Biology 1 – Lesson 8: Cellular Respiration – The Citric Acid Cycle and Oxidative Phosphorylation

Overview

Building on our exploration of glycolysis and fermentation (Lesson 7), this lesson delves into the aerobic stages of cellular respiration—the Citric Acid (Krebs) Cycle and Oxidative Phosphorylation. We will investigate how eukaryotic cells use mitochondria to generate large amounts of ATP, how electron carriers (NADH and FADH₂) donate electrons to the electron transport chain (ETC), and how a proton gradient drives ATP synthesis.

1. The Citric Acid (Krebs) Cycle

1.1 Mitochondrial Structure

• Double-Membrane Organelle

  • Outer Membrane: Relatively permeable to small molecules, thanks to porin proteins.

  • Inner Membrane: Highly selective, folded into cristae to increase surface area for oxidative phosphorylation.

• Matrix

  • Enclosed by the inner membrane.

  • Contains enzymes for the citric acid cycle, along with mitochondrial DNA and ribosomes.

1.1 Mitochondrial Structure

Double-Membrane Organelle

• Outer Membrane: Relatively permeable to small molecules, thanks to porin proteins.
• Inner Membrane: Highly selective, folded into cristae to increase surface area for oxidative phosphorylation.

Matrix

• Enclosed by the inner membrane.
• Contains enzymes for the citric acid cycle, along with mitochondrial DNA and ribosomes.

[Diagram: Mitochondrion Cross-Section Highlighting Matrix, Inner, and Outer Membranes]

1.2 Steps of the Citric Acid Cycle

1. Acetyl-CoA Formation

   • Pyruvate (from glycolysis) is transported into the mitochondrial matrix.

   • Pyruvate is converted to acetyl-CoA, releasing CO₂ and generating NADH.

2. Citrate Synthesis

   • Acetyl-CoA (2C) joins oxaloacetate (4C) → citrate (6C).

3. Oxidation and Decarboxylation

   • Sequential steps remove CO₂ molecules, generating NADH and FADH₂ as electrons are transferred to these carriers.

4. Regeneration of Oxaloacetate

   • The cycle regenerates oxaloacetate, allowing another acetyl-CoA to enter.

1.3 Products and Regulation

• Per Turn of the Cycle (per 1 acetyl-CoA):

  • 2 CO₂ molecules released

  • 3 NADH and 1 FADH₂ produced

  • 1 ATP (or GTP) generated via substrate-level phosphorylation

• Regulation Points

  • High NADH or ATP levels signal sufficient energy, inhibiting key enzymes (e.g., isocitrate dehydrogenase).

  • ADP or low energy states activate the cycle to accelerate ATP production.

2. Oxidative Phosphorylation

2.1 The Electron Transport Chain (ETC)

• Location: Embedded in the inner mitochondrial membrane.

• Electron Flow

  • NADH and FADH₂ donate electrons to protein complexes.

  • Electrons pass through complexes I, II, III, and IV, each step releasing energy.

  • O₂ is the final electron acceptor, forming H₂O.

• Proton Pumping

  • As electrons flow, protons (H⁺) are pumped from the matrix to the intermembrane space, creating an electrochemical gradient.

2.1 The Electron Transport Chain (ETC)

Location: Embedded in the inner mitochondrial membrane.

Electron Flow

• NADH and FADH₂ donate electrons to protein complexes.
• Electrons pass through complexes I, II, III, and IV, each step releasing energy.
• O₂ is the final electron acceptor, forming H₂O.

Proton Pumping

• As electrons flow, protons (H⁺) are pumped from the matrix to the intermembrane space, creating an electrochemical gradient.

₂ → H₂O FADH₂

[Diagram: Electron Transport Chain Complexes in the Inner Mitochondrial Membrane]

2.2 Chemiosmosis and ATP Synthase

• Proton Gradient

  • The high concentration of protons in the intermembrane space exerts a strong driving force to re-enter the matrix.

• ATP Synthase

  • This enzyme harnesses the energy of proton flow (H⁺ traveling down its gradient) to synthesize ATP from ADP + Pi.

  • Often described as a molecular “turbine.”

2.3 Coupling Electron Flow and ATP Production

• Oxidative Phosphorylation:

  • The ETC oxidizes NADH and FADH₂, releasing electrons.

  • The resulting proton gradient powers phosphorylation of ADP → ATP.

• Total ATP Yield

  • Up to ~30–34 ATP can be generated per glucose under optimal conditions in eukaryotic cells.

  • Actual yield can vary due to shuttle costs, proton leak, and other factors.

3. Real-Life Applications

3.1 Human Physiology

• Muscle Activity: Muscle cells heavily rely on oxidative phosphorylation for sustained energy demands (e.g., long-distance running).

• Brain Function: Neurons require a continuous supply of ATP for membrane potentials and neurotransmitter release.

3.2 Pathologies

• Mitochondrial Disorders: Defects in ETC complexes can cause severe diseases (e.g., Leigh syndrome).

• Cyanide Poisoning: Cyanide blocks electron flow at complex IV, halting ATP production and leading to cell death.

3.3 Industrial Biotech

• Bioreactors: Knowing how microbes use respiration helps optimize fermentation or respiration conditions for maximum yield (e.g., biofuel production).

• Drug Targets: Many antiparasitic or antimicrobial agents target differences in mitochondrial or bacterial respiration pathways.

4. Exercise: Measuring Respiratory Activity with a Simple Yeast Setup

Objective

Observe oxygen consumption and CO₂ production in aerobic respiration using yeast.

Materials

• Dry baker’s yeast

• Glucose or sugar

• Warm water (~30–35°C)

• A small respirometer or any airtight container with a tube to measure gas exchange

• Limewater (or bromothymol blue solution) to detect CO₂

Procedure

1. Yeast Suspension

   • Dissolve 1 packet of yeast and 1 tablespoon of sugar in ~200 mL of warm water in a container.

2. Set Up the Respirometer

   • Close the container but allow the gas to pass into a test tube containing limewater.

3. Observe

   • As aerobic respiration proceeds, CO₂ produced will bubble through the limewater, turning it cloudy.

4. Analysis

   • Record time until the limewater becomes noticeably cloudy.

   • Relate the rate of gas production to the metabolic activity of yeast under aerobic conditions.

5. Additional Learning Components

5.1 Historical Anecdote: Hans Adolf Krebs

Hans Krebs discovered the Krebs Cycle in 1937, unraveling the fundamental pathway of aerobic energy metabolism. His groundbreaking insights eventually earned him the Nobel Prize in Physiology or Medicine (1953).

5.2 Researcher Spotlight: Peter Mitchell

Peter Mitchell proposed the Chemiosmotic Theory in the 1960s, explaining how proton gradients drive ATP synthesis. He won the Nobel Prize in Chemistry (1978) for this revolutionary concept.

5.3 Advanced Reading Suggestions

• “Lehninger Principles of Biochemistry” (Nelson & Cox): Offers an in-depth look at the Krebs cycle, ETC, and regulatory mechanisms.

• Journal Articles: Cell, Nature, and Science often feature research on mitochondrial dynamics and disease-related mutations.

5.4 Notable Breakthrough: ATP Synthase Structure via Cryo-EM

Recent cryo-electron microscopy techniques provide high-resolution structures of ATP synthase, revealing its rotating subunits and offering insights for drug design against pathogens’ energy machinery.

5.5 Interactive Concept

Online 3D animations of the Krebs cycle and ETC can help visualize molecular transformations and how the electron carriers shuttle electrons through complexes to produce ATP.

6. Recall Questions

1. Location: Where do the citric acid cycle and oxidative phosphorylation take place in eukaryotic cells, and why is this compartmentalization important?

2. Krebs Cycle Regulation: Name two key regulatory steps in the citric acid cycle and describe how they are allosterically regulated.

3. ETC: What role do NADH and FADH₂ play in the electron transport chain, and why is O₂ essential?

4. Chemiosmosis: How does the proton gradient created by the electron transport chain lead to ATP production via ATP synthase?

5. Applications: Briefly explain how defects in oxidative phosphorylation can affect human health and name one example.

Use these questions to test your understanding of how eukaryotic cells efficiently generate ATP through the coordinated steps of the Krebs cycle and oxidative phosphorylation.