Simultaneous presence of p47(phox) and flavocytochrome b(-245), are required for the activation of NADPH oxidase by anionic amphiphiles - Evidence for an intermediate state of oxidase activation

We have examined the kinetics of NADPH oxidase activation induced by arachi donic acid or SDS in a cell-free system using mixtures of recombinant Phox proteins and purified flavocytochrome b(-245). Activation of oxidase activi ty required the simultaneous presence of p47(phox), flavocytochrome b(-245) , and the anionic amphiphile. The activation of electron transfer reactions was much more rapid when iodonitrotetrazolium violet was used as electron acceptor than when oxygen alone was the acceptor. We propose that this diff erence represents an intermediate activation state of NADPH oxidase in whic h electron flow can proceed hom NADPH to enzyme flavin (and hence to iodoni trotetrazolium violet) but not from flavin to heme (or not between the heme s). A model for NADPH oxidase activation is presented that is consistent wi th these observations.