Oxidative Phosphorylation — AQA A-level Biology

SPEC 3.5.2 - RESPIRATION

Krebs Cycle & Oxidative Phosphorylation

Krebs cycle, chemiosmosis, ATP synthase, the electron transfer chain and other respiratory substrates

Respiration: The Big Picture

Aerobic respiration has four stages. In Lesson 5.03 you covered glycolysis, anaerobic respiration and the link reaction. This lesson covers the final two stages — the Krebs cycle and oxidative phosphorylation — where most ATP is made.

StageLocationKey outputs

GlycolysisCytoplasm2 pyruvate, 2 ATP (net), 2 NADH

Link reactionMitochondrial matrix2 acetyl CoA, 2 NADH, 2 CO₂

Krebs cycleMitochondrial matrix6 NADH, 2 FADH₂, 2 ATP, 4 CO₂

Oxidative phosphorylationInner mitochondrial membrane~26–28 ATP, H₂O

Key principle

The Krebs cycle and link reaction do not produce much ATP directly. Their main role is to produce reduced NAD (NADH) and reduced FAD (FADH₂) — the coenzymes that power oxidative phosphorylation.

Mitochondrial Structure & Respiration Locations

The structure of the mitochondrion is precisely adapted for aerobic respiration.

Examiner Alert

Pyruvate is transported into the mitochondrial matrix by active transport. The Krebs cycle occurs in the matrix. Oxidative phosphorylation occurs on the inner mitochondrial membrane. These locations are regularly tested.

Krebs Cycle: Key Definitions

Krebs Cycle

A cyclical metabolic pathway in the mitochondrial matrix in which acetyl CoA is completely oxidised, releasing CO₂, reducing NAD and FAD, and producing ATP by substrate-level phosphorylation.

FAD (Flavin Adenine Dinucleotide)

A hydrogen carrier; accepts hydrogen atoms (electrons + protons) and is reduced to FADH₂ during the Krebs cycle. Donates electrons to the electron transfer chain.

Substrate-Level Phosphorylation

The direct synthesis of ATP from ADP + Pᵢ by transfer of a phosphate group from a metabolic intermediate — without the electron transfer chain. Occurs in glycolysis and the Krebs cycle.

Carbon tracking

The 2-carbon acetate of acetyl CoA enters the Krebs cycle. Both carbons are released as CO₂ by the end of one turn. This accounts for all the carbon originally in glucose being released as CO₂.

Krebs Cycle: Step by Step

The Krebs cycle takes place in the mitochondrial matrix and turns twice per glucose (once per acetyl CoA).

1

Acetyl CoA (2C) + oxaloacetate (4C) → citrate (6C) — coenzyme A is released and recycled.

2

Citrate (6C) is decarboxylated and oxidised → 5C compound — CO₂ released; NAD reduced to NADH.

3

5C compound → 4C compound — further decarboxylation and oxidation; CO₂ released; NADH produced; FADH₂ produced; 1 ATP by substrate-level phosphorylation.

4

4C compound is regenerated as oxaloacetate — cycle continues. NADH produced in this final step.

Products per turn (per acetyl CoA)

2 CO₂ | 3 NADH | 1 FADH₂ | 1 ATP

Per glucose (×2 turns): 4 CO₂ | 6 NADH | 2 FADH₂ | 2 ATP

Krebs Cycle: Diagram

Exam Tip

Name the 4C compound as oxaloacetate and the 6C compound as citrate. Examiners award marks for these specific names. The 5C intermediate does not need to be named at A-level.

Oxidative Phosphorylation: Definitions

Oxidative Phosphorylation

The synthesis of ATP from ADP + Pᵢ, driven by the movement of electrons down the electron transfer chain and the flow of protons through ATP synthase in the inner mitochondrial membrane.

Chemiosmosis

The movement of protons (H⁺) down their electrochemical gradient, through the enzyme ATP synthase, which catalyses the synthesis of ATP from ADP + Pᵢ. First proposed by Peter Mitchell (Mitchell's chemiosmotic theory).

Final Electron Acceptor

Oxygen (O₂) is the final electron acceptor in the electron transfer chain. Electrons combine with O₂ and H⁺ to form water. Without O₂, the chain stops and ATP synthesis by oxidative phosphorylation cannot occur.

Electron Transfer Chain: Step by Step

The electron transfer chain (ETC) is located on the inner mitochondrial membrane.

1

NADH and FADH₂ donate electrons to carrier proteins (protein complexes) in the inner mitochondrial membrane.

2

Electrons pass along the carrier proteins in a series of redox reactions, releasing energy at each transfer.

3

Released energy is used to actively pump H⁺ (protons) from the matrix into the intermembrane space, building an electrochemical (proton) gradient.

4

H⁺ diffuse back into the matrix down their electrochemical gradient through the enzyme ATP synthase — this is chemiosmosis. ATP synthase uses this flow to synthesise ATP from ADP + Pᵢ.

5

At the end of the chain, electrons + H⁺ + O₂ → water. Oxygen is the final electron acceptor. Without O₂, the chain stops.

Electron Transfer Chain: Diagram

Why Aerobic Respiration Requires Oxygen

Oxygen's role is not to burn glucose directly. Its precise role is as the final electron acceptor in the electron transfer chain.

The chain reaction of no oxygen

O₂ absent → electrons cannot be passed to the final acceptor

Electron transfer chain stops

Protons cannot be pumped into intermembrane space

No proton gradient → no chemiosmosis → no ATP synthase activity

NADH and FADH₂ cannot donate electrons → NAD⁺ and FAD cannot be regenerated

Without NAD⁺, the Krebs cycle and glycolysis slow/stop

Common Error

Students often write "oxygen is used to break down glucose." This is incorrect. Oxygen acts as the final electron acceptor and combines with electrons and H⁺ to form water at the end of the ETC. CO₂ is produced in the link reaction and Krebs cycle — not by oxygen reacting with carbon.

Three Molecules — Three Locations

CO₂ produced: link reaction + Krebs cycle

H₂O produced: end of electron transfer chain

O₂ consumed: end of electron transfer chain (final electron acceptor)

ATP Yield Per Glucose

The approximate ATP yield from complete aerobic respiration of one glucose molecule:

StageDirect ATPReduced coenzymesATP via ETC (approx.)

Glycolysis2 ATP (net)2 NADH~4–5

Link reaction (×2)02 NADH~5

Krebs cycle (×2)2 ATP6 NADH + 2 FADH₂~16–18

Total4 ATP~26–28

Examiner Alert

Do not state a precise ATP yield as absolute fact. AQA acknowledges the yield varies (~30–32 per glucose) depending on the organism, membrane conditions and coupling efficiency. State "approximately 30–32 ATP" or "around 30 ATP". Writing "exactly 38 ATP" will lose marks.

Why NADH vs FADH₂ differ

NADH donates electrons at an earlier (higher-energy) point in the ETC than FADH₂, so NADH drives more H⁺ pumping and produces more ATP (~2.5 ATP) than FADH₂ (~1.5 ATP) per molecule.

Other Respiratory Substrates

Glucose is the primary respiratory substrate, but lipids and amino acids can also be respired when glucose is scarce (e.g. during prolonged exercise or starvation).

Lipids as Respiratory Substrates

The breakdown products of lipids (fatty acids and glycerol) enter the Krebs cycle. Fatty acids are broken down to produce acetyl CoA, which enters the Krebs cycle directly.

ATP yield from lipids > glucose per gram — lipids contain more C–H bonds (more reduced) and therefore yield more reduced NAD and more ATP per gram than carbohydrates.

Amino Acids as Respiratory Substrates

Transamination removes the amino group (–NH₂), which is excreted as urea. The remaining carbon skeleton enters the Krebs cycle at various points (e.g. as acetyl CoA, oxaloacetate, or other intermediates) or enters glycolysis.

Exam Tip

For lipids: the key points are (1) breakdown products of lipids enter the Krebs cycle; (2) fatty acids → acetyl CoA → Krebs cycle; (3) higher ATP yield per gram than glucose. For amino acids: (1) amino group removed; (2) carbon skeleton enters Krebs at various points. You do not need to name specific entry points.

Required Practical 9: Rate of Respiration

Required Practical 9

Investigation into the effect of a named variable on the rate of respiration of cultures of single-celled organisms (e.g. yeast).

Example variable: temperature

Variable typeExampleHow measured/controlled

IndependentTemperature (°C)Water baths at different temperatures

DependentRate of respirationVolume of CO₂ produced per unit time (e.g. using a manometer/gas syringe, or volume of O₂ consumed); or colour change of redox indicator (e.g. DCPIP or methylene blue — decolourisation indicates dehydrogenase activity)

ControlConcentration of glucose substrateKeeps substrate availability constant — prevents this affecting rate

ControlVolume/mass of yeast suspensionKeeps number of respiring cells constant

ControlpHUse a buffer solution — pH affects enzyme activity

Redox indicator method

A redox indicator (e.g. DCPIP or methylene blue) is reduced (decolourised) by the hydrogen released from reduced NAD/FAD during dehydrogenase reactions. A faster decolourisation indicates higher dehydrogenase activity and higher respiration rate.

DEEP Framework: Extended Answer

AQA extended response questions on respiration often ask you to "explain how ATP is produced during aerobic respiration." Use DEEP:

DEEP Chain — ATP Synthesis by Oxidative Phosphorylation

D

Define: Oxidative phosphorylation is the synthesis of ATP from ADP + Pᵢ, driven by the movement of electrons down the electron transfer chain and the flow of protons through ATP synthase in the inner mitochondrial membrane.

E

Explain mechanism: Reduced NAD and reduced FAD (produced in glycolysis, link reaction and Krebs cycle) donate electrons to carrier proteins in the inner mitochondrial membrane. As electrons pass along the chain, energy is released and used to pump H⁺ from the matrix to the intermembrane space. This creates an electrochemical (proton) gradient. H⁺ diffuse back through ATP synthase by chemiosmosis, driving ATP synthesis. At the end of the chain, electrons combine with H⁺ and O₂ (the final electron acceptor) to form water.

E

Evidence/Example: If cyanide is added (blocks electron transfer), or if oxygen is removed, the ETC stops, the proton gradient cannot be maintained, and ATP synthesis by oxidative phosphorylation ceases. Cells switch to anaerobic respiration producing only 2 ATP per glucose from glycolysis.

P

Practical/Quantitative: Approximately 26–28 ATP are produced per glucose from oxidative phosphorylation, compared with only 4 ATP from direct (substrate-level) phosphorylation. The overall yield is approximately 30–32 ATP per glucose under aerobic conditions.

Practice Question

Explain how the Krebs cycle and oxidative phosphorylation are linked, and explain what would happen to ATP production if oxygen were removed. [6 marks]

D:

The Krebs cycle produces reduced coenzymes — reduced NAD (NADH) and reduced FAD (FADH₂) — which are used in oxidative phosphorylation. ATP is synthesised by oxidative phosphorylation: the flow of protons through ATP synthase driven by the electron transfer chain.

E:

NADH and FADH₂ donate electrons to carrier proteins in the inner mitochondrial membrane. Electron flow releases energy to pump H⁺ into the intermembrane space, building a proton gradient. H⁺ flow back through ATP synthase by chemiosmosis, driving ATP synthesis.

E:

If O₂ is removed: O₂ is the final electron acceptor. Without O₂, electrons cannot be passed to the final acceptor → electron transfer chain stops → H⁺ cannot be pumped → no proton gradient → no chemiosmosis → ATP synthase inactive → oxidative phosphorylation stops. NAD⁺ cannot be regenerated from NADH → Krebs cycle also stops.

P:

Only 2 ATP (net) from substrate-level phosphorylation in glycolysis remains available. This falls from ~30–32 ATP under aerobic conditions — a reduction of approximately 93% in ATP yield.

Examiner Alerts: Common Errors

Error 1: CO₂, H₂O and O₂ locations

CO₂ is produced in the link reaction and Krebs cycle. H₂O is produced at the end of the ETC. O₂ is consumed at the end of the ETC. Do not write that O₂ is needed for the Krebs cycle, or that CO₂ is produced by the ETC.

Error 2: Precise ATP yield

Never write "38 ATP per glucose" as a definitive figure. The AQA mark scheme accepts "approximately 30–32 ATP" or similar. The theoretical maximum of 38 ATP from older textbooks is not used by AQA.

Error 3: What chemiosmosis is

Chemiosmosis is the movement of H⁺ (protons) through ATP synthase down their gradient. It is not "the movement of electrons." Electrons move down the ETC; protons move through ATP synthase.

Error 4: Where NADH and FADH₂ go

NADH and FADH₂ do not "make ATP directly." They donate electrons to the ETC. It is the proton gradient generated by this electron flow that drives ATP synthesis via ATP synthase.

Synoptic Links

Synoptic Link — Photosynthesis (3.5.1)

The light-dependent reaction of photosynthesis also uses chemiosmosis and ATP synthase to produce ATP — but protons are pumped across the thylakoid membrane, not the inner mitochondrial membrane. The principles are identical: electron flow → proton pumping → proton gradient → ATP synthase. AQA Paper 3 essays frequently ask students to compare these two systems.

Synoptic Link — Protein Structure (3.1.4)

ATP synthase is an enzyme with a specific tertiary structure. Changes in pH (e.g. disruption of the proton gradient) affect the function of ATP synthase — synoptic link to enzyme denaturation and active site specificity.

Synoptic Link — Muscle Contraction (3.6.5)

Muscle fibres require large amounts of ATP for contraction. Slow-twitch (Type I) muscle fibres are densely packed with mitochondria and rely on aerobic respiration (Krebs + oxidative phosphorylation). Fast-twitch (Type II) fibres respire anaerobically when O₂ is limited — linking to the consequences of stopping the ETC.

Summary

Key Points: Krebs Cycle & Oxidative Phosphorylation

ProcessLocationKey products per glucose

Krebs cycle (×2)Mitochondrial matrix4 CO₂, 6 NADH, 2 FADH₂, 2 ATP

Oxidative phosphorylationInner mitochondrial membrane~26–28 ATP, H₂O

Chemiosmosis

H⁺ flow through ATP synthase down their electrochemical gradient → ATP synthesised from ADP + Pᵢ. Driven by electron flow along ETC.

Oxygen's role

Final electron acceptor at end of ETC. Combines with e⁻ + H⁺ → water. Without O₂, ETC stops and ~93% of ATP cannot be made.

Exam Tip

For any "explain ATP synthesis" question: name NADH/FADH₂ → electrons → ETC → proton pumping → gradient → chemiosmosis → ATP synthase → ATP. Never skip steps. Always state O₂ is the final electron acceptor and H₂O is formed at the end of the ETC — not CO₂.