In contrast to many unicellular organisms including yeast, which displayed evolved mechanisms allowing them to grow and survive in the absence of oxygen, animals fully rely on cell respiration that takes place in the mitochondria. The oxidative phosphorylation (OxPhos) machinery is a key functional unit, located on the inner mitochondrial membrane that combines electron transport with cell respiration and ATP synthesis.

The energy given to the electrons of the reduced coenzyme NADH and to succinate by the TCA cycle is transferred in small steps in the inner membrane of the mitochondrion through a chain of five protein complexes. This series of coupled reactions is often referred to as oxidative phosphorylation. OXPHOS consists of the electron transport chain (ETC), which comprises NADH-dehydrogenase (complex I), succinate dehydrogenase (complex II), ubiquinone (Coenzyme Q10 (CoQ10)), bc1 complex (complex III), cytochrome c (Cyt c), and cytochrome c oxidase (complex IV).

The movement of electrons through complexes I-IV causes protons (hydrogen atoms) to be pumped out of the intramitochondrial matrix into intermembrane space.  As a result, a net negative charge (from the electrons) builds up in the matrix space while a net positive charge (from the proton pumping) builds up in the intermembrane space. This differential electrical charge establishes an electrochemical gradient.

ATP synthase (complex V) functions like a molecular nanomotor. The synthesis of ATP requires several steps, including the binding of ADP and phosphate, the formation of the new phosphate-phosphate bond, and release of ATP. As the axle turns, it forces the motor into three different conformations that assist these difficult steps.