The TCA Cycle & Oxidative Phosphorylation

The tricarboxylic acid (TCA) cycle, also known as the citric acid cycle or Krebs cycle, is the central metabolic hub of aerobic organisms. It oxidises acetyl-CoA derived from carbohydrates, fatty acids, and amino acids, capturing energy as NADH and FADH2 that fuel oxidative phosphorylation and ATP synthesis.

The TCA Cycle: Eight Reactions

The cycle begins when acetyl-CoA (2 carbons) condenses with oxaloacetate (OAA, 4 carbons) to form citrate (6 carbons), catalysed by citrate synthase. This is the entry point and a key regulatory step. Citrate is isomerised to isocitrate via aconitase (through the intermediate cis-aconitate), requiring dehydration then rehydration to reposition the hydroxyl group.

Isocitrate dehydrogenase (IDH) catalyses the first oxidative decarboxylation: isocitrate + NAD+ → alpha-ketoglutarate + CO2 + NADH. This is a major regulatory point — IDH is allosterically activated by ADP and Ca2+, and inhibited by ATP and NADH. Alpha-ketoglutarate dehydrogenase complex (analogous to PDC, requiring TPP, lipoic acid, FAD, CoA, NAD+) catalyses the second oxidative decarboxylation: alpha-ketoglutarate + NAD+ + CoA → succinyl-CoA + CO2 + NADH.

Succinyl-CoA synthetase converts succinyl-CoA to succinate, with substrate-level phosphorylation generating GTP (equivalent to ATP). This is the only step of the TCA cycle that directly yields a high-energy phosphate. Succinate dehydrogenase (Complex II of the ETC) oxidises succinate to fumarate, reducing FAD to FADH2 — uniquely, this enzyme is embedded in the inner mitochondrial membrane. Fumarase adds water across the double bond to form L-malate. Malate dehydrogenase oxidises malate to OAA, producing the third NADH of the cycle. OAA is regenerated, completing the cycle.

Per acetyl-CoA turn: 3 NADH, 1 FADH2, 1 GTP, 2 CO2 released.

Regulation of the TCA Cycle

Three key enzymes are regulated: (1) Citrate synthase — inhibited by its product citrate, by NADH (high energy), and ATP; activated when OAA and acetyl-CoA levels are high. (2) Isocitrate dehydrogenase — inhibited by NADH and ATP; activated by ADP and Ca2+. (3) Alpha-ketoglutarate dehydrogenase — inhibited by succinyl-CoA and NADH (product inhibition); activated by Ca2+. The cycle accelerates when ADP is high and NADH/NAD+ ratio is low (energy demand), and decelerates when ATP and NADH are abundant (energy sufficiency). Ca2+ signals (e.g., during muscle contraction) stimulate all three regulatory enzymes simultaneously, matching energy production to demand.

Anaplerosis and Cataplerosis

TCA intermediates are continuously withdrawn for biosynthesis (cataplerosis): oxaloacetate → phosphoenolpyruvate (gluconeogenesis); alpha-ketoglutarate and oxaloacetate → glutamate and aspartate (transamination); succinyl-CoA → haem synthesis; citrate → cytoplasmic acetyl-CoA for fatty acid synthesis. Anaplerotic reactions replenish TCA intermediates: pyruvate carboxylase (pyruvate + CO2 + ATP → OAA, requires biotin), transamination of glutamate → alpha-ketoglutarate, propionyl-CoA → succinyl-CoA (from odd-chain fatty acid and branched-chain amino acid catabolism, requiring vitamin B12).

The Electron Transport Chain

The ETC is located in the inner mitochondrial membrane (IMM). NADH donates 2 electrons to Complex I (NADH:ubiquinone oxidoreductase, ~45 subunits), which pumps 4 H+ per electron pair from matrix to intermembrane space (IMS). FADH2 donates electrons to Complex II (succinate dehydrogenase) — no proton pumping. Both Complex I and II pass electrons to ubiquinone (coenzyme Q, CoQ), a lipid-soluble electron carrier in the IMM. CoQH2 transfers electrons to Complex III (cytochrome bc1 complex), which pumps 4 H+ via the Q cycle. Cytochrome c (a peripheral protein on the outer surface of the IMM) shuttles electrons from Complex III to Complex IV (cytochrome c oxidase), which reduces O2 to 2 H2O and pumps 2 H+. Total H+ pumped per NADH: ~10; per FADH2: ~6.

Chemiosmosis and ATP Synthesis

The proton-motive force (Δp) generated by ETC proton pumping comprises a chemical gradient (ΔpH, ~0.75 units) and an electrical gradient (Δψ, ~−180 mV), together equivalent to ~200 mV. ATP synthase (Complex V, F0F1-ATPase) spans the IMM; F0 is the membrane-embedded proton channel, F1 is the catalytic headpiece in the matrix. Proton flow through F0 rotates the γ-subunit, driving conformational changes in the three β-subunits of F1 (Boyer's binding-change mechanism): each β-subunit cycles through Open (O), Loose (L), and Tight (T) states, synthesising ATP at the T state. Approximately 8 H+ flow per 3 ATP synthesised (c-ring stoichiometry ~8:3). The phosphate carrier and ATP/ADP translocase consume additional H+, making the actual cost ~3.7 H+ per cytoplasmic ATP. Modern P/O ratios: NADH ≈ 2.5 ATP; FADH2 ≈ 1.5 ATP.

Uncouplers and Inhibitors

Uncouplers dissipate the proton gradient as heat without ATP synthesis. 2,4-Dinitrophenol (DNP, a lipid-soluble weak acid) carries protons across the IMM, uncoupling electron flow from phosphorylation — historically misused for weight loss with fatal outcomes. Thermogenin (UCP1) is the physiological uncoupler in brown adipose tissue, activated by fatty acids and norepinephrine, generating heat (non-shivering thermogenesis) — critical for neonatal and hibernating animal thermoregulation. ETC inhibitors: rotenone (Complex I), antimycin A (Complex III), cyanide/CO/azide (Complex IV — bind haem a3, prevent O2 reduction). Oligomycin inhibits ATP synthase F0 subunit. ATP synthase inhibition blocks the ETC as the proton gradient builds to oppose further pumping.

Clinical Correlations

Cyanide poisoning: inhibits Complex IV, halting electron flow and O2 consumption. Cells switch to anaerobic glycolysis → severe lactic acidosis. Treatment: hydroxocobalamin (scavenges CN−) or sodium thiosulfate + sodium nitrite. Metformin: inhibits Complex I, reducing NADH oxidation and lowering gluconeogenesis (reduced ATP in hepatocytes). Leigh syndrome: mitochondrial disease from mutations in ETC subunits or assembly factors → neurodegeneration. MELAS: mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes — mtDNA mutation impairs ETC subunit translation.