5.2 Respiration

• Why is respiration important?

  • ● Respiration produces ATP (to release energy)
  • ● For active transport, protein synthesis etc.
• Summarise the stages of aerobic respiration

  1. Glycolysis - cytoplasm (anaerobic)
  2. Link reaction - mitochondrial matrix
  3. Krebs cycle - mitochondrial matrix
  4. Oxidative phosphorylation - inner mitochondrial membrane
• Summarise the stages of anaerobic respiration

  1. Glycolysis - cytoplasm
  2. NAD regeneration - cytoplasm
• Describe the process of glycolysis

  1. Glucose phosphorylated to glucose phosphate
  2. Using inorganic phosphates from 2 ATP
  3. Hydrolysed to 2 x triose phosphate
  4. Oxidised to 2 pyruvate
  5. 2 NAD reduced
  6. 4 ATP regenerated (net gain of 2)
• Explain what happens after glycolysis if respiration is anaerobic

  1. Pyruvate converted to lactate (animals &some bacteria) or ethanol (plants & yeast)
  2. Oxidising reduced NAD → NAD regenerated
  3. 3. So glycolysis can continue (which needs NAD) allowing continued production of ATP
• Suggest why anaerobic respiration produces less ATP per molecule of glucose than aerobic respiration

  • Only glycolysis involved which produces little ATP (2 molecules)
  • No oxidative phosphorylation which forms majority of ATP (around 34 molecules)
• What happens after glycolysis if respiration is aerobic?

  Pyruvate is actively transported into the mitochondrial matrix
• Describe the link reaction

  1. Pyruvate oxidised (and decarboxylated) to acetate
  2. CO2 produced
  3. Reduced NAD produced (picks up H)
  4. Acetate combines with coenzyme A, forming Acetyl Coenzyme A
• What are the products per glucose molecule made in the link reaction?

  2 x Acetyl Coenzyme A,

  2 X CO2,

  2 X reduced NAD
• Describe the Krebs cycle

  1. Acetyl coenzyme A (2C) reacts with a 4C molecule
  2. Releasing coenzyme A
  3. Producing a 6C molecule that enters the Krebs cycle
  4. In a series of oxidation-reduction reactions, the 4C molecule is regenerated and:
  5. 2 x CO2 lost
  6. ○ Coenzymes NAD & FAD reduced
  7. Substrate level phosphorylation (direct transfer of Pi from intermediate compound to ADP) → ATP produced
• What are the products per glucose molecule made in the Krebs cycle?

  6 x reduced NAD,

  2 x reduced FAD,

  2 x ATP,

  4 x CO2
• Describe the process of oxidative phosphorylation (steps 1-2 out of 4)

  1. Reduced NAD/FAD oxidised to release H atoms → split into protons (H+) and electrons (e-)
  2. Electrons transferred down electron transfer chain (chain of carriers at decreasing energy levels)
  3. By redox reactions
• Describe the process of oxidative phosphorylation (steps 4+5 out of 5)

  1. (3) Energy released by electrons used in the production of ATP from ADP + Pi (chemiosmotic theory):
  2. Energy used by electron carriers to actively pump protons from matrix → intermembrane space
  3. Protons diffuse into matrix down an electrochemical gradient, via ATP synthase (embedded)
  4. Releasing energy to synthesise ATP from ADP + Pi
  5. (4) In matrix at end of ETC, oxygen is final electron acceptor (electrons can’t pass along otherwise)
  6. So protons, electrons and oxygen combine to form water
• Give examples of other respiratory substrates

  • Breakdown products of lipids and amino acids, which enter the Krebs cycle. For example:
  • Fatty acids from hydrolysis of lipids → converted to Acetyl Coenzyme A
  • Amino acids from hydrolysis of proteins → converted to intermediates in Krebs cycle
• RP9: Describe how a respirometer can be used to measure the rate of aerobic respiration (by measuring oxygen uptake)

  1. Add set mass of single-celled organism eg. yeast to set volume / conc. of substrate eg. glucose
  2. Add a buffer to keep pH constant
  3. Add a set volume / conc. of a chemical that absorbs CO2 eg. sodium hydroxide
  4. Place in water bath at a set temperature and allow to equilibrate
  5. Measure distance moved by coloured liquid in a set time

  
• RP9: Explain why the liquid moves. (4)

  • Organisms aerobically respire so take in O2
  • CO2 given out but absorbed by sodium hydroxide solution
  • So volume of gas and pressure in container decrease
  • Fluid in tube moves down pressure gradient towards organism
• RP9: Explain why the respirometer apparatus is left open for 10 minutes. (1)

  • Allow apparatus to equilibrate
  • Allow for overall pressure expansion/change throughout
  • Allow respiration rate of organisms to stabilise
• RP9: Explain why the apparatus must be airtight. (2)

  • Prevent air entering or leaving
  • Would change volume and pressure, affecting movement of liquid
• RP9: Describe a more accurate way to measure volume of gas. (1)

  ● Use a gas syringe
• RP9: Suggest a suitable control experiment and explain why it is necessary. (2)

  • No organisms OR use inert objects OR use dead organisms AND all other conditions / apparatus / equipment the same
  • To show that (respiring) organisms are causing liquid to move / taking up oxygen / causing the change in volume / pressure
• Describe how a respirometer can be used to measure the rate of anaerobic respiration (by measuring carbon dioxide release)

  • Repeat experiment as above but remove chemical that absorbs CO2
  • Make conditions anaerobic, for example:
  • Layer of oil / liquid paraffin above yeast to stop O2 diffusing in
  • Add a chemical that absorbs O2
  • Leave for an hour to allow O2 to be respired and used up

  
• Anaerobic respirometer - Explain why the liquid moves. (3)

  • Yeast anaerobically respire so release CO2
  • So volume of gas and pressure in container increase
  • So fluid in capillary tube moves down a pressure gradient away from organism
• Anaerobic respirometer - Explain why the apparatus is left for an hour after the culture has reached a constant temperature. (1)

  ● Allow time for oxygen to be used / respired
• Respirometer - Describe how rate of respiration can be calculated

  1. Calculate volume of O2 / CO2 consumed / released (calculate area of a cylinder)
  2. Calculate cross-sectional area of capillary tube using $πr^2$
  3. Multiply by distance liquid has moved
  4. Divide by mass of organism and time taken
  5. Units - unit for volume per unit time per unit mass eg. $cm^3min^-1g^-1$
• Describe how redox indicator dyes such as Methylene blue can be used to measure rate of respiration

  1. Add a set volume of organism eg. yeast and a set volume of respiratory substrate eg. glucose to tubes
  2. Add a buffer to keep pH constant
  3. Place in water bath at a set temperature and allow to equilibrate for 5 mins
  4. Add a set volume of methylene blue, shake for a set time (do not shake again)
  5. Record time taken for colour to disappear in tube
  6. Rate of respiration (s-1) = 1 / time (sec)
• How does redox indicator dyes such as Methylene blue work to be used to measure rate of respiration

  • Redox indicators (eg. methylene blue) change colour when they accept electrons becoming reduced
  • Redox indicators take up hydrogens and get reduced instead of NAD / FAD → modelling their reactions
• RP9 Methylene Blue: Give two examples of variables that could be controlled. (2)

  • Volume of single-celled organism
  • Volume / conc. / type of respiratory substrate
  • Temperature (with a water bath)
  • pH (with a buffer)
  • Volume of redox indicator (only control)
• RP9 Methylene Blue: Why leave tubes in the water bath for 5 minutes? (1)

  ● Allow for solutions to equilibrate and reach the same temperature as the water bath
• RP9 Methylene Blue: Suggest a suitable control experiment and explain why it is necessary. (3)

  • Add methylene blue to boiled / inactive / dead yeast (boiling denatures enzymes)
  • All other conditions the same
  • To show change is due to respiration in organisms
• RP9 Methylene Blue: Suggest and explain why you must not shake tubes containing methylene blue. (3)

  • Shaking would mix solution with oxygen
  • Which would oxidise methylene blue / cause it to lose its electrons
  • So methylene blue would turn back to its original blue colour
• RP9 Methylene Blue: Suggest one source of error in

  using methylene blue. Explain how this can be reduced. (2)

  • Subjective as to determination of colour change / end point
  • Compare results to a colour standard (one that has already changed) OR use a colorimeter for quantitative results