electrons from ferredoxin to NADP+, producing the NADPH needed for carbon reduction and assimilation • The enzyme responsible is ferredoxin- NADP+ reductase (FNR) Noncyclic electron flow • The components of the chloroplast ETS provide a continuous unidirectional flow of electrons from water to NADP+ Photosynthetic Energy Transduction III: ATP Synthesis  In the final stage of photosynthetic energy transduction the potential energy stored in a proton gradient is used to synthesize ATP  This process is called photophosphorylation  The thylakoid membrane is virtually impermeable to protons, so a substantial proton gradient can develop The ATP Synthase Complex Couples Transport of Protons Across the Thylakoid Membrane to ATP Synthesis • The movement of protons back across the membrane to regions of lower concentration drives the synthesis of ATP by an ATP synthase • The ATP synthase complex found in chloroplasts is called the CF0CF1 complex, very similar to the F0F1 complex of mitochondria The CF0CF1 complex • CF1 is a hydrophylic group of polypeptides protruding from the stromal side of the thylakoid membrane, and containing three catalytic sites for ATP synthesis • CF0 is a hydrophobic assembly of polypeptides anchored to the thylakoid membrane Components of CF0  Subunits I and II form a stalk that connects CF0 and CF1  Subunit IV is the proton translocator, through which protons flow back to the stroma  Subunit III is a ring of polypeptides next to subunit IV, the rotation of which is coupled to ATP synthesis, similar to mitochondria Four protons per ATP  Recent evidence suggests that four protons are translocated for every ATP generated  There are 14 copies of subunit III, and 3 ATP are generated by one complete rotation, leading to estimates of more than four protons per ATP • 4. Ferredoxin-NADP+reductase - Enzyme on stromal side of thylakoid membrane  When NADPH consumption is low, or more ATP is needed, cyclic electron flow can divert the power of PSI into ATP synthesis rather than NADP+ reduction A Summary of the Complete Energy Transduction System • The component parts of the complete system can be summarized as follows: • 1. Photosystem II complex - Assembly of chlorophyll, pigments, proteins - Oxidization of water, evolving O2 - P680 becomes photoexcited, enabling it to reduce plastoquinone • 2. Cytochrome b6 /f complex  – Accepts electrons from plastoquinone (noncyclic) or ferredoxin (cyclic)  – Pumps protons unidirectionally into thylakoid lumen • 3. Photosystem I complex  – Assembly of chlorophyll, pigments, proteins  – P700 becomes photoexcited, enabling it to reduce ferredoxin (stromal protein) - Catalyzes transfer of electrons from two reduced ferredoxins to NADP+ - NADPH product essential reducing agent in many anabolic pathways • 5. ATP synthase complex (CF0CF1)  - CFoCF1 proton channel and ATP synthase  - Uses energy from exergonic flow of protons to synthesize ATP in the stroma  - ATP essential for carbon fixation/assimilation Cyclic Photophosphorylation Allows a Photosynthetic Cell to Balance NADPH and ATP Synthesis  This is called cyclic photophosphorylation  No water is oxidized nor O2 released, because PSII is not involved Photosynthetic Carbon Assimilation I: The Calvin Cycle • The main pathway for movement of inorganic carbon into the biosphere is the Calvin cycle • In plants and algae, the cycle is confined to the chloroplast stroma, where ATP and NADPH accumulate Entry of CO into plants 2 • In plants, CO2enters the leaves through special pores called stomata  Once inside a leaf, CO 2iffuses into mesophyll cells and usually travels into the stroma  The stroma is the site of carbon fixation Three stages of the Calvin cycle 1. The carboxylation of ribulose-1,5- bisphosphate, and generation of two 3- phosphoglycerate molecules 2. Reduction of 3-phosphoglycerate into glyceraldehyde-3-phosphate 3. Regeneration of the original acceptor to allow continued carbon assimilation Carbon Dioxide Enters the Calvin Cycle by Carboxylation of Ribulose-1,5-Bisphosphate • The first stage begins with the covalent attachment of CO2 to ribulose-1,5-bisphosphate (CC-1) • This leads to production of two 3-carbon molecules, 3-phosphoglycerate • Ribulose-1,5-bisphosphate carboxylase/oxygenase (“rubisco”) is the most abundant protein on the planet 3-Phosphoglycerate Is Reduced to Form Glyceraldehyde-3-Phosphate  The reduction of 3-phosphoglycerate to form glyceraldehyde-3-phosphate is essentially the reverse of the oxidative sequence of glycolysis  The coenzyme is NADPH instead of NADH • Phosphoglycerokinase catalyzes reaction CC- 2 and glyceraldehyde-3-phosphate dehydrogenase catalyzes CC-3 Energy is consumed in the first stages of carbon fixation • For every CO m2lecule fixed by rubisco two ATP molecules must be hydrolyzed and two NADPH molecules are oxidized Regeneration of Ribulose-1,5- Bisphosphate Allows Continuous Carbon Assimilation  One of six triose phosphate molecules generated is used for biosynthesis of organic molecules  The remaining five are used to regenerate three molecules of the (five-carbon) acceptor ribulose- 1,5- bisphosphate (CC-4) • The reactions are catalyzed by aldolases, transketolases, phosphatases, and isomerases Regeneration of ribulose-1,5- bisphosphate requires energy • Threemoleculesofribulose-5-phosphateare converted to ribulose-1, bisphosphate by phosphoribulokinase (PRK) • Regeneration of the three ribulose-1,5- bisphosphate consumes three more ATPs Regulation of the Calvin Cycle • In the dark, phototrophs must meet a steady demand for energy and carbon using the surplus accumulated when light is available • Several regulatory systems are used to ensure that the Calvin cycle does not operate unless light is available The Calvin Cycle Is Highly Regulated to Ensure Maximum Efficiency  The first level of control is regulation of key enzymes in the Calvin cycle  These enzymes are not synthesized in tissues that are not exposed to light  Also, reduced ferredoxin, ATP, and NADPH, act as signals to activate Calvin cycle enzymes Rubisco and other enzymes are points for metabolic control • Rubisco is an obvious control point as it catalyzes the carboxylation reaction of the Calvin cycle • All three of the regulated enzymes - Are unique to the Calvin cycle - Catalyze reactions that are essentially irreversible Photosynthetic Carbon Assimilation II: Carbohydrate Synthesis  The most abundant protein in the chloroplast is a phosphate translocator, which catalyzes the exchange of triose phosphates in the stroma for Pi in the cytosol  This antiport system only exports triose phosphates if Pi for making new triose phosphates returns to the stroma  The triose phosphates that remain in the stroma are used for starch synthesis Glucose-1-Phosphate Is Synthesized from Triose Phosphates  Two triose phosphates undergo a condensation reaction catalyzed by aldolase, to generate fructose-1,6-bisphosphate  This is dephosphorylated by fructose-1,6- bisphosphatase to form fructose-6-phosphate (S-1)  This is catalyzed in both stroma and cytosol by distinct forms of the enzyme, called isoenzymes S-2 and S-3  Fructose-6-phosphate can be converted to glucose-6-phosphate (S-2)  This is then converted to glucose-1-phosphate (S-3) There are separate stromal and cytosolic isoenzymes for these reactions too, as the hexoses involved cannot be transported between the cytosol and stroma The Biosynthesis of Sucrose Occurs in the Cytosol  Sucrose synthesis is located in the cytosol of a photosynthetic cell  Triose phosphates exported from the stroma that are not used in other metabolic pathways are converted to glucose-1-phosphate  Glucose is then produced by reaction with UTP (uridine triphosphate) to produce UDP-glucose (S- 4c) • The glucose of UDP-glucose is transferred to fructose-6-phosphate to form sucrose-6- phosphate (S-5c) • The hydrolytic removal of the phosphate group generates free sucrose (S-6c) Control of sucrose biosynthesis  Sucrose synthesis is controlled to prevent conflict with degradation pathways  Cytosolic fructose-1,6-bisphosphatase is inhibited by fructose-2,6-bisphosphate, a regulator of glycolysis and gluconeogenesis  Sucrose phosphate synthase is stimulated by glucose-6-phosphate and inhibited by sucrose-6- phosphate, UDP, and Pi The Biosynthesis of Starch Occurs in the Chloroplast Stroma • Starch synthesis is confined to plastids, where triose phosphates are converted to glucose-1-phosphate, which is then used for starch synthesis  Glucose-1-phosphate reacts with ATP to generate ADP-glucose (S-4s)  The activated glucose is added to a growing starch chain by starch synthase (S-5s) Control of starch biosynthesis • Starch synthesis is regulated to prevent conflict with degradation pathways Chemotrophic Energy Metabolism: Aerobic Respiration • Some cells meet their energy needs through anaerobic fermentation • However, fermentation yields only modest amounts of energy due to the absence of electron transfer Cellular Respiration: Maximizing ATP Yields • Cellular respiration (or respiration) uses an external electron acceptor to oxidize substrates completely to CO 2 • External electron acceptor: one that is not a by-product of glucose catabolism • ATP yield is much higher in cellular respiration Cellular respiration defined • Respiration is the flow of electrons through or within a membrane, from reduced coenzymes to an external electron acceptor usually accompanied by the generation of ATP • Coenzymes such as FAD (flavin adenine dinucleotide) and coenzyme Q (ubiquinone) are involved • The terminal electron acceptor • In aerobic respiration, the terminal electron acceptor is oxygen and the reduced form is water • Other terminal electron acceptors (sulfur, protons, and ferric ions) are used by other organisms, especially bacteria and archaea. These are examples of anaerobic respiration. Mitochondria • Most aerobic ATP production in eukaryotic cells takes place in the mitochondrion • In bacteria, the plasma membrane and cyotoplasm are analogous to the mitochondrial inner membrane and matrix with respect to energy metabolism Aerobic Respiration Yields Much More Energy than Fermentation Does • With O a2 the terminal electron acceptor, pyruvate can be oxidized completely to CO 2 • Aerobic respiration has the potential of generating up to 38 ATP molecules per glucose • Oxygen provides a means of continuous reoxidation of NADH and other reduced coenzymes Respiration Includes Glycolysis, Pyruvate Oxidation, the TCA Cycle, Electron Transport, and ATP Synthesis Respiration will be considered in five stages: • Stage 1: the glycolytic pathway • Stage 2: pyruvate is oxidized to generate acetyl CoA • Stage 3: acetyl CoA enters the tricarboxylic acid cycle (TCA cycle), where it is completely oxidized to CO 2 • Stage 4: electron transport, the transfer of electrons from reduced coenzymes to oxygen coupled to active transport of protons across a membrane • Stage 5: The electrochemical proton gradient formed in step 4 is used to drive ATP synthesis (oxidative phosphorylation) The Mitochondrion: Where the Action Takes Place • The mitochondrion is called the “energy powerhouse” of the eukaryotic cell • These organelles are thought to have arisen from bacterial cells • Mitochondria have been shown to carry out all the reactions of the TCA cycle, electron transport, and oxidative phosphorylation Mitochondria Are Often Present Where the ATP Needs Are Greatest • Mitochondria are found in virtually all aerobic cells of eukaryotes • They are present in both chemotrophic and phototrophic cells • Mitochondria are frequently clustered in regions of cells with the greatest need for ATP, e.g., muscle cells Are Mitochondria Interconnected Networks Rather than Discrete Organelles? • In electron micrographs, mitochondria usually appear as oval structures. However they can take various shapes and sizes, depending on the cell type. • Their appearance under EM suggests that they are large, and numerous discrete entities • The work of Hoffman and Avers suggests that the oval profiles in electron micrographs of yeast cells reflect slices through a single large branched mitochondrion • This work suggests that mitochondria may be larger than previously thought, and less numerous The Outer and Inner Membranes Define Two Separate Compartments and Three Regions • A distinctive feature of mitochondria is the presence of both outer and inner membranes • The outer membrane contains porins that allow passage of solutes with molecular weights up to 5000 • The intermembrane space between the inner and outer membranes is continuous with the cytosol The inner membrane • The inner membrane of the mitochondria is impermeable to most solutes, partitioning the mitochondrion into two separate compartments • The intermembrane space • The interior of the organelle, or mitochondrial matrix The inner boundary membrane and cristae • The portion of the inner membrane adjacent to the intermembrane space is called the inner boundary membrane • The inner membrane is about 75% protein by weight; the proteins include those involved in solute transport, electron transport, and ATP synthesis The cristae • The inner membrane of most mitochondria has many infoldings called cristae • They increase surface area of the inner membrane, and provide more space for electron transport to take place • The cristae provide localized regions, intracristal spaces, where protons can accumulate during electron transport • The cristae (continued) • The cristae are thought to be tubular structures that associate in layers • They have limited connections to the inner boundary membrane through small openings, crista junctions • Cells with high metabolic activity seem to have more cristae in their mitochondria The mitochondrial matrix • The interior of the mitochondrion is filled with a semi-fluid matrix • The matrix contains many enzymes involved in mitochondrial function as well as DNA molecules and ribosomes • Mitochondria contain proteins encoded by their own DNA as well as some that are encoded by nuclear genes Mitochondrial Functions Occur in or on Specific Membranes and Compartments • Specific functions and pathways have been localized within mitochondria by fractionation studies • Most of the enzymes involved in pyruvate oxidation, the TCA cycle, and catabolism of fatty acids and amino acids are found in the matrix • Most electron transport intermediates are integral inner membrane components Localization of Specific Mitochondrial Functions • Knoblike spheres called F comp1exes protrude from the inner membrane into the matrix • These are involved in ATP synthesis • Each complex is an assembly of several different polypeptides, and can be seen in an electron micrograph using negative staining F1complexes • Each F c1mplex is attached by a short protein stalk to an F complex o • This is an assembly of hydrophobic polypeptides embedded in the mitochondrial inner membrane • This F o c1mplex is an ATP synthase that is responsible for most of the ATP generation in the mitochondria (and in bacterial cells as well) • Knoblike spheres called F comp1exes protrude from the inner membrane into the matrix • These are involved in ATP synthesis • Each complex is an assembly of several different polypeptides, and can be seen in an electron micrograph using negative staining The Tricarboxylic Acid Cycle: Oxidation in the Round • In the presence of oxygen pyruvate is oxidized fully to carbon dioxide with the released energy used to drive ATP synthesis • This involves the TCA (tricarboxylic acid) cycle, in which citrate is an important intermediate • The TCA cycle is also called the Krebs cycle after Hans Krebs, whose lab played a key role in elucidating the cycle Pyruvate Is Converted to Acetyl Coenzyme A by Oxidative Decarboxylation • The glycolytic pathway ends with pyruvate, which is small enough to enter the intermembrane space of the mitochondrion • At the inner mitochondrial membrane, a specific symporter transports pyruvate into the matrix, along with a proton • Then, pyruvate is converted to acetyl CoA by pyruvate dehydrogenase complex (PDH) Conversion of pyruvate • The conversion is a decarboxylation because one carbon is liberated as CO 2 + • It is also an oxidation because two electrons (and one proton) are transferred to NAD to form NADH The Tricarboxylic Acid Cycle • The TCA cycle metabolizes acetyl CoA, produced from pyruvate decarboxylation • Acetyl CoA can also arise from fatty acid oxidation • Acetyl CoA transfers its acetate group to a four-carbon acceptor called oxaloacetate, generating citrate The fate of citrate • After its formation, citrate undergoes two successive decarboxylations • It also goes through several oxidation steps • Eventually oxaloacetate is regenerated, and can accept two more carbons from acetyl CoA and the cycle begins again The overall TCA cycle: • Each round of the TCA cycle involves the entry of two carbons, the release of two CO , and the 2 regeneration of oxaloacetate • Oxidation occurs at five steps, four in the cycle itself and one when pyruvate is converted to acetyl CoA • In each case, electrons are accepted by coenzymes • The fate of citrate: After its formation, citrate undergoes two successive decarboxylations. • It also goes through several oxidation steps. Eventually oxaloacetate is regenerated, and can accept two more carbons from acetyl CoA and the cycle begins again. The TCA Cycle Begins with the Entry of Acetate as Acetyl CoA • With each round of the TCA cycle, two carbon atoms enter in organic form as acetate and leave in inorganic form as carbon dioxide • In the first reaction, TCA-1, the two-carbon acetate group is transferred from acetyl CoA to oxaloacetate (4C) to form citrate (6C) • This reaction is catalyzed by citrate synthetase Direct Generation of GTP (or ATP) Occurs at One Step in the TCA Cycle • So far in the cycle, two carbon atoms have entered and two have left (but not the same two carbons), and two molecules of NADH have been generated • Succinyl CoA has been generated; like acetyl CoA it has a high-energy thioester bond • The energy from hydrolysis of this bond is used to generate one ATP (bacteria, plants) or GTP(animals) (TCA-5) The Final Oxidative Reactions of the TCA Cycle Generate FADH and NADH 2 • Of the remaining three steps in the cycle, two are oxidations • Succinate i+ oxidized to fumarate (TCA-6); this transfers electrons to FAD, a lower-energy coenzyme than NAD • In the next step, fumarate is hydrated to produce malate (TCA-7) by fumarate hydratase • In the final step of the TCA cycle, the hydroxyl group of malate becomes the target of the final oxidation in the cycle • Electrons are transferred to NAD+, producing NADH as malate is converted to oxaloacetate (TCA-8) Summing Up: The Products of the TCA Cycle Are CO , ATP, NADH, and FADH 2 2 • The TCA cycle accomplishes the following: 1. Two carbons enter the cycle as acetyl CoA, which joins oxaloacetate to form the six-carbon citrate 2. Decarboxylation occurs at two steps to balance the input of two carbons by releasing two CO 2 3. Oxidation occurs at four steps, with NAD the electron acceptor in three steps and FAD in one The Products of the TCA Cycle Are CO , ATP,2NADH, and FADH (continued)2 4. ATP is generated at one point, with GTP as an intermediate in the case of animal cells 5. One turn of the cycle is completed as oxaloacetate, the original 4C acceptor, is regenerated Several TCA Cycle Enzymes Are Subject to Allosteric Regulation • Like all metabolic pathways, the TCA cycle must be carefully regulated to meet cellular needs • Most of the control of the cycle involves allosteric regulation of four key enzymes by specific effector molecules • Effector molecules may be activators or inhibitors Fat as a Source of Energy • Fats are highly reduced compounds that liberate more energy per gram upon oxidation than do carbohydrates • They are a long-term energy storage form for many organisms • Most fat is stored as deposits of triacylglycerols, neutral triesters of glycerol and long-chain fatty acids Catabolism of triacylglycerols • Triacylglycerol catabolism begins with their hydrolysis to glycerol and free fatty acids • The glycerol is channeled into the glycolytic pathway by oxidative conversion to dihydroxyacetone phosphate • Fatty acids are linked to coenzyme A, to form fatty acyl CoAs, then degraded by b-oxidation b-oxidation • b-oxidation is a catabolic process that generates acetyl CoA and the reduced coenzymes NADH and FADH 2 Fatty acid degradation • Most fatty acids are oxidatively converted to acetyl CoA in the mitochondrion • These can be further catabolized in the TCA cycle • The fatty acids are degraded in a series of repetitive cycles, which removes two carbons at a time until the fatty acid is completely degraded • The result is the production of one FADH , one 2ADH, and one acetyl CoA per cycle Protein as a Source of Energy and Amino Acids • Besides their other numerous functions, proteins can be catabolized to produce ATP if necessary when carbohydrate and lipid stores are depleted • Eventually cells undergo turnover of proteins and protein-containing structures • The resulting amino acids can be used to generate new proteins or degraded for energy • These are converted into intermediates of mainstream catabolism in as few steps as possible • The pathways differ for individual amino acids, but all eventually lead to acetyl CoA, pyruvate, or a few key TCA cycle intermediates Electron Transport: Electron Flow from Coenzymes to Oxygen • Chemotrophic energy metabolism through the TCA cycle accounts for synthesis of 4 ATP per glucose (2 from glycolysis and 2 from the TCA cycle) • This accounts for only a small portion of the energy in the original glucose molecule • The remainder is stored in NADH and FADH 2 The Electron Transport System Conveys Electrons from Reduced Coenzymes to Oxygen • Coenzyme reoxidation by transfer of electrons to oxygen is called electron transport • Electron transport and ATP generation are not independent processes; they are functionally linked to each other Electron Transport and Coenzyme Oxidation • Electron transport involves the highly exergonic oxidation of NADH and FADH with O as the2 2 terminal electron acceptor and so accounts for the formation of water The Electron Transport System • Electron transfer is carried out as a multistep process involving an ordered series of reversibly oxidized electron carriers functioning together • This is called the electron transport system, ETS • The ETS contains a number of integral membrane proteins that are found in the inner mitochondrial membrane (or plasma membrane of bacteria) The Electron Transport System Consists of Five Kinds of Carriers • Flavoproteins • Iron-sulfur proteins • Cytochromes • Copper-containing cytochromes • Coenzyme Q Coenzyme Q • The only nonprotein component of the ETS is coenzyme Q (CoQ), a quinone • Because of its ubiquitous occurrence in nature, it is also called ubiquinone • CoQ is reduced in two successive (one electron plus one proton) steps to semiquinone (CoQH) and then dihydroquinone (CoQH ) 2 • Coenzyme Q (continued) • Unlike the proteins of the ETS, most of the CoQ is freely mobile in the inner mitochondrial membrane • They serve as a collection point for electrons from the reduced FMN and FAD-linked dehydrogenases in the membrane • A portion of the CoQ is tightly bound to specific respiratory complexes • Coenzyme Q (continued) • CoQ accepts both protons and electrons when it is reduced and releases both protons and electrons when it is oxidized • This is vital to its role in the active transport of protons across the inner mitochondrial membrane • It accepts protons on one side of the membrane, diffuses across and releases the protons Most of the Carriers Are Organized into Four Large Respiratory Complexes • Although many electron carriers are part of the ETS, most are organized into multiprotein complexes • Most are thought to be organized into four different kinds of respiratory complexes *******Properties of the Respiratory Complexes******* Each respiratory complex consists of distinctive assembly of polypeptides and prosthetic groups • Complex I transfers electrons from NADH to CoQ and is called the NADH-coenzyme Q oxidation complex (or NADH dehydrogenase complex) • The respiratory complexes • Complex II transfers to CoQ the electrons derived from succinate in Reaction TCA-6 and it is called the succinate-coenzyme Q oxidoreductase complex, or succinate dehydrogenase • Complex III is called the coenzyme Q-cytochrome oxidoreductase complex because it accepts electrons from coenzyme Q and passes them to cytochrome c • Complex III is also called cytochrome b/c comp1ex • Complex IV transfers electrons from cytochrome c to oxygen and is called cytochrome c oxidase • For each pair of electrons transported through complexes I through IV, 10 protons are pumped from the matrix to the intermembrane space The Role of Cytochrome c Oxidase • Cytochrome c oxidase (complex IV) is the terminal oxidase, transferring electrons directly to oxygen • Cyanide and azide are toxic to most aerobic cells because they bind the Fe-Cu center of cytochrome c oxidase, blocking electron transport Electron Transport and ATP Synthesis Are Coupled Events • The crucial link between electron transport and ATP production is an electrochemical proton gradient • It is established by the directional pumping of protons across the membrane in which electron transport is occurring • ATP synthesis is coupled to electron transport Respiratory Control of Electron Transport • The availability of ADP regulates the rate of oxidative phosphorylation and thus of electron transport • This is called respiratory control • Electron transport and ATP generation will be favored when ADP concentration is high and inhibited when ADP concentration is low The Chemiosmotic Model: The “Missing Link” Is a Proton Gradient • In 1961 Peter Mitchell proposed the chemiosmotic coupling model • The essential feature of the model is that the link between electron transport and ATP formation is the electrochemical potential across a membrane • The electrochemical potential is created by the pumping of protons across a membrane as electrons are transferred through the respiratory complexes Coenzyme Oxidation Pumps Enough Protons to Form 3 ATP per NADH and 2 ATP per FADH 2 • The transfer of two electrons from NADH is accompanied by the pumping of a total of 10 protons (12 if the Q cycle is operating) • The number of protons required per molecule of ATP is thought to be 3 or 4, with 3 regarded as most likely • So, about 3 molecules of ATP are synthesized per NADH oxidized • Number of ATP generated is an estimate • FADH do2ates electrons to complex II with higher reduction potential, pumping 6 protons (8 if the Q cycle is operating) • So about 2 ATP are synthesized per FADH 2 • These values are estimates, affected by an organism’s specific ATP synthase and other factor ATP Synthesis: Putting It All Together • Some of the energy of glucose is transferred to reduced coenzymes during glycolysis and the TCA cycle • This energy is used to generate an electrochemical proton gradient across the inner mitochondrial membrane • The proton motive force (pmf) of that gradient is harnessed to make ATP • The F FoA1P synthase • F orovides a channel for exergonic flow of protons across the membrane • F 1arries out the ATP synthesis, driven by the energy of the proton gradient • Together, they form a complete ATP synthase • Synthesis of ATP without thermodynamic cost? • ATP synthesis does not proceed without thermodynamic cost • The needed input of energy occurs elsewhere in the cycle • Energy comes from the proton gradient generated by electron transport and transmitted through rotation of the g subunit The Chemiosmotic Model Involves Dynamic Transmembrane Proton Traffic • There is continuous, dynamic two-way proton traffic across the inner membrane • NADH sends 10 protons across via complexes I, III, and IV; FADH sends 6 acro2s, via complexes II, III, and IV • Assuming that 3 protons must return through F F per ATP oe1erated, this means 3 ATP per NADH and 2 per FADH are 2enerated Aerobic Respiration: Summing It All Up • As carbohydrates and fats are oxidized to generate energy, coenzymes are reduced • These reduced coenzymes represent a storage form of the energy released during oxidation • This energy can be used to drive ATP synthesis as the enzymes are reoxidized by the ETS • As electrons are transported from NADH or FADH to O , they2pass 2hrough respiratory complexes where proton pumping is coupled to electron transport • The resulting electrochemical gradient exerts a pmf that serves as the driving force for ATP synthesis *The Maximum Yield of Aerobic Respiration Is 38 ATPs per glucose


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