21.3 TCA Cycle, Electron Transport & Oxidative Phosphorylation

Pyruvate Dehydrogenase

Before entering the TCA cycle, pyruvate is oxidatively decarboxylated to acetyl-CoA by the pyruvate dehydrogenase complex (PDHC) in the mitochondrial matrix. The net reaction: pyruvate + CoA + NAD+ → acetyl-CoA + CO2 + NADH. PDHC is a multi-enzyme complex of three enzymes (E1 pyruvate dehydrogenase, E2 dihydrolipoyl transacetylase, E3 dihydrolipoyl dehydrogenase) requiring five cofactors: thiamine pyrophosphate (B1), lipoic acid, CoA (pantothenate), FAD (B2), NAD+ (B3). This cofactor list — "thiamine, lipoate, CoA, FAD, NAD" — is a high-yield PA-CAT fact per the Bulletin of Information, rev. 20240815. PDHC is regulated by covalent modification: PDH kinase phosphorylates and inhibits E1, activated by ATP, NADH, acetyl-CoA and inhibited by ADP, pyruvate; PDH phosphatase dephosphorylates and activates, stimulated by Ca2+ (signaling muscle contraction). Thiamine deficiency (beriberi, Wernicke-Korsakoff) impairs PDHC and the TCA cycle.

TCA Cycle (Krebs Cycle)

The tricarboxylic acid (TCA) cycle oxidizes acetyl-CoA to two CO2, producing 3 NADH, 1 FADH2, and 1 GTP per acetyl-CoA. The eight steps, all mitochondrial:

1. Citrate synthase (oxaloacetate + acetyl-CoA → citrate)
2. Aconitase (citrate → isocitrate)
3. Isocitrate dehydrogenase (rate-limiting; → α-ketoglutarate, first NADH, first CO2)
4. α-Ketoglutarate dehydrogenase (→ succinyl-CoA, second NADH, second CO2; uses the same five cofactors as PDHC)
5. Succinyl-CoA synthetase (→ succinate, GTP via substrate-level phosphorylation)
6. Succinate dehydrogenase (→ fumarate, FADH2; the only membrane-bound TCA enzyme — Complex II of the ETC)
7. Fumarase (→ malate)
8. Malate dehydrogenase (→ oxaloacetate, third NADH)

Regulation: citrate synthase (inhibited by citrate, succinyl-CoA, ATP), isocitrate dehydrogenase (activated by ADP, Ca2+; inhibited by ATP, NADH), and α-ketoglutarate dehydrogenase (inhibited by succinyl-CoA, NADH, ATP) are the three regulated steps. The cycle is amphibolic — it provides intermediates for gluconeogenesis (oxaloacetate), amino acid synthesis (α-ketoglutarate, oxaloacetate), heme synthesis (succinyl-CoA), and fatty acid synthesis (citrate exported to cytosol). Anaplerotic reactions replenish intermediates; pyruvate carboxylase (pyruvate → oxaloacetate) is the most important.

Electron Transport Chain

The electron transport chain (ETC) in the inner mitochondrial membrane transfers electrons from NADH and FADH2 to O2, using the released energy to pump protons. Four multiprotein complexes:

• Complex I (NADH dehydrogenase) — oxidizes NADH, passes electrons via FMN and Fe-S centers to ubiquinone (CoQ); pumps 4 H+.
• Complex II (succinate dehydrogenase) — oxidizes FADH2 (from succinate), passes electrons to CoQ; pumps 0 H+.
• Complex III (cytochrome bc1 complex) — passes electrons from CoQH2 to cytochrome c; pumps 4 H+.
• Complex IV (cytochrome c oxidase) — passes electrons to O2, reducing it to H2O; pumps 2 H+.

Ubiquinone (CoQ) is the mobile lipid-soluble carrier between Complexes I/II and III; cytochrome c is the mobile water-soluble carrier between III and IV.

Chemiosmosis and ATP Synthase

The proton gradient generated by the ETC (higher [H+] in the intermembrane space, lower in the matrix) stores electrochemical energy — the proton-motive force. ATP synthase (Complex V) harnesses this gradient: protons flow through the F0 transmembrane channel, rotating the c-ring and γ-stalk; conformational changes in the F1 catalytic β-subunits condense ADP + Pi → ATP. Approximately 4 H+ are required per ATP (3 for synthesis, 1 for ADP/ATP and Pi transport). Oxidative phosphorylation is the coupled process of ETC electron transfer plus ATP synthase ATP production; it depends on the inner membrane being intact and impermeable to protons.

Energy Yield

NADH entry at Complex I pumps enough protons to generate ~2.5 ATP; FADH2 entry at Complex II (skipping Complex I) generates ~1.5 ATP. Complete glucose oxidation yields ~30–32 ATP: glycolysis 2 ATP (substrate-level) + 2 NADH (→ ~5 ATP via the malate-aspartate shuttle) + 2 pyruvate → 2 acetyl-CoA (2 NADH, ~5 ATP) + 2 TCA turns (6 NADH ~15 ATP, 2 FADH2 ~3 ATP, 2 GTP = 2 ATP). Older "3 ATP per NADH / 2 per FADH2" figures (~36–38 ATP) reflect pre-chemiosmotic bookkeeping.

Inhibitors and Uncouplers

Inhibitors block electron transfer at specific sites: rotenone (Complex I), malonate (competitive inhibitor of succinate dehydrogenase, Complex II), antimycin A (Complex III), cyanide, carbon monoxide, and azide (Complex IV). Uncouplers dissipate the proton gradient, allowing electron transport to continue without ATP synthesis: 2,4-dinitrophenol (DNP, a lipophilic proton carrier) and thermogenin (UCP1 in brown adipose tissue, activated by free fatty acids for non-shivering thermogenesis). Uncoupling increases O2 consumption and heat production. Oligomycin directly inhibits ATP synthase. These inhibitors are clinically and toxicologically important — cyanide and CO poisoning are classic PA-CAT ties between biochemistry and physiology.

ATP Accounting, TCA Control, and Shuttle Yields

The modern P/O ratio (ATP per pair of electrons reaching O2) explains why the older values of 3 ATP per NADH and 2 per FADH2 are obsolete. Each NADH drives roughly 10 protons across the inner membrane (4 at Complex I, 4 at III, 2 at IV); ATP synthase plus the ADP/ATP and Pi transporters consume about 4 protons per ATP, giving a P/O near 2.5. FADH2 skips Complex I, pumps only 6 protons, and yields ~1.5. Proton leak (through UCP1 in brown fat and the adenine nucleotide translocase) lowers the realized yield further, which is why whole-body glucose oxidation lands at ~30 to 32 ATP rather than the older 36 to 38. PA-CAT items quoting 36 to 38 test whether you recognize the chemiosmotic correction.

Calcium ties the TCA cycle to muscle contraction: Ca2+ released from the sarcoplasmic reticulum activates isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, and PDH phosphatase at once, accelerating fuel oxidation when ATP demand spikes. The three rate-limiting TCA enzymes—citrate synthase, isocitrate dehydrogenase, and alpha-ketoglutarate dehydrogenase—are activated by ADP and Ca2+ and inhibited by ATP, NADH, and their own products (citrate, succinyl-CoA). Succinyl-CoA synthetase is not rate-limiting but is the only TCA step that makes ATP by substrate-level phosphorylation—GTP forms directly from succinyl-CoA thioester cleavage, independent of the ETC and proton gradient. Every other TCA ATP arrives only through oxidative phosphorylation, so a poisoned ETC still leaves this one GTP intact per acetyl-CoA.

Cytosolic NADH from glycolysis cannot cross the inner membrane, so its reducing power arrives via two shuttles with different ATP payouts. The glycerol-3-phosphate shuttle transfers electrons to FAD at Complex II, yielding ~1.5 ATP per cytosolic NADH and dominating in fast-twitch muscle and brain. The malate-aspartate shuttle transfers electrons to NAD+ in the matrix, yielding ~2.5 ATP and dominating in liver, kidney, and heart. The shuttle choice, not glycolysis itself, decides whether the two cytosolic NADH contribute ~3 or ~5 ATP to the glucose total.

A classic PA-CAT inhibitor-discrimination question contrasts three failure modes. Rotenone (Complex I) and antimycin A (Complex III) block electron flow, so the chain backs up, proton pumping stops, and O2 consumption falls. Oligomycin directly blocks ATP synthase: electron transport continues briefly but halts as the gradient saturates—no ATP is made, yet the block is downstream of the pumps. Uncouplers such as DNP carry protons back across the membrane, dissipating the gradient: electron flow and O2 consumption accelerate, heat rises, but no ATP is synthesized. The discriminator is whether electron flow stops, ATP synthesis stops, or the two diverge.

Bioenergetics: Why Any of This Runs

Bioenergetics is its own Bulletin objective under Intermediary Metabolism, and it supplies the accounting that makes the pathways above make sense. The governing quantity is the Gibbs free energy change, ΔG = ΔH − TΔS. A reaction is exergonic and spontaneous when ΔG < 0 and endergonic when ΔG > 0; ΔG = 0 defines equilibrium. Distinguish ΔG°′, the standard value at pH 7, from the actual ΔG in the cell, which depends on real concentrations through ΔG = ΔG°′ + RT ln Q. This distinction resolves a common confusion: the ATP-to-ADP hydrolysis standard value is about −30.5 kJ/mol (≈7.3 kcal/mol), but because cells hold ATP far above and ADP and Pi far below equilibrium, the effective in-cell value is nearer −50 kJ/mol.

Cells drive endergonic work by energy coupling: an unfavorable reaction is run alongside a favorable one, and only the sum of the two ΔG values must be negative. Hexokinase illustrates the pattern — phosphorylating glucose alone is endergonic at about +13.8 kJ/mol, but coupled to ATP hydrolysis the net is roughly −16.7 kJ/mol and the reaction proceeds. ATP works as the cell’s energy currency not because its bonds are unusually strong but because ADP and Pi are more stable than ATP: charge repulsion among the phosphates is relieved, the products resonance-stabilize, and both are better solvated. That is why ATP sits in the middle of the phosphoryl-transfer scale — phosphoenolpyruvate (−61.9 kJ/mol) and creatine phosphate (−43.1 kJ/mol) can phosphorylate ADP, while glucose-6-phosphate (−13.8 kJ/mol) cannot — making it an effective intermediate carrier in both directions rather than a dead end.

The same logic governs oxidative phosphorylation itself. Electron transfer down the chain is exergonic because each carrier has a more positive standard reduction potential than the last, ending at O2 at +0.82 V; the relationship ΔG°′ = −nFΔE°′ converts that voltage drop into roughly −220 kJ/mol for the NADH-to-O2 span. The proton-motive force stores part of that energy as an electrochemical gradient, and ATP synthase spends it. Nothing in metabolism violates the second law: the cell maintains its local order by exporting heat and disorder to its surroundings.