explain that reactions in the Krebs cycle involve decarboxylation and dehydrogenation and the reduction of the coenzymes NAD and FAD

Learning Outcomes (Cambridge International AS & A Level Biology 9700 – Syllabus 12)

• Describe the location and main features of glycolysis, the link (pyruvate) reaction, the Krebs cycle and oxidative phosphorylation.
• Explain how ATP is formed by substrate‑level phosphorylation and by chemiosmosis (ATP‑synthase).
• Analyse the decarboxylation and dehydrogenation steps of the Krebs cycle and the reduction of the co‑enzymes NAD⁺ and FAD.
• Interpret the energy values of carbohydrates, fats and proteins and calculate respiratory quotients (RQ).
• Discuss the regulation of the Krebs cycle and the role of mitochondrial structure in respiration.
• Design and evaluate practical investigations related to cellular respiration.

Respiration – Overview

Aerobic respiration in eukaryotic cells occurs in four linked stages (Figure 1). Each stage occurs in a specific cellular compartment and contributes to the overall production of ATP.

1. Glycolysis – cytosol
2. Link (pyruvate) reaction – mitochondrial matrix
3. Krebs (citric‑acid) cycle – mitochondrial matrix
4. Oxidative phosphorylation – inner mitochondrial membrane (electron‑transport chain + chemiosmosis)

Figure 1 – Schematic of aerobic respiration

(Insert a circular diagram showing glucose → pyruvate → acetyl‑CoA → Krebs cycle → NADH/FADH₂ → ETC → ATP)

1. Glycolysis

• Location: Cytosol
• Purpose: Convert one molecule of glucose (6 C) into two molecules of pyruvate (3 C each), generating ATP and NADH.
• Overall net reaction (per glucose):

  Glucose + 2 NAD⁺ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H⁺ + 2 ATP + 2 H2O

Key steps (summary)

Energy yield from glycolysis (per glucose)

• 2 ATP (substrate‑level phosphorylation)
• 2 NADH → 2 × 2.5 ≈ 5 ATP (via ETC)
• Total ≈ 7 ATP equivalents

2. Link (Pyruvate) Reaction

• Location: Mitochondrial matrix
• Overall reaction:

  Pyruvate + CoA‑SH + NAD⁺ → Acetyl‑CoA + CO₂ + NADH + H⁺

• Enzyme complex: Pyruvate dehydrogenase complex (PDH)
• Key points: One NADH and one CO₂ are produced per pyruvate; the resulting acetyl‑CoA enters the Krebs cycle.

3. Krebs (Citric‑Acid) Cycle

Overall reaction (per acetyl‑CoA)

Acetyl‑CoA + 3 NAD⁺ + FAD + GDP + Pi + H₂O → 2 CO₂ + 3 NADH + FADH₂ + GTP + CoA‑SH

This net equation summarises three fundamental transformations:

• Two decarboxylations (release of CO₂)
• Four dehydrogenations (reduction of NAD⁺ three times and FAD once)
• One substrate‑level phosphorylation (formation of GTP, equivalent to ATP)

Step‑by‑step reactions

Key Concepts

Decarboxylation

Steps 3 and 4 each remove a carbon atom as CO₂. This shortens the carbon chain, releases energy, and creates a more oxidised intermediate that can undergo further dehydrogenation.

Dehydrogenation

Hydrogen atoms (as H⁺ + 2 e⁻) are transferred from the substrate to the co‑enzymes:

• NAD⁺ + 2H → NADH + H⁺
• FAD + 2H → FADH₂

The reduced co‑enzymes (NADH, FADH₂) carry high‑energy electrons to the electron‑transport chain.

Substrate‑level phosphorylation

Step 5 converts the high‑energy thioester bond of succinyl‑CoA into a phospho‑high‑energy bond, producing GTP (readily converted to ATP by nucleoside‑diphosphate kinase).

Energy yield from one turn of the Krebs cycle (per acetyl‑CoA)

• 3 NADH → 3 × 2.5 ≈ 7.5 ATP (via ETC)
• 1 FADH₂ → 1 × 1.5 ≈ 1.5 ATP (via ETC)
• 1 GTP → 1 ATP (substrate‑level)
• Total ≈ 10 ATP equivalents

4. Oxidative Phosphorylation – Electron Transport Chain & Chemiosmosis

Location

Inner mitochondrial membrane (highly folded cristae increase surface area).

Mitochondrial structure (relevant to respiration)

Electron‑transport chain (ETC)

• Complex I (NADH → CoQ): NADH donates two electrons; 4 H⁺ are pumped from matrix to inter‑membrane space.
• Complex II (FADH₂ → CoQ): Succinate dehydrogenase feeds electrons from FADH₂; no proton pumping.
• Coenzyme Q (ubiquinone): Mobile electron carrier between Complex I/II and Complex III.
• Complex III (CoQ → cytochrome c): Transfers electrons; pumps 4 H⁺.
• Cytochrome c: Small soluble carrier that shuttles electrons to Complex IV.
• Complex IV (cytochrome c → O₂): Reduces molecular oxygen to water; pumps 2 H⁺.

Chemiosmosis – ATP synthesis

Proton pumping creates an electrochemical gradient (proton‑motive force). Protons flow back into the matrix through ATP synthase (Complex V), driving the phosphorylation of ADP to ATP:

ADP + Pi + 4 H⁺(out) → ATP + H₂O + 4 H⁺(in)

Yield (per NADH) ≈ 2.5 ATP; per FADH₂ ≈ 1.5 ATP.

Energy Values of Major Substrates (Cambridge Syllabus 12.1)

Respiratory Quotient (RQ)

Definition: RQ = (moles of CO₂ produced) ÷ (moles of O₂ consumed) for a given substrate.

Worked example (glucose):

C₆H₁₂O₆ + 6 O₂ → 6 CO₂ + 6 H₂O ⇒ RQ = 6 CO₂ / 6 O₂ = 1.00

Anaerobic Respiration (Fermentation)

• Lactic‑acid fermentation (muscle, some bacteria)

  Pyruvate + NADH → Lactate + NAD⁺

• Alcoholic fermentation (yeast, some plants)

  Pyruvate → Acetaldehyde + CO₂

  Acetaldehyde + NADH → Ethanol + NAD⁺

Fermentation regenerates NAD⁺, allowing glycolysis to continue, but yields only 2 ATP per glucose.

Regulation of the Krebs Cycle (Cambridge Syllabus 12.2)

• Key regulatory enzymes: Citrate synthase, isocitrate dehydrogenase, α‑ketoglutarate dehydrogenase.
• Allosteric effectors:

  • ATP & NADH – inhibit isocitrate dehydrogenase and α‑ketoglutarate dehydrogenase (high‑energy status).
  • ADP & NAD⁺ – activate isocitrate dehydrogenase (low‑energy status).
  • Acetyl‑CoA – activates citrate synthase (substrate availability).

• Calcium ions (Ca²⁺): In muscle and liver cells, Ca²⁺ released during contraction stimulates isocitrate dehydrogenase and α‑ketoglutarate dehydrogenase, matching ATP production to demand.

Practical Investigations (Suggested Classroom Activities)

1. Respirometer experiment: Compare the RQ of germinating beans (carbohydrate‑rich) with that of yeast undergoing alcoholic fermentation. Measure O₂ consumption and CO₂ production in a sealed system and calculate RQ.
2. DCPIP assay for NADH production: Mix isolated mitochondria with substrates for the link reaction or the Krebs cycle and the redox indicator DCPIP. The colour change (blue → colourless) indicates NADH‑mediated reduction of DCPIP, providing a visual measure of dehydrogenation activity.
3. Effect of respiratory inhibitors: Add malonate (succinate dehydrogenase inhibitor) or cyanide (Complex IV inhibitor) to isolated mitochondria and record O₂ consumption with a Clark‑type electrode. Relate the observed changes to the role of FADH₂ and the ETC.
4. Measuring ATP synthesis: Use a luciferin‑luciferase assay to quantify ATP produced by mitochondria before and after addition of ADP (state 3 respiration) and after adding oligomycin (ATP‑synthase inhibitor) to illustrate chemiosmotic coupling.

Key Take‑aways

• Glycolysis, the link reaction, the Krebs cycle and oxidative phosphorylation together convert the chemical energy of glucose (or other substrates) into ATP.
• The Krebs cycle features two decarboxylation steps and four dehydrogenation steps, reducing NAD⁺ to NADH and FAD to FADH₂.
• Reduced NADH and FADH₂ deliver high‑energy electrons to the electron‑transport chain, where chemiosmosis via ATP synthase produces the bulk of cellular ATP.
• Energy yields differ between carbohydrates, fats and proteins, reflected in their characteristic respiratory quotients.
• Regulatory mechanisms (allosteric effectors, Ca²⁺, substrate availability) ensure that respiration matches the cell’s energy demand.
• Understanding the integration of these pathways fulfills the Cambridge International AS & A Level Biology (9700) syllabus requirements.

Suggested diagram

Insert a circular schematic of the Krebs cycle showing each intermediate, the two CO₂‑releasing steps, and the points where NAD⁺/FAD are reduced to NADH/FADH₂.