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OCR A2 UNIT F214 RESPIRATION Specification: 1. Outline why plants, animals and microorganisms need to respire, with reference to active transport and metabolic reactions; 2. Describe, with the aid of diagrams, the structure of ATP; 3. State that ATP provides the immediate source of energy for biological processes; 4. Explain the importance of coenzymes in respiration, with reference to NAD and coenzyme A; 5. State that glycolysis takes place in the cytoplasm; 6. Outline the process of glycolysis beginning with the phosphorylation of glucose to hexose bisphosphate, splitting of hexose bisphosphate into two triose phosphate molecules and further oxidation to pyruvate, producing a small yield of ATP and reduced NAD; 7. State that, during aerobic respiration in animals, pyruvate is actively transported into mitochondria; 8. Explain, with the aid of diagrams and electron micrographs, how the structure of mitochondria enables them to carry out their functions; 9. State that the link reaction takes place in the mitochondrial matrix; 10. Outline the link reaction, with reference to decarboxylation of pyruvate to acetate and the reduction of NAD; 11. Explain that acetate is combined with coenzyme A to be carried to the next stage; 12. State that the Krebs cycle takes place in the mitochondrial matrix; 13. Outline the Krebs cycle, with reference to the formation of citrate from acetate and oxaloacetate and the reconversion of citrate to oxaloacetate (names of intermediate compounds are not required); 14. Explain that during the Krebs cycle, decarboxylation and dehydrogenation occur, NAD and FAD are reduced and substrate level phosphorylation occurs; 1 15. Outline the process of oxidative phosphorylation, with reference to the roles of electron carriers, oxygen and the mitochondrial cristae; 16. Outline the process of chemiosmosis, with reference to the electron transport chain, proton gradients and ATPsynthase; 17. State that oxygen is the final electron acceptor in aerobic respiration; 18. Evaluate the experimental evidence for the theory of chemiosmosis; 19. Explain why the theoretical maximum yield of ATP per molecule of glucose is rarely, if ever, achieved in aerobic respiration; 20. Explain why anaerobic respiration produces a much lower yield of ATP than aerobic respiration; 21. Compare and contrast anaerobic respiration in mammals and in yeast; 22. Define the term respiratory substrate; 23. Explain the difference in relative energy values of carbohydrate, lipid and protein respiratory substrates. Definition of Cell Respiration Cell respiration is the process whereby energy, stored in complex organic molecules*, is transferred to ATP in living cells. ATP provides the immediate source of energy in cells, for biological processes. *The complex organic molecules referred to above are the respiratory substrates and include carbohydrates, lipids and proteins Summary of Energy Transfer between Organisms Photoautotrophs use sunlight energy to synthesise complex organic molecules from simple inorganic molecules and ions in photosynthesis Photoautotrophs are the producers in many food chains – plants, some protoctists and some bacteria Heterotrophs are the consumers or decomposers in food chains – animals, fungi and most bacteria . 2 Heterotrophs obtain their complex organic molecules and energy, by feeding on photoautotrophs or other heterotrophs that have fed on photoautotrophs. Heterotrophs have to feed on other organisms All organisms must respire to transfer the chemical potential energy of carbohydrates, lipids and proteins to ATP The summary below also shows that some of the potential chemical energy in respired organic molecules is converted to thermal energy. This is important to maintain cell temperatures suitable for enzyme reactions 3 Metabolism and Metabolic Reactions in Cells Metabolism refers to all the chemical reactions that occur in cells Most metabolic reactions occur within metabolic pathways A metabolic pathway has the following features: A sequence of chemical reactions where the product of one reaction is the substrate for the next. Each reaction is catalysed by a particular enzyme. Enzyme a Enzyme b Enzyme c A B C D Two Types of Metabolic Pathway - Catabolic and Anabolic CATABOLIC REACTIONS ANABOLIC REACTIONS Involve the break down of larger molecules into smaller molecules Involve the synthesis of larger molecules from smaller molecules Release energy Require energy Examples include: Breakdown of glucose to pyruvate in glycolysis in respiration Digestion of starch to maltose Examples include: Protein synthesis from amino acids in all living cells The synthesis of carbohydrate, protein and lipid molecules in photosynthesis Advantages of metabolic pathways include: Greater control over the release of energy from catabolic reactions, preventing cell damage Intermediate products may be useful themselves, or be substrates for other pathways The final product may act as an inhibitor of an earlier enzyme in the pathway leading to end product inhibition, an important method of regulating the pathway 4 Why Organisms Need Energy and ATP Energy in the form of ATP is required to drive metabolic reactions in all living cells. These metabolic reactions include the following: Active transport of molecules and ions across plasma membranes, against a concentration gradient Secretion of large molecules by exocytosis Endocytosis Anabolic reactions as detailed in the table on page 4 DNA replication and organelle synthesis in interphase Movement such as movement of bacterial flagella, eukaryotic cilia and flagella, muscle contraction and microtubule motors that move organelles within cells Phosphorylation of metabolites to activate them – such as phosphorylation of glucose in glycolysis so that it can be broken down to release energy Some energy released from reactions is in the form of thermal energy and this is important to maintain cell temperatures suitable for enzyme controlled reactions ATP (Adenosine Triphosphate) Structure of ATP ATP is a mononucleotide consisting of the nitrogenous base adenine, the pentose sugar ribose, (adenine + ribose = adenosine) and three phosphate groups (rather than the usual one in a nucleotide). Number the carbon atoms of ribose in the diagram above 5 Draw and label the components of a molecule of ATP Features of ATP ATP is one of the main products of aerobic and anaerobic respiration in cells ATP has a universal role as an energy source in living cells. ATP is often referred to as the universal energy currency of a cell ATP is an immediate source of energy in a cell When hydrolysed, a small amount of energy is released that will not damage the cell ATP can release energy very quickly in the cell as required In cells, the release of energy by hydrolysis of ATP is coupled with a metabolic reaction that requires energy eg protein synthesis To release energy, ATP is broken down by hydrolysis into ADP (adenosine diphosphate) and inorganic phosphate (Pi). This reaction is exergonic (releases energy) and is catalysed by the enzyme ATPase (ATP synthase) ATP + H2O ADP + Pi + energy (30.6kJmol-1) In this reaction, 30.6kJ of energy is released per mole of ATP. This small amount of energy is often sufficient for an energy requiring process This reaction is reversible, the reverse reaction is also catalysed by ATPase (ATPsynthase). ATP is synthesised from ADP and Pi in 6 respiration (oxidative phosphorylation and substrate level phosphorylation reactions) More energy is released if the ADP product is hydrolysed further ADP + H2O AMP + Pi + energy (30.6kJmol-1) AMP stands for adenosine monophosphate ATP is an efficient energy donor molecule because the covalent bonds between the phosphate groups are unstable – the phosphate groups themselves are negatively charged and repel each other ATP is a small water-soluble molecule and easily diffuses within the cell to the site of use ATP remains in cells in low concentrations and is not normally transported from one cell to another (although companion cells in phloem tissue do transport ATP to the phloem sieve tube elements). ATP can only be stored for a few minutes and therefore, it must be continuously produced as it provides an immediate source of energy in the cell. This means that all cells must continually carry out cell respiration Coenzymes in Respiration Coenzymes are molecules that are required for the activity of some enzymes Two vital coenzymes in respiration are needed for oxidationreduction reactions. These molecules are NAD and FAD Many reactions in respiration, involve oxidation of substrates by removal of electrons and protons (hydrogen atoms). If the oxidized substrate is to remain in this form, the electrons/hydrogen atoms must be removed from the substrate. This can be done by passing the electrons and protons (hydrogen atoms) to an electron carrier molecule such as NAD or FAD To summarise, the oxidation of a substrate in respiration is coupled to the reduction of NAD or FAD, as two hydrogen atoms are removed from the substrate and transferred to the coenzyme NAD or FAD 7 NAD and FAD are called coenzymes because they are needed for the activity of dehydrogenase enzymes that catalyse oxidation/reduction reactions NAD (nicotinamide adenine dinucleotide) NAD is a di-nucleotide made of two linked nucleotides Both nucleotides of NAD contain ribose as the pentose sugar and one phosphate group One nucleotide has adenine as the nitrogenous base; the other contains nicotinamide (derived from vitamin B3). Nicotinamide is a ring structure that can accept hydrogen atoms When a molecule of NAD has accepted two hydrogen atoms it is reduced. When it donates the two electrons and protons, it is re-oxidised Reaction to show the reduction of NAD+ NAD+ + 2H+ + oxidized 2e- NADH + H+ reduced This reaction is reversible and cataysed by an oxidoreductase enzyme called dehydrogenase. Note that NADH + H+ may also be written as reduced NAD 8 FAD (flavine adenine dinucleotide) FAD is a di-nucleotide containing ribose, two phosphate groups, adenine and flavine (a derivative of the B vitamin riboflavine) Reaction to show the reduction of FAD FAD + 2H+ + oxidised 2e- FADH2 reduced This reaction is reversible and catalysed by a dehydrogenase enzyme FADH2 may also be written as reduced FAD Coenzyme A (CoA) Coenzyme A is not a coenzyme linked to oxidation-reduction reactions The function of CoA is to transfer ethanoate (acetate) groups from pyruvate, fatty acids and some amino acids into the Krebs cycle in respiration. Coenzyme A is used in the link reaction A Summary of Aerobic and Anaerobic Respiration 9 Locations of Stages of Aerobic and Anaerobic Respiration STAGE Glycolysis Link reaction Krebs cycle Oxidative phosphorylation Anaerobic respiration LOCATION IN CELL Cytoplasm Mitochondrial matrix Mitochondrial matrix Inner mitochondrial membrane/cristae Cytoplasm Glycolysis Location - Cytoplasm of the cell Glycolysis is a metabolic pathway involving ten enzyme controlled reactions. For the A level examination, you only need to know the pathway in outline Glycolysis is the first pathway of aerobic respiration. It also occurs in anaerobic respiration in which it is the sole source of ATP Summary: one molecule of Glucose (6C) is converted to two molecules of pyruvate (3C) There are four main events that take place in glycolysis: 1. Phosphorylation of Glucose to Hexose bis-phosphate The addition of a phosphate group to glucose forms glucose phosphate (also referred to as hexose phosphate) The source of phosphate is ATP, which is hydrolysed to ADP and Pi Energy is also released for the phosphorylation reaction, during ATP hydrolysis A second phosphorylation of the hexose phosphate, also using ATP, produces hexose bis-phosphate (bis means that the hexose sugar has 2 phosphate groups on different carbon atoms) 10 Reasons for phosphorylation of glucose Phosphorylation makes the glucose more reactive Phosphorylation also prevents the glucose from leaving the cell (glucose phosphate is too large to leave the cell) Summary of the reaction GLUCOSE HEXOSE BIS-PHOSPHATE Complete the detail of each reaction summary, including the number of carbon atoms within each substrate and product 2. Splitting of Hexose bis-phosphate Each hexose bis-phosphate molecule is split into two triose phosphate (3C) molecules. Triose phosphate may be abbreviated to TP Summary of the reaction HEXOSE BIS-PHOSPHATE TRIOSE PHOSPHATE 3. Oxidation of Triose Phosphate and ATP formation by Substrate Level Phosphorylation Each triose phosphate molecule loses 2 hydrogen atoms and becomes oxidized and dehydrogenated The oxidation of triose phosphate is catalysed by a dehydrogenase enzyme. NAD+ accepts the 2 hydrogen atoms from triose phosphate and becomes reduced to NADH +H+ 11 Two molecules of reduced NAD are produced from the 2TP GP conversions The product of triose phosphate dehydrogenation is (3C) Glycerate-3phosphate (glycerate phosphate may be abbreviated to GP) During the conversion of TP to GP, enough energy is released to directly phosphorylate ADP to ATP. ATPase/ATP synthase catalyses this reaction Two molecules of ATP are produced from the 2 TP 2 GP conversions. This method of ATP synthesis is called substrate level phosphorylation Summary of the reaction TRIOSE PHOSPHATE GLYCERATE PHOSPHATE 4. Conversion of Glycerate-3-Phosphate to Pyruvate Glycerate phosphate is finally converted to pyruvate During this conversion, there is again, sufficient energy released to phosphorylate ADP to ATP by substrate level phosphorylation, catalysed by ATPase/ATP synthase 2 molecules of ATP are produced when 2 molecules of GP are converted to 2 molecules of pyruvate 12 Summary of glycolysis GLYCERATE PHOSPHATE PYRUVATE ATP Yield from One Molecule of Glucose in Glycolysis 4 molecules of ATP produced from substrate level phosphorylation 2 molecules of ATP used up in the initial phosphorylation steps Overall yield of ATP from 1 molecule of glucose in glycolysis: 4 - 2 = 2 ATP molecules per molecule of glucose Products of Glycolysis from one molecule of glucose: 2 x pyruvate (3C) 2 x NADH +H+ Net gain of 2 x ATP 13 Summary of Glycolysis Number of molecules Number of carbon atoms GLUCOSE HEXOSE BIS-PHOSPHATE TRIOSE PHOSPHATE GLYCERATE PHOSPHATE PYRUVATE Mitochondrial Structure Mitochondria are usually 1.0µm in diameter and up to 5µm long. More active cells will have larger mitochondria and more of them All mitochondria have inner and outer mitochondrial membranes. Together they form the mitochondrial envelope The outer membrane is smooth and has a similar phospholipid bilayer structure to other organelle membranes, with channel and carrier proteins and some enzymes The inner membrane is folded into cristae. These increase the membrane surface area. Electron carriers and the stalked particles with ATP synthase molecules are located within the inner membrane 14 The inter-membrane space is formed between the outer and inner mitochondrial membranes The matrix is surrounded by the inner membrane. The matrix is gel-like and contains a loop of mitochondrial DNA and small (70S) ribosomes. Mitochondrial Structure to Function Mitochondria are the site of aerobic respiration in eukaryotic cells, specifically, the site of the Link reaction, Krebs cycle and oxidative phosphorylation The mitochondrial membranes compartmentalize the reactions that occur in aerobic respiration and confine the enzymes involved within a small area The smooth outer mitochondrial membrane is partially permeable and has carrier proteins for the active transport of pyruvate into the mitochondrion, from the cytoplasm 15 The matrix is the site of the link reaction and Krebs cycle. For these reactions, the matrix contains the substrates (eg oxaloacetate), NAD, FAD, coenzyme A and the enzymes required The loop of DNA has genes that code for proteins. The 70S ribosomes are the site of synthesis of proteins such as ATP synthase, proteins acting as electron carriers and enzymes involved in the link reaction and Krebs cycle The inner mitochondrial membrane is the site of oxidative phosphorylation by which ATP is synthesised The electron transport chain carriers and ATP synthase (in the stalked particles) are within the inner mitochondrial membrane. The stalked particles have a protein channel that allows protons to pass through them by chemiosmosis. This proton flow is linked to ATP synthesis Apart from the protein channels in the stalked particles, the inner membrane is impermeable to small ions such as protons. This allows the protons to accumulate in the inter-membrane space, building up a proton gradient between the space and the matrix. This is the source of potential energy used to synthesise ATP The increased surface area of the cristae increases the number of electron carriers and the number of ATP synthase enzymes, for increased ATP synthesis 16 X 200,000 17 The Link Reaction Location - The matrix of the mitochondrion. Pyruvate (produced in the cytoplasm from glycolysis) must first pass into the mitochondrion matrix. It passes across the mitochondrial membranes, by active transport The link reaction requires coenzyme A (CoA) to transfer a 2 carbon acetate group from pyruvate into the Krebs cycle reactions Summary of the link reaction 2 Pyruvate + 2 CoA + 2 NAD+ 2 acetyl CoA + 2 CO2 + 2 NADH + H+ The link reaction involves 1. Pyruvate losing CO2 by de-carboxylation, catalysed by a decarboxylase enzyme 2. Pyruvate transferring an acetyl group to coenzyme A to form acetylCoA 3. Pyruvate losing 2 hydrogen atoms by dehydrogenation, catalysed by a dehygrogenase. The 2 hydrogen atoms are accepted by NAD+ , producing reduced NAD+ /NADH+H+ 4. Pyruvate is oxidized and dehydrogenated in the link reaction 5. The link reaction takes place twice for each molecule of glucose as two molecules of pyruvate are produced from one molecule of glucose. The overall final products of the Link Reaction, from one molecule of glucose, are as follows: 2 x Acetyl Coenzyme A 2 x CO2 2 x NADH + H+ 18 Krebs Cycle Location: the matrix of the mitochondrion Summary of the Krebs Cycle reactions Oxaloacetate (4C) combines with acetate (2C) from acetyl CoA to form citrate (6C) The cycle involves a series of enzyme controlled reactions that convert citrate (6C) back to oxaloacetate (4C), so that it may be re-used in the cycle During the reactions, CO2 is produced as a waste product Several molecules of reduced coenzymes are produced in oxidation/reduction reactions, involving the transfer of hydrogen atoms from intermediates in the cycle, to NAD+ or FAD A little ATP is also produced directly by substrate level phosphorylation Three main processes involved in one cycle 1. Removal of carbon dioxide from intermediates by decarboxylation Decarboxylation occurs twice in each cycle, once when a 6C molecule is being converted to a 5C molecule and again when a 5C molecule is converted to a 4C molecule. Two molecules of CO2 are formed per turn of the cycle. These decarboxylation reactions are catalysed by decarboxylase enzymes 2. Oxidation/Dehydrogenation Removal of 2 hydrogen atoms from an intermediate occurs four times in each cycle producing three molecules of NADH/H+ and one molecule of FADH2. The intermediate is oxidised/dehydrogenated and NAD+ and FAD are reduced 3. Phosphorylation of ADP This happens once in each cycle producing one molecule of ATP directly, by substrate level phosphorylation. The conversion of one intermediate to another releases sufficient energy for ATP synthesis from ADP and Pi. 19 Krebs Cycle occurs Twice per Molecule of Glucose Remember that the cycle is performed twice for each molecule of glucose as two pyruvate molecules, and therefore, two acetyl CoA, molecules are produced from each molecule of glucose. Final products of Krebs cycle, for one molecule of glucose, are as follows: 4 x CO2 6 x NADH + H+ 2 x FADH2 2 x ATP (directly) Summary of Krebs Cycle 2C acetyl-CoA CoA acetate 4C oxaloacetate 6C citrate 4C intermediate 5C intermediate 4C intermediate 4C intermediate 20 Oxidative Phosphorylation Location: Cristae/inner mitochondrial membrane Aims of Oxidative Phosphosphorylation To synthesise ATP (most of the ATP produced in aerobic respiration is synthesised in this process) To re-oxidise NADH + H+ and FADH2 so that the processes of glycolysis, link reaction and Krebs cycle may continue Processes occurring in oxidative phosphorylation In the process of re-oxidising NADH + H+ and FADH2, electrons are passed from NADH + H+ and FADH2 to a series of electron carriers in the electron transport chain (ETC). These electron carriers are located in the inner membrane of the mitochondrion and are enzyme complexes The first electron carrier to accept electrons from reduced NAD is a protein complex called complex I. This complex contains reduced NAD dehydrogenase enzyme. At complex I, reduced NAD releases 2 protons and 2 electrons. The protons are released into solution in the matrix and the electrons are donated to complex I which becomes reduced. The electrons are transferred along a series of complexes in the inner mitochondrial membrane. Each complex becomes reduced as it accepts the electrons and then re-oxidised as it transfers the electrons to the next carrier in the chain The final electron acceptor in the chain is oxygen, which is reduced to water. Both electrons and protons are required to reduce water, as shown below O2 + 4H+ + 4e- 2H2O (or ½ O2 + 2H+ + 2e H2O) The transfer of electrons along the electron carriers releases free energy, since each carrier is at a lower energy level than the previous carrier. This energy is used to synthesise ATP from ADP and Pi As re-oxidation of NADH + H+ and FADH2 only takes place in the presence of oxygen, and is coupled with phosphorylation of ADP, the processes occurring on the cristae are known as oxidative phosphorylation 21 A Simplified Version of the Electron Transport Chain ATP Synthesis in Oxidative Phosphorylation and the Chemiosmosis Theory The energy released from the electrons as they are transferred along the electron transport chain, is used to pump the protons (H+) out of the matrix and into the inter-membranal space. This is not active transport since ATP is not required. The energy required for the proton pumps is released during electron flow in the electron transport chain A high concentration of protons accumulate in the inter-membranal space, creating an electrochemical gradient of protons between the intermembranal space and the matrix 22 There are (protein) proton channels in the stalked particles, located in the cristae membrane, that allow diffusion of these protons from the intermembranal space back into the matrix, down a proton gradient The stalked particles have ATP synthase located in their structure. When protons diffuse through the protein channel of the stalked particles back into the matrix, the kinetic energy released due to the proton motive force is used to generate ATP from ADP and Pi The diffusion of protons through the stalked particles is called chemiosmosis (although nothing to do with water potentials!) Error! Yield of ATP from Oxidative Phosphorylation Re-oxidation of one molecule of NADH/H+ releases 2.6 ATP molecules Re-oxidation of one molecule of FADH2 releases 1.6 ATP molecules (this is because FADH2 transfers its electrons to an electron transport carrier further down the chain than NADH + H+ - check the top diagram on page 20 23 Overall Yield of ATP from One Molecule of Glucose in Aerobic Respiration Name of Stage Source of ATP Glycolysis Substrate level phosphorylation Substrate level phosphorylation 2 x NADH + H+ 2 x NADH + H+ 6 x NADH + H+ 2 x FADH2 Krebs Cycle Glycolysis Link Reaction Krebs Cycle Krebs Cycle Number of ATP molecules per glucose molecule 2 2 5.2 5.2 15.6 3.2 Total is approx. 33 ATP molecules Why is the theoretical yield of ATP from aerobic respiration rarely achieved? According to the table above, approximately 33 ATP molecules should be synthesised from each glucose molecule in aerobic respiration. However, this total of ATPs is rarely achieved because: Some protons leak across the outer mitochondrial membrane, reducing the proton concentration for generating the proton motive force Some ATP is used to actively transport pyruvate into the mitochondrion from the cytoplasm Some ATP is used to bring hydrogen from reduced NAD made in glycolysis, into the mitochondrial matrix (the inner membrane is impermeable to reduced NAD) Efficiency of Aerobic Respiration using Glucose as the Respiratory Substrate Aerobic respiration is a relatively efficient process for releasing energy from a fuel, such as glucose Less than 40% of the energy in glucose is used to synthesise ATP The remaining energy is lost as heat, which in mammals warms the cells and the blood. Much heat is lost eventually to the atmosphere. 24 Evaluation of the Evidence for Chemiosmosis The chemiosmotic theory was devised by Peter Mitchell in 1961 and at first,his theory was treated with scepticism. Now there is much evidence in support and he was awarded the Nobel Prize for Chemistry in 1978 Scientists now know: That the stalked particle is ATP synthase That some of the complexes in the electron transport chain have coenzymes that can use the energy released from electron transport to pump protons across the inner membrane into the inter-membrane space When mitochondria are placed into a solution of higher water potential, the outer membrane ruptures releasing the contents of the inter-membranal space. Electron transport in these mitochondria did not produce ATP suggesting that the space was important for ATP synthesis ATP synthesis does not occur in the presence of oligomycin, an antibiotic that blocks the flow of protons through ATP synthase ATP is not synthesised if the head of the stalked particle is removed. 25 In intact mitochondria, the potential difference across the inner membrane is -200mV, being more negative on the matrix side of the membrane (lower concentration of positive ions) Also, in intact mitochondria, the pH of the inter-membrane space is lower than that of the matrix The outer mitochondrial membrane is permeable to protons. If isolated mitochondria are supplied with ADP and inorganic phosphate and placed in a solution of pH 8, no ATP is produced. If however, these mitochondria are placed in an acidic solution, ATP is produced. This indicates that the protons must accumulate in the inter-membrane space Anaerobic Respiration Anaerobic respiration is respiration in the absence of oxygen Consequences of a lack of oxygen The electron transport chain cannot function as there is no oxygen to act as the final electron acceptor NADH + H+ and FADH2 produced from the link reaction or Krebs cycle, cannot be re-oxidized, without the ETC The link reaction and Krebs cycle cannot take place, without a supply of NAD+ and FAD Oxidative phosphorylation cannot occur and therefore. ATP cannot be synthesized in this process 26 The only stage that does generate ATP, in anaerobic respiration, is glycolysis, although far less ATP ( 2 ATP molecules per molecule of glucose) is produced in this pathway on its own However, for glycolysis to continue, NAD+ must be regenerated from NADH + H+ and this occurs by another mechanism, not involving the ETC . Two alternative mechanisms for the re-oxidation of NADH + H+ are used by living organisms: Anaerobic Respiration in Yeast and Green Plants Site of anaerobic respiration: in the cytoplasm First of all, glycolysis occurs to produce pyruvate and NADH + H+ Pyruvate is then decarboxylated (CO2 is removed) to produce ethanal. This reaction is catalysed by pyruvate decarboxylase The ethanal is then reduced to ethanol, using 2 hydrogen atoms from NADH + H+. this reaction is catalysed by ethanol dehydrogenase This means that the NADH + H+ is re- oxidized to NAD+ that can be reused in glycolysis, so that glycolysis can continue The energy yield from glycolysis and hence anaerobic respiration is much lower than from aerobic respiration. 2 ATP molecules are released per molecule of glucose substrate. Some energy remains in the ethanol product Ethanol is toxic and if allowed to build up will eventually kill the yeast/plant cells Anaerobic fermentation in yeast cells is an important process exploited in the production of beer and wine. It is therefore also known as alcoholic fermentation. Complete the equation below PYRUVATE ETHANAL 27 ETHANOL Note the error in the flow diagram above Anaerobic respiration in Animals (in muscle) Site of anaerobic respiration: the cytoplasm Again, glycolysis continues in the absence of sufficient oxygen, to produce pyruvate and NADH + H+ in the muscle cells Pyruvate is reduced directly by the transfer of 2 hydrogen atoms from NADH + H+. the product is lactate and NAD+ is regenerated The regenerated NAD+ can then be re-used in glycolysis Complete the equation below PYRUVATE LACTATE 28 Lactate will build up in muscles during heavy exercise, causing muscle fatigue, as lactate is toxic However the reaction described above is reversible and the lactate can be metabolized when more oxygen becomes available. The lactate is transported to the liver where it is oxidized back to pyruvate, which can then be aerobically respired. Liver cells can tolerate lactate/low pH and have the enzymes to metabolise lactate, unlike muscle cells. Also, the conversion of lactate requires oxygen which is deficient in muscle cells during anaerobic respiration Alternatively the pyruvate can be converted to glycogen and stored. in liver cells The oxygen needed to fully oxidize the lactate produced by anaerobic respiration is called the oxygendebt. REMEMBER THAT ANAEROBIC RESPIRATION ALWAYS STARTS WITH GLYCOLYSIS. IF YOU ARE ASKED TO DESCRIBE ANAEROBIC RESPIRATION IN MAMMALS OR YEAST, YOU MUST DESCRIBE THE DETAILS OF GLYCOLYSIS FIRST 29 Comparison of Anaerobic respiration in Yeast and Mammals YEAST MAMMALS Hydrogen acceptor Is carbon dioxide produced? Is ATP produced? Is NAD re-oxidised? End products Enzymes involved Respiratory Substrates A respiratory substrate is an organic molecule that can be used for respiration Energy Values of Different Respiratory Substrates Most ATP is produced in oxidative phosphorylation as protons flow through ATP synthase It follows that the more hydrogen atoms a substrate has per molecule, the more protons will flow and the more ATP will be produced in respiration It also follows that a substrate containing more hydrogen atoms per mole will require more oxygen per mole of substrate for respiration Carbohydrate Glucose is the main respiratory substrate in mammalian cells. Brain cells can only respire glucose Animal cells store glycogen and plant cells store starch. Both polysaccharides can be hydrolysed to glucose as a respiratory substrate Fructose and galactose, both hexose sugars are converted to glucose for respiration The theoretical energy yield from glucose is 2870kJmol-1 30 Therefore, the theoretical ATP yield is 94mol per mol of glucose (the hydrolysis of ATP to ADP + Pi produces 30.6kJmol-1) The actual ATP yield is around 30 moles of ATP; an efficiency of 32% The remaining energy is lost as heat but this is important to maintain enzyme activity Protein Excess amino acids are deaminated in the liver. The keto acids produced can be metabolised to glycogen or fat for storage When an individual is fasting or undergoing vigorous exercise, amino acids from muscle tissue can be respired Some amino acids can be converted to pyruvate, some to acetate and some enter the Krebs cycle directly One mol of protein contains slightly more hydrogen atoms than one mol of glucose. Therefore slightly more ATP is produced per mol of protein Lipids Lipids are important respiratory substrates particularly in muscle Glycerol from triglycerides can be converted to glucose and then respired 31 Fatty acids have hydrocarbon chains, some with many hydrogen atoms These hydrogen atoms are a source of many protons for chemiosmosis, so fatty acids produce many ATP molecules Each fatty acid combines with CoA. This requires energy from the hydrolysis of ATP to AMP + 2Pi The fatty acid-CoA complex enters the mitochondrial matrix where 2C acetate groups are successively removed from the fatty acid, and combined with CoA. This pathway is called β-oxidation The acetyl CoA complexes enter Krebs cycle The β-oxidation pathway produces reduced NAD and reduced FAD (one molecule of each for each acetate group released). A long chain fatty acid therefore produces many reduced NAD and reduced FAD molecules both in β-oxidation and Krebs cycle Energy Values of Different Respiratory Substrates Respiratory Substrate Carbohydrate Lipid Protein Energy Content kJg-1 15.8 39.4 17.0 32 33 Measuring Rates of Respiration using Respirometers The rate of respiration of small organisms can be determined by measuring the volume of oxygen taken in over a known time, using a respirometer. One type of respirometer is shown below The respirometer consists of: Two identical chambers – one containing the respiring organisms (woodlice in this experiment) and the other a control chamber containing an equal volume of non-respiring material such as glass beads. the glass beads must be put into the control chamber (A) otherwise A would contain more air than B The two chambers are connected by a U-shaped manometer tube containing a coloured fluid The point of the control chamber is to ensure that any temperature/pressure fluctuations affect both chambers equally and therefore cancel each other out 34 An equal volume of a substance that absorbs CO2 – such as soda lime, is added to each chamber Use of this respirometer to measure the effects of temperature on the rate of respiration Method: The apparatus is left in the water bath for 10min to allow it to reach the required temperature. Screw clips A and B are left open during this time to allow expanded air to escape After 10 min the screw clips A and B are closed. The level of the fluid in the manometer tube is equal on both sides The woodlice respire, using up O2 and releasing CO2 The CO2 released from the respiring woodlice in chamber B is absorbed by the soda lime Since the woodlice are removing O2 from the air in chamber B, there is a reduction in B’s air volume Since chamber B is air-tight, there is a reduction in air pressure in chamber B The air pressure in chamber A is now greater than that in chamber B, so air moves from chamber A to chamber B causing the fluid in the U-tube to move towards tube B The distance moved by the manometer fluid in a given time is measured Clip A is opened and the syringe plunger above tube A is raised to equate the air pressures in both A and B (fluid is at equal height ib both sides of the manometer tube) The method is repeated twice more at the same temperature to collect replicate results Both clips A and B are opened to allow oxygen to diffuse back into chamber B The procedure is repeated at a different temperature 35 Calculating the volume of oxygen used up per unit time Volume = πr2h Where r is the internal radius of the manometer tube and h is the distance moved by the fluid per unit time What effect will temperature have on respiration rate? As temperature increases, respiration rate will increase. This is because at higher temperatures, enzyme and substrate molecules have increased kinetic energy and energetically favourable collisions are more likely The important enzymes affected by temperature in respiration are the decarboxylases, dehydrogenases and ATP synthase At higher temperatures, enzymes may be denatured and respiration rate will decrease A simpler respirometer used in the laboratory 36 The simple respirometer shown on page 35 contains pea seeds previously soaked in water to start their germination When soaked seeds germinate, they start to grow a root and a shoot. Growth requires repeated mitosis and cell division, DNA replication and protein synthesis. All of these processes require more ATP – hence soaked seeds have an increased respiration rate A control could be set up with an equivalent volume of glass beads or dry pea seeds. Dry pea seeds will have a very slow growth rate (or none at all) and reduced/no respiration because water is required as a medium for metabolic reactions – water allows enzymes and substrates to move and collide so that reactions can occur Calculating Respiratory Quotients This is not strictly on the syllabus but is an application of using respirometers. Therefore, it is worthwhile you being familiar with the concept of respiratory quotients The respiratory quotient (RQ) is a measure of the ratio of carbon dioxide given out by a respiring organism to the oxygen consumed over a given time period: RQ = volume of CO2 given out / volume of O2 taken in Different RQ values indicate the type of respiratory substrate Glucose gives an RQ of 1.0. Lipids contain more carbon and less oxygen and therefore give out more CO2. Their RQs are lower than carbohydrates (approx. 0.7) Proteins give variable RQ values depending upon the types of amino acids they contain. Most proteins give an RQ value of approx.. 0.9 Most respiring organisms have an RQ of between 0.8 to 0.9. This suggests that protein is the main respiratory substrate but this would only be the case in starvation. Therefore, values of 0.8-0.9 suggest a mixture of lipids and carbohydrates in the diet as respiratory substrates 37 Studying respiration using redox indicators A redox indicator is one that changes colour when it accepts electrons and becomes reduced One example is TTC (2,3,5-triphenyl-tetrazolium chloride) which is colourless when oxidised and pink when reduced TTC will diffuse into cells and accept electrons from the electron transport chain if the cells are actively respiring The speed of the colour change is an indication of the speed of metabolic activity. A faster change in colour indicates more reduced NAD production in glycolysis, link reaction and Krebs cycle There will then be more electron transfer to TTC and a faster TTC colour change to pink Examination tip OCR has a habit of including applied questions in their papers, often based on data from an experiment. In your answers you must include A level factual information so think hard about the scientific background to the data and include this information 38