When electrons go through the electron transport chain, the protons in the matrix of the mitochondrion are pumped into the intermembrane space (the space between inner and outer membranes). This creates a proton gradient that is used by ATP synthase to create ATP through oxidative phosphorylation, not substrate-level phosphorylation. Remember that substrate-level phosphorylation is used by glycolysis and the Krebs cycle to generate ATP.

When electrons travel down the series of molecules in the electron transport chain they go from molecules of low electron affinity to molecules high electron affinity. The next molecule in the series must have higher affinity so that it can pull the electron away from its predecessor.

Electrons from electron carriers, such as NADH and FADH₂, go through the electron transport chain, which involves a series of molecules that accept and donate electrons. Transfer to the electron through these proteins results in the net movement of protons across the inner mitochondrial membrane and into the intermembrane space, generating the proton gradient that will drive ATP synthase.

The final molecule in the electron transport chain is oxygen. The oxygen molecule accepts the electron from the final protein in the chain and becomes water, one of the final products of metabolism. Remember that each subsequent molecule in the electron transport chain has a higher affinity for electrons than the molecule before it; therefore, the final electron acceptor will have the highest affinity for electrons. Oxygen has a very high electronegativity, making it a good electron acceptor.

The direct purpose of moving electrons down the electron transport chain is to pump protons (hydrogen ions) into the intermembrane space. This creates a chemiosmotic gradient that the cell uses to generate ATP by selectively allowing hydrogen ions to move back into the mitochondrial matrix.

Energy is not directly captured as electrons travel down the electron transport chain to synthesize ATP. GTP is a product of the Krebs cycle and can be used to generate cellular energy, but is not involved in synthesizing ATP or the electron transport chain. Other metabolic processes are often used to regulate glucose concentrations in the blood, indirectly influencing the rate of glycolysis and cellular respiration, but these processes do not directly provide energy for the electron transport chain.

ATP synthase, the enzyme responsible for ATP production on the inner mitochondrial membrane, depends on the proton gradient produced by the electron transport chain (ETC). If the proton gradient is disrupted, not as many ATP can be produced.

NADH and FADH₂ are essential to the function of the electron transport chain as electron donors, and are produced during glycolysis and the Krebs cycle to facilitate this process. Electron donation from these compounds is what fuels the formation of the proton gradient, while decreases in these compounds can cause uncoupling.

Oxidative phosphorylation is composed of electron transport and chemiosmosis. Chemiosmosis occurs when ions cross a selectively permeable membrane down their concentration gradient. In cellular respiration, hydrogen ions (protons) move down their concentration gradient through a membrane protein to produce ATP. The gradient of protons is established by the electron transport portion of oxidative phosphorylation, which is used to transfer protons into the intermembrane space. Protein complexes I, II, III, and IV help protons to cross the membrane.

Substrate-level phosphorylation occurs during glycolysis, and does not utilize chemiosmosis.

Both NADH and FADH₂ donate two electrons to the electron transport chain, so theoretically they should make the same amount of ATP. However, NADH donates its electrons to complex I while FADH₂ donates its electrons further "downstream" at complex II. Because complex I is a site for pumping protons into the intermembrane space, FADH₂'s electrons will not pump as many protons as those from NADH. This results in more ATP being generated from a single molecule of NADH than a single molecule of FADH₂.

The Krebs cycle creates a proton gradient between the intermembrane space and the inner mitochondrial membrane. This proton gradient provides the energy necessary to drive the proton through the ATP synthase. As the protons are passively diffusing through the ATP synthase, the energy is coupled to phosphorylate ADP to ATP. If a base were injected into this space, then it would would consume these protons due to its electronegativity and decrease ATP synthase’s ability to transform ADP to ATP.

The enzyme ATP synthase uses the electromotive force generated by the unequal concentrations of protons across both sides of the membrane to attach a phosphate group to ADP, generating ATP. The passing of a proton from a high concentration to low concentration permits the formation of the ATP molecule. Cytochrome c is an enzyme embedded in the inner mitochondrial membrane, but is not directly associated with ATP synthesis. Succinate dehydrogenase and aldolase are enzymes involved in the Krebs cycle.

The final electron acceptor in the electron transport chain is oxygen. It gets reduced by accepting two electrons and two protons from the ATP synthase to form water via the following equation:

The most acidic environment, or the lowest pH, would be found in the intermembrane space. This is because as  and  pass their electrons to the enzymes in the electron transport chain, protons are pumped into the intermembrane space. This is where a high concentration protons is generated, which is considered acidic. The low concentration of protons is generated in the mitochondrial matrix rendering it basic.