During electron transport, electrons are released from NADH and FADH₂ and eventually transferred to O₂, forming H₂O, according to the following overall reactions:

NADH + H⁺ + ½ O₂ → NAD⁺ + H₂O,

ΔG = −52.6 kcal/mol

FADH₂ + ½ O₂ → FAD + H₂O,

ΔG = −43.4 kcal/mol

Recall that the conversion of 1 glucose molecule to CO₂ via the glycolytic pathway and citric acid cycle yields 10 NADH and 2 FADH₂ molecules (see Table 12-3). Oxidation of these reduced coenzymes has a total ΔG°′ of −613 kcal/mol [10(−52.6) + 2(−43.4)]. Thus of the total potential free energy present in the chemical bonds of glucose (−686 kcal/mol), about 90 percent is conserved in the reduced coenzymes. Why should there be two different coenzymes, NADH and FADH₂? Although many of the reactions involved in glucose and fatty acid oxidation are sufficiently energetic to reduce NAD⁺, several are not. To capture the energy released by those reactions, they are coupled to reduction of FAD, which requires less energy.

The energy carried in the reduced coenzymes can be released by oxidizing them. The biochemical challenge faced by the mitochondrion is to transfer, as efficiently as possible, the energy released by this oxidation into the energy in the terminal phosphoanhydride bond in ATP.

P_i²⁻ + H⁺ + ADP³⁻ → ATP⁴⁻ + H₂O,

ΔG = +7.3 kcal/mol

A relatively simple one-to-one reaction involving reduction of one coenzyme molecule and synthesis of one ATP molecule would be terribly inefficient because the ΔG°′ for ATP generation from ADP and P_i is substantially less than that for the coenzyme oxidation, and much energy would be lost as heat. To efficiently recover that energy, the mitochondrion converts the energy of coenzyme oxidation into a proton-motive force using a series of electron carriers, all but one of which are integral components of the inner membrane (see stage III in Figure 12-14). The proton-motive force can then be used to generate ATP very efficiently.