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Biochemistry Unit 9: Citric Acid Cycle and Oxidative Phosphorylation

The citric acid cycle oxidizes acetyl-CoA to CO₂ and loads electrons onto NADH and FADH₂, and oxidative phosphorylation turns those electrons into most of the cell's ATP. This unit covers the reactions, energy accounting and control of aerobic metabolism.

Covers: Pyruvate dehydrogenase complex, Reactions of the citric acid cycle, Regulation and anaplerosis, Electron transport chain complexes, Redox potentials and free energy, Chemiosmosis and ATP synthase, ATP yield, shuttles, inhibitors and uncouplers

Study guide

Pyruvate enters the mitochondrial matrix and is converted to acetyl-CoA by the pyruvate dehydrogenase (PDH) complex, releasing CO₂ and making NADH. PDH uses five cofactors: thiamine pyrophosphate, lipoic acid, coenzyme A, FAD and NAD⁺. It is irreversible and tightly controlled: products (acetyl-CoA, NADH) inhibit it, and PDH kinase phosphorylates and inactivates it when ATP and NADH are high. PDH phosphatase, activated by Ca²⁺ and insulin, turns it back on. Thiamine deficiency impairs PDH and causes pyruvate and lactate to accumulate.

In the citric acid cycle, citrate synthase joins acetyl-CoA (2C) to oxaloacetate (4C) to make citrate (6C). Aconitase forms isocitrate; isocitrate dehydrogenase and α-ketoglutarate dehydrogenase each release CO₂ and make NADH. Succinyl-CoA synthetase makes GTP, succinate dehydrogenase (Complex II) makes FADH₂, fumarase adds water, and malate dehydrogenase makes the third NADH while regenerating oxaloacetate. Each turn yields 3 NADH, 1 FADH₂, 1 GTP and 2 CO₂. Isocitrate dehydrogenase and α-ketoglutarate dehydrogenase are activated by ADP and Ca²⁺ and inhibited by ATP and NADH. Anaplerotic reactions, such as pyruvate carboxylase, refill intermediates drawn off for biosynthesis.

The electron transport chain in the inner membrane passes electrons from NADH and FADH₂ to O₂. Complex I (NADH dehydrogenase) passes electrons to ubiquinone and pumps 4 H⁺. Complex II feeds electrons from succinate to ubiquinone and pumps none. Complex III transfers electrons from ubiquinol to cytochrome c through the Q cycle, pumping 4 H⁺. Complex IV reduces O₂ to water and pumps 2 H⁺. Electrons flow from carriers with low (negative) reduction potentials to higher ones, and ΔG°′ = −nFΔE°′. NADH to O₂ releases about −219 kJ/mol.

Mitchell's chemiosmotic theory explains how this energy makes ATP. Pumped protons create a proton-motive force across the inner membrane, made of a membrane potential (matrix negative) and a pH gradient (matrix basic). Protons flow back through ATP synthase: they turn the F₀ c-ring, which rotates the γ stalk inside F₁, driving conformational changes (binding change mechanism) that make ATP. In mammals, about 2.7 H⁺ per ATP drive synthesis and about 1 more is used to import phosphate, so roughly 3.7 H⁺ per cytosolic ATP. This gives P/O ratios of about 2.5 for NADH and 1.5 for FADH₂.

Complete oxidation of glucose yields about 32 ATP with the malate-aspartate shuttle or 30 with the glycerol 3-phosphate shuttle, which feeds cytosolic NADH electrons to ubiquinone. Inhibitors reveal the chain's order: rotenone blocks Complex I, antimycin A blocks Complex III, and cyanide, azide and CO block Complex IV. Oligomycin blocks ATP synthase, which also slows electron flow because the two are coupled. Uncouplers such as DNP and the brown-fat protein UCP1 let protons leak back without making ATP, so O₂ consumption rises and the energy is released as heat.

Key ideas

• PDH links glycolysis to the citric acid cycle and is controlled by product inhibition and phosphorylation.
• Each acetyl-CoA yields 3 NADH, 1 FADH₂, 1 GTP and 2 CO₂.
• Electrons flow from low to high reduction potential; ΔG°′ = −nFΔE°′.
• Complexes I, III and IV pump protons; Complex II does not.
• ATP synthase uses the proton-motive force through rotary catalysis.
• Electron transport and ATP synthesis are coupled; uncouplers break the link.
• Glucose yields about 30 to 32 ATP aerobically versus 2 anaerobically.

Formulas and rules

Common mistakes

• Using old ATP values (3 per NADH, 2 per FADH₂, 36 to 38 per glucose) when the course uses 2.5 and 1.5.
• Thinking Complex II pumps protons.
• Saying oligomycin raises O₂ use; it lowers it, while uncouplers raise it.
• Subtracting reduction potentials in the wrong order and getting the wrong sign of ΔG.
• Assuming acetyl-CoA carbons are released as CO₂ in the same turn they enter.

Before the test, make sure you can

• Describe the PDH reaction, its cofactors and its regulation.
• List citric acid cycle steps that produce NADH, FADH₂, GTP and CO₂.
• Calculate ATP yield per acetyl-CoA and per glucose.
• Calculate ΔG°′ from reduction potentials.
• Describe each electron transport complex and which ones pump protons.
• Explain chemiosmosis and how ATP synthase works.
• Predict effects of inhibitors and uncouplers on O₂ use and ATP synthesis.
• Explain anaplerosis and why acetyl-CoA cannot make net glucose.

Words to know

Pyruvate dehydrogenase complex

Converts pyruvate to acetyl-CoA, CO₂ and NADH using TPP, lipoate, CoA, FAD and NAD⁺.

Citrate synthase

Joins acetyl-CoA and oxaloacetate to form citrate.

Isocitrate dehydrogenase

Oxidizes isocitrate to α-ketoglutarate, releasing CO₂ and NADH; a key control point.

α-Ketoglutarate dehydrogenase

Makes succinyl-CoA, CO₂ and NADH; similar to PDH.

Succinyl-CoA synthetase

Makes GTP by substrate-level phosphorylation.

Succinate dehydrogenase

Oxidizes succinate to fumarate, making FADH₂; also Complex II.

Anaplerotic reaction

A reaction that refills citric acid cycle intermediates, such as pyruvate carboxylase.

Ubiquinone (Q)

A lipid-soluble electron carrier between Complexes I/II and III.

Cytochrome c

A small soluble protein carrying electrons from Complex III to IV.

Complex IV

Cytochrome c oxidase; reduces O₂ to H₂O and pumps protons.

Chemiosmotic theory

Mitchell's idea that a proton gradient couples electron transport to ATP synthesis.

Proton-motive force

The energy stored in the membrane potential plus pH gradient across the inner membrane.

ATP synthase

A rotary enzyme (F₀F₁) that uses proton flow to make ATP.

P/O ratio

ATP made per pair of electrons (per O atom) passed to O₂.

Uncoupler

A molecule like DNP that dissipates the proton gradient without making ATP.

Oligomycin

An inhibitor of ATP synthase's F₀ proton channel.

Malate-aspartate shuttle

Moves cytosolic NADH electrons into the matrix as NADH, preserving 2.5 ATP.

UCP1 (thermogenin)

A brown-fat proton channel that makes heat by uncoupling.

Sample practice questions

30 more questions like these, from level 1 to 5, are in the practice set.