Generation of superoxide by purified and relipidated cytochrome b559 in the absence of cytosolic activators.

Purified cytochrome b559 from guinea pig macrophages was relipidated with several phospholipid mixtures. Relipidated cytochrome b559 was found capable of NADPH-dependent superoxide (O2-) production in the absence of the cytosolic components of the NADPH oxidase complex. The rate of O2- generation by cytochrome b559 varied with the type of phospholipid utilized for relipidation, was absolutely dependent on exogenous FAD, and was enhanced by a critical concentration of anionic amphiphile. It is demonstrated that exogenous FAD acts by binding to cytochrome b559. These results provide firm experimental evidence for the proposal that cytochrome b559 comprises the complete electron transporting apparatus of the O2- forming NADPH oxidase and that the cytosolic components function merely as activators.