During the electron transport chain, electrons from NADH and FADH₂ are first transferred to complexes I and II, respectively. These two complexes then transfer the electrons to ubiquinol, which carries them further to complex III. Complex III passes the electrons across the intermembrane space to Cyt c, which carries them further to complex IV. Complex IV donates electrons to oxygen and reduces it to water. As electrons pass through complexes I, III, and IV, the energy released aids the pumping of protons into the intermembrane space, creating a proton gradient. This proton gradient drives the synthesis of ATP from ADP and inorganic phosphate in complex V or ATP synthase and helps fulfill the cell's energy requirements.

Superoxide Generation in Complex III

The electron transport chain complexes located on the mitochondrial membrane are the major sites of non-enzymatic superoxide generation within a cell. These superoxides are the primary cause of cellular oxidative damage that underlies various degenerative diseases as well as aging. While complexes I and II generate superoxides within the mitochondrial matrix, complex III produces superoxides either inside the matrix or the intermembrane space.

The actual source of superoxides in complex III is the ubiquinone or Q cycle, where an unstable radical ubisemiquinone (Q-) is generated. This radical can donate its unpaired electron to oxygen to generate superoxide anions. Drugs such as stigmatellin obstruct the electron flux from ubiquinone to iron-sulfur proteins and prevents the oxidation of ubiquinone to ubisemiquinone, thereby diminishing the generation of superoxides. In contrast, drugs such as Antimycin A can increase the generation of superoxides within the Q-cycle by increasing the steady-state concentration of ubisemiquinone.

Complex IV acts as the Regulatory Center

Cytochrome c oxidase (COX) or Complex IV acts as the final oxygen accepting complex as well as the regulatory center of oxidative phosphorylation in eukaryotic cells. It is regulated through various mechanisms, including allosteric-ATP inhibition. When the cells' ATP/ADP ratio is high, the phosphorylated COX undergoes feedback inhibition by ATP. This allosteric inhibition helps sense the cells' energy levels and adjust ATP synthesis in the mitochondria according to the energy demand.