The cytosolic component p47(phox) is not a sine qua non participant in the activation of NADPH oxidase but is required for optimal superoxide production

The superoxide (O-2(radical anion))-generating NADPH oxidase of phagocytes is a multicomponent complex consisting of a membrane-associated flavocytochrome (cytochrome b(559)), bearing the NADPH binding site and two redox centers (FAD and heme) and three cytosolic activating components: p47(phox), p67(phox), and the small GTPase Rac (1 or 2). The canonical view is that the induction of OX generation involves the stimulus-dependent assembly of all three cytosolic components with cytochrome b(559), a process mimicked in vitro by a cell-free system activated by anionic amphiphiles. We studied the requirement for individual cytosolic components in the activation of NADPH oxidase in a cell-free system consisting of purified and relipidated cytochrome b(559), recombinant p47(phox), p67(phox), and Rac1, and the amphiphile, lithium dodecyl sulfate. We found that pronounced activation of NADPH oxidase can be achieved by exposing cytochrome b(559) to p67(phox) and Rac1, in the total absence of p47(phox) (turnover = 60 mol O-2((radical anion))/mol cytochrome b(559)). However, maximal activation (turnover = 153 mol O-2((radical anion))/s/mol cytochrome b(559)) could only be obtained in the presence of p47(phox). O-2((radical anion)) production, in the absence of p47(phox), was dependent on: high molar ratios of p67(phox) and Rac1 to cytochrome b(559), Rac1 being in the GTP-bound form, cytochrome b(559) being saturated with FAD, and an optimal concentration of amphiphile. Single cytosolic components or combinations of two cytosolic components, other than p67(phox) and Rac1, were incapable of activation. We conclude that p67(phox) and Rac1 are the only cytosolic components directly involved in the induction of electron transport in cytochrome b(559). p47(phox) appears to facilitate or stabilize the interaction of p67(phox) and, possibly, Rac1 with cytochrome b(559), and is required for optimal generation of O-2((radical anion)) under physiological conditions.