The electron transport chain generates the chemiosmotic gradient by pumping protons from the mitochondrial matrix into the intermembrane space as it passes electrons down the electron transport chain. NADH and FADH₂ donate electrons to the first protein complex in the chain, which subsequently passes the electrons on to other complexes until the electrons are donated to an oxygen molecule. With each electron transfer between transport complexes, protons are translocated into the intermembrane space.

Protons are not imported or exported from the cytoplasm. There are no scaffold proteins that actually carry protons between the mitochondrial matrix and the intermembrane space.

The electron transport chain (ETC) consists of several membrane proteins that are used to carry electrons along the membrane and, by harnessing this energy, generate a proton gradient across the inner membrane of the mitochondria.

The reason that NADH has a higher production of ATP is because it enters the ETC at an earlier point than FADH₂. This allows the cell to derive more energy from the electrons because they are moved further in the chain. FADH₂ does not provide fewer electrons and the size of the molecule does not come into play at all. It also does not matter where the FADH₂ is generated, especially because both NADH and FADH₂ are produced during the Krebs cycle in the mitochondrial matrix.

Aerobic respiration has three main stages: glycolysis, the Krebs cycle, and the electron transport chain. Glycolysis functions to convert a six-carbon glucose molecule into two three-carbon pyruvate molecules, and can occur in either aerobic or anaerobic environments. The net yield from glycolysis is two ATP per glucose. The two pyruvate molecules then enter the Krebs cycle, which serves to produce the electron donor NADH. The Krebs cycle produces two GTP molecules per glucose, which carry energy similar to ATP. The NADH from the Krebs cycle is transported to the electron transport chain and used to generate the chemiosmotic proton gradient that exists between the two mitochondrial membranes. The electron transport itself does not generate any ATP. Oxidative phosphorylation occurs on the inner mitochondrial membrane and uses the energy of the proton gradient to power ATP synthase. Through oxidative phosphorylation, ATP synthase is able to produce approximately 36 ATP per glucose.

Although all stages of respiration result in ATP production, oxidative phosphorylation produces much more ATP than any other step.

Oxygen is not directly needed for the completion of the Krebs cycle. Electron carriers, such as NADH and FADH₂, are responsible for bringing electrons to the electron transport chain (ETC), not oxygen. Oxygen is incredibly important, however, in acting as the final electron acceptor of the electron transport chain. During this process, the oxygen reacts with the electrons and free hydrogen to form water. Keep in mind that, though the electron transport chain is used to power oxidative phosphorylation, the two are essentially separate processes. Oxygen accepts electrons that were used to pump protons in the electron transport chain, but does not interact with ATP synthase or oxidative phosphorylation.

Cytochromes are structurally similar to hemoglobin molecules in that they contain a central iron atom. Iron can go from an oxidation state of  to  after receiving an electron, and back to  after the electron has been passed on to the next carrier. Thus cytochromes are enzymes that catalyze redox reactions.

Oxygen is the final electron acceptor in the electron transport chain. The electrons and two hydrogen atoms are picked up by oxygen in order to make water.

 is capable of generating more ATP through the electron transport chain. This is because  donates its electrons to the  dehydrogenase complex while  donates it electron to ubiquinone, a later step in the transport chain. In summation, more protons are pumped across the membrane in the case of , resulting in greater ATP production per molecule. In other words,  has a higher reduction potential (less negative) than , and thus  does not give up its electrons as easily as does .  instead skips down the electron transport chain to ubiquinone, which has a high enough reduction potential to spontaneously strip the electron from . This can be seen by remembering that the more positive the reduction potential, the more spontaneous the reaction:

NADH dehydrogenase, also referred to as enzyme complex I, is found in the inner mitochondrial membrane. NADH produced during glycolysis (in the cytoplasm), during pyruvate dehydrogenation (in the mitochondrial matrix), and the Krebs cycle (in the mitochondrial matrix) can then donate two electrons to NADH dehydrogenase to begin the electron transportation and subsequent ATP production through the process known as chemiosmosis. This involves pumping hydrogen ions into the intermembrane space then allowing them to flow down their established electrochemical gradient through ATP synthetase, which is anchored in the inner mitochondrial membrane, into the mitochondrial matrix to combine with oxygen and electrons to form water.