cellular respiration

• TCA Cycle (Tricarboxylic Acid Cycle)


  Also known as the Krebs cycle or citric acid cycle
• TCA Cycle


  • Central metabolic pathway that oxidizes acetyl-CoA to CO₂ and generates NADH and FADH₂, which are essential for the electron transport chain (ETC)
• TCA Cycle


  1. Acetyl-CoA combines with oxaloacetate to form citrate

  2. Citrate is converted into isocitrate

  3. Isocitrate is oxidized to alpha-ketoglutarate, producing NADH

  4. Alpha-ketoglutarate is oxidized to succinyl-CoA, producing NADH

  5. Succinyl-CoA is converted to succinate, generating GTP (or ATP)

  6. Succinate is oxidized to fumarate, producing FADH₂

  7. Fumarate is hydrated to malate

  8. Malate is oxidized to oxaloacetate, producing NADH
• Chemiosmotic Theory


  Explains how ATP is synthesized in mitochondria using the energy of the proton gradient created by the electron transport chain (ETC)
• Chemiosmotic Theory


  • Protons are pumped across the inner mitochondrial membrane, creating an electrochemical gradient (proton motive force)
  • ATP synthase uses this gradient to synthesize ATP from ADP and Pi
• Electron Transport Chain (ETC)


  Series of complexes that transfer electrons from NADH and FADH₂ to oxygen, the final electron acceptor, forming water
• Electron Transport Chain (ETC)


  • Complex I (NADH:ubiquinone oxidoreductase): Transfers electrons from NADH to ubiquinone (Q), pumping protons into the intermembrane space
  • Complex II (Succinate dehydrogenase): Transfers electrons from succinate to ubiquinone without proton pumping
  • Complex III (Cytochrome bc₁ complex): Transfers electrons from reduced ubiquinone (ubiquinol) to cytochrome c, pumping protons
  • Complex IV (Cytochrome c oxidase): Transfers electrons from cytochrome c to oxygen, forming water and pumping protons
  • Complex V (ATP Synthase): Uses the proton gradient to synthesize ATP
• Rotenone


  Inhibits Complex I (NADH:ubiquinone oxidoreductase)
• Malonate


  Competitive inhibitor of Succinate Dehydrogenase (Complex II)
• Antimycin A


  Inhibits Complex III (Cytochrome bc₁ complex)
• Cyanide (CN⁻)


  Irreversible inhibitor of Cytochrome c oxidase (Complex IV)
• Carbon Monoxide (CO)


  Competitive inhibitor of oxygen at the active site of Cytochrome c oxidase (Complex IV)
• Nitric Oxide (NO)


  Competitive inhibitor of oxygen at the active site of Cytochrome c oxidase (Complex IV)
• Oligomycin


  Inhibitor of ATP Synthase (Complex V)
• FCCP (Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone)


  Uncoupler of oxidative phosphorylation
• Clark Electrode


  Measures the rate of oxygen consumption by cells
• Clark Electrode


  1. Contains a platinum cathode and a silver anode

  2. Oxygen is reduced at the cathode, producing a current proportional to the oxygen concentration
• Seahorse Analyzer


  Measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in real time
• Basal Respiration: Measure oxygen consumption under baseline conditions without any inhibitors or uncouplers
• ATP Production: Calculate oxygen consumption associated with ATP synthesis by adding oligomycin to inhibit ATP synthase and measuring the decrease in OCR
• Proton Leak: Determine the integrity of the mitochondrial membrane by measuring OCR after adding an uncoupler like FCCP
• Maximal Respiration: Measure the maximum capacity of the electron transport chain by adding FCCP to dissipate the proton gradient
• Spare Respiratory Capacity: Calculate the difference between maximal respiration and basal respiration
• Non-Mitochondrial Oxygen Consumption: Measure the residual oxygen consumption after inhibiting the electron transport chain with rotenone and antimycin A
• Practical Session Tasks


  1. Collect and record OCR values under different conditions (e.g., basal respiration, ATP production, proton leak)

  2. Use the collected data to generate a comprehensive table summarizing the results

  3. Create a summary graph to visually represent the collected data, ensuring clarity and accuracy

  4. Interpret the data and draw appropriate conclusions about mitochondrial respiration and the effects of different inhibitors

  5. Discuss the implications of the findings and suggest potential improvements for future experiments