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 phagoc ytes is a multicomponent complex consisting of a membrane-associated f lavocytochrome (cytochrome b(559)), bearing the NADPH binding site and two redox centers (FAD and heme) and three cytosolic activating compo nents: p47(phox), p67(phox), and the small GTPase Rac (1 or 2). The ca nonical view is that the induction of OX generation involves the stimu lus-dependent assembly of all three cytosolic components with cytochro me b(559), a process mimicked in vitro by a cell-free system activated by anionic amphiphiles. We studied the requirement for individual cyt osolic components in the activation of NADPH oxidase in a cell-free sy stem consisting of purified and relipidated cytochrome b(559), recombi nant p47(phox), p67(phox), and Rac1, and the amphiphile, lithium dodec yl sulfate. We found that pronounced activation of NADPH oxidase can b e 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))/mo l cytochrome b(559)). However, maximal activation (turnover = 153 mol O-2((radical anion))/s/mol cytochrome b(559)) could only be obtained i n the presence of p47(phox). O-2((radical anion)) production, in the a bsence 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, cytoc hrome b(559) being saturated with FAD, and an optimal concentration of amphiphile. Single cytosolic components or combinations of two cytoso lic components, other than p67(phox) and Rac1, were incapable of activ ation. We conclude that p67(phox) and Rac1 are the only cytosolic comp onents directly involved in the induction of electron transport in cyt ochrome b(559). p47(phox) appears to facilitate or stabilize the inter action of p67(phox) and, possibly, Rac1 with cytochrome b(559), and is required for optimal generation of O-2((radical anion)) under physiol ogical conditions.