Glycolysis and the citric acid cycle feed electrons (as NADH/FADH₂) into the electron transport chain (Complexes I–IV) on the inner mitochondrial membrane. Their energy pumps H⁺ from the matrix into the intermembrane space, building an electrochemical gradient. Protons flow back through ATP synthase, spinning its rotor and phosphorylating ADP into ATP.
NADH + ½O₂ + H⁺ → NAD⁺ + H₂O (drives H⁺ pumping)
ADP + Pi --[ATP synthase, H⁺ flux]--> ATP
- Substrate supply — how fast glycolysis/Krebs deliver electrons to the chain; scales pumping rate.
- Oxygen availability — O₂ is the final electron acceptor at Complex IV; low O₂ backs up the whole chain (hypoxia).
- Cyanide — blocks Complex IV outright, so electron flow and proton pumping halt almost immediately.
- Uncoupler (DNP-like) — lets H⁺ leak straight back across the membrane, bypassing ATP synthase: the gradient collapses and the energy is released as heat instead of ATP.
Real-world relevance: cyanide poisoning kills by stopping Complex IV; brown-fat thermogenesis and some banned diet drugs (2,4-dinitrophenol) work by deliberately uncoupling this same gradient to burn energy as heat.