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F214 - Respiration 1. Outline why plants, animals and microorganisms need to respire, with reference to active transport and metabolic reactions. Respiration is the process in which energy, that is stored in complex organic molecules in living cells, is used to make ATP. ATP is necessary to drive all the necessary metabolic process inside the body, such as: Active transport - much of an organism’s energy is used for this Secretion- large molecules made in some cells are released by exocytosis Endocytosis- bulk movement of larger molecules into the cell Metabolic reactions- synthesis of large molecules from smaller ones e.g. proteins from Amino Acids, steroids from cholesterol, cellulose from β-glucose = anabolic reactions Catabolic reactions = hydrolysis of larger molecules to produce smaller ones. Replication of DNA and synthesis of organelles before a cell divides Movement of Bacterial flagella, Eukaryotic cilia and undulipodia. Muscle contractions Activation of chemicals e.g. phosphorylation of glucose 2. Describe, with the aid of diagrams, the structure of ATP ATP is a phosphorylated nucleotide found in both prokaryotic and eukaryotic cells. Each molecule consists of adenosine (adenine and ribose sugar) plus 3 phosphate/phosphoryl groups. ATP can be hydrolysed to ADP and inorganic phosphate (Pi). This releases immediate energy to cells, in adequate amounts, for biological processes. 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 (CoA). Coenzymes aid enzymes in oxidation and reduction reactions of respiration: > NAD combines with hydrogen atoms in oxidation, taking them to the mitochondrial membrane where they later split into hydrogen ions and electrons for the election transport chain and the production of ATP in oxidative phosphorylation. When NAD accepts 2 hydrogen ions it is reduced. NAD is important to most stages of respiration. >CoA carries acetate groups from the link reaction, or those made from fatty/amino acids onto the Krebs cycle. F214 - Respiration 5. State that Glycolysis occurs in the cytoplasm of cells 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. 1) An ATP molecule is hydrolysed and the phosphate lost attaches to a glucose molecule at C6 2) = Glucose-6-Phosphate, which becomes fructose 6 phosphate 3) Another ATP is hydrolysed, and the phosphate attaches to C-1 4) The hexose sugar is activated by the energy release from the hydrolysed ATP molecules, preventing it from leaving the cell. This becomes Hexose1,6-bisphosphate 5) Hexose-1,6-bisphosphate is split into 2 molecules of Triose Phosphate (3 carbon sugar molecules) 6) 2 hydrogen atoms are removed from each Triose Phosphate (the substrate). This involves dehydrogenase enzymes. 7) NAD combines with the removed hydrogen atoms to form reduced NAD 8) At this stage 2 molecules of ATP are formed (substrate level phosphorylation). 9) 4 enzyme-catalysed reactions convert each triose phosphate molecule to a 3C molecule of pyruvate. 10) 2 more molecules of ATP are formed so there is a net gain of 2 ATP. ATP is used in the phosphorylation stages and produced in oxidation and the production on pyruvate. NAD is reduced during oxidation. 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 Allows protons to pass through inner membrane The ETC contain 100s of oxidoreductase enzymes - involved in oxidation and reduction reactions. Some of the electron carriers also have a coenzyme that pumps protons from the matrix to the intermembrane space. F214 - Respiration 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 The link reaction converts pyruvate (produced during glycolysis) into acetate (a 2 carbon compound) by decarboxylation and dehydrogenation, catalysed by enzymes. > Pyruvate dehydrogenase removes hydrogen atoms from pyruvate. > Pyruvate decarboxylation removes a carboxyl group from pyruvate. > NAD accepts the hydrogen atoms to become reduced NAD and carries the hydrogen to the mitochondrial matrix for the production of ATP in oxidative phosphorylation. 11. Explain that acetate is combined with coenzyme A to be carried to the next stage Coenzyme A accepts acetate to become acetyl coenzyme A. The function of coenzyme A is to carry acetate to the Krebs cycle. 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) 1) Acetate is released from CoA and joins to oxaloacetate to form citrate. 2) Citrate is decarboxlyated (carboxyl group becomes CO2) and dehydrogenated (H is then accepted by NAD to form reduced NAD and taken to the electron transport chain) = forms a 5C compound. 3) The 5C compound is decarboxylated and dehydrogenated to form a 4C compound and another molecule of reduced NAD. 4) The 4C compound is changed into another 4C compound, and a molecule of ADP is phosphorylated to form ATP. 5) The second 4C compound is changed into a third 4C compound. A pair of hydrogen atoms are removed and accepted by FAD, reducing FAD. There is one cycle for each molecule of acetate (which is made from one molecule of pyruvate) Therefore the cycle occurs twice for each molecule of glucose. Therefore 6x Reduced NAD, 2x Reduced FAD, 4x CO2 and 2x ATP is produced by Krebs for each molecule of glucose. 6) The third 4C compound is further dehydrogenated and regenerates oxaloacetate. Another molecule of NAD is reduced. F214 - Respiration 14. Explain that during the Krebs cycle, decarboxylation and dehydrogenation occur, NAD and FAD are reduced and substrate level phosphorylation occurs > Decarboxylation and dehydrogenation occurs in 2), 3) and 6) > NAD is reduced 3 times, when dehydrogenation occurs in 2), 3) and 6) > FAD is reduced when a pair of hydrogen atoms are removed in 5) > Substrate level phosphorylation occurs when ADP is phosphorylated in 4) 15. Outline the process of oxidative phosphorylation, with reference to the roles of electron carriers, oxygen and the mitochondrial cristae Reduced NAD and reduced FAD need to be oxidised again in order for reactions to continue, this occurs during oxidative phosphorylation and transfers energy to ATP. Oxidative phosphorylation is known as the formation of ATP by the addition of a phosphate group to ADP, in the presence of oxygen, and occurs as follows: Reduced NAD/FAD provide hydrogen atoms and, when oxidised, protons and electrons. These electrons pass along the electron transport chain in the inner mitochondrial. This provides the energy for the active transport of protons, pumping from the matrix into the intermembrane space before they flow through ATP synthase enzymes and drive the rotation part of the enzyme to join ADP and Pi to make ATP. The electrons are passed from the final electron carrier to oxygen, which is the final electron acceptor and can be reduced to water with hydrogen: 4H+ + 4e- + O2 2H2O 16. Outline the process of chemiosmosis, with reference to the electron transport chain, proton gradients and ATP synthase. This is the process by which protons are pumped only through protein complexes in the ETC (in the inner membrane), increasing the concentration in the intermembrane space to create a proton gradient and a potential difference across the inner membrane. Protons can only diffuse through channels in ATP synthase across the gradient. This proton gradient and potential difference are a ‘proton-motive force’, which provides energy to synthesise ATP in the chloroplasts, mitochondria or bacteria. In chloroplasts: ATP is made when protons move from the thylakoid space, through the thylakoid membrane, into the stroma. In mitochondria: ATP is made when protons move from the intermembrane space, through the inner mitochondrial membrane/cristae, into the matrix. F214 - Respiration 17. State that oxygen is the final electron acceptor in aerobic respiration 18. Evaluate the experimental evidence for the theory of chemiosmosis Peter Mitchell realised that the movement of ions across an electrochemical membrane potential could provide the energy needed to produce ATP. His theory was confirmed by the discovery of ATP synthase, a membrane-bound protein that uses the potential energy of the electrochemical gradient to make ATP. The pH in the intermembrane space is lower than in the mitochondrial matrix and is lower in the thylakoid spaces than in the stroma. Protons can lower the pH of a solution, thus showing that protons are of higher concentration in the intermembrane spaces. When isolated chloroplasts are illuminated, the medium in which they are suspended becomes alkaline - as we would predict if protons were being removed from the medium and pumped into the thylakoids. Isolated grana (stacks of thylakoids) kept in an acid medium can make ATP when transferred into an alkaline solution in the dark and given ADP and phosphate. The acid solution creates a proton gradient, which protons move down, into the thylakoid spaces. The alkaline solution provides an environment like in the stroma (of low proton concentration), meaning that there is now a proton gradient from the thylakoid space to the stroma. Here, ATP is formed from added phosphate and ADP by diffusing from the thylakoid space through ATP Synthase, in the thylakoid membrane, into the stroma. 19. Explain why the theoretical maximum yield of ATP per molecule of glucose is rarely, if ever, achieved in aerobic respiration Some protons leak across the mitochondrial membrane, meaning there are fewer protons to generate the proton-motive force. Some ATP is used to actively transport pyruvate into the mitochondria Some ATP is used to bring Hydrogen from reduced NAD made during glycolysis, into the mitochondria from the cytoplasm. 20. Explain why anaerobic respiration produces a much lower yield of ATP than aerobic respiration Anaerobic respiration is the release of energy from substrates, such as glucose, in the absence of oxygen. In anaerobic respiration, glycolysis is the only process that occurs. The electron transport chain cannot occur, as there is no oxygen to act as the final electron acceptor. Thus NAD is all reduced, meaning the Krebs cycle stops, which in turn prevents the link reaction from occurring. Anaerobic respiration takes the pyruvate, and by reducing it, frees up the NAD, so glycolysis can continue, producing two molecules of ATP per glucose molecule respired. This is a much lower yield of ATP compared to what aerobic respiration can produce. F214 - Respiration 21. Compare and contrast anaerobic respiration in mammals and in yeast MAMMALS Pyruvate combines with a hydrogen atom, which is provided by reduced NAD. This reaction forms lactate and oxidised NAD in the muscles and some tissues YEAST Pyruvate converts into ethanal by decarboxylation, producing CO2. Ethanal combines with hydrogen from reduced NAD, forming ethanol Net gain of ATP per molecule of glucose is 2 ATP Net gain of ATP per molecule of glucose is 2 ATP Reduced NAD is oxidised, pyruvate is the hydrogen acceptor Reduced NAD is oxidised, ethanal is the hydrogen acceptor Decarboxylation isn’t involved Decarboxylation is involved Lactate is recycled in the liver to glucose/glycogen, which preserves the energy and can make pyruvate again. So, the reaction is reversible. Yeast and plants cannot metabolise ethanol into another substance. Therefore this form of anaerobic respiration is irreversible. 22. Define the term respiratory substrate An organic substance that can be used for respiration. 23. Explain the difference in relative energy values of carbohydrate, lipid and protein respiratory substrates. The higher the number of hydrogen atoms per mole, the higher the relative energy value, as more NAD molecules can be reduced and used in the Electron Transport Chain. Lipids have the most number of hydrogen atoms per mole, followed by proteins, and then carbohydrates. About twice as much ATP can be made from the complete oxidation of one gram of lipid compared with one gram of either carbohydrate or protein.