Noxa1 as a moderate activator of Nox2-based NADPH oxidase

► We developed a pure cell-free system of Nox2 with noncanonical cytosolic factors. ► The system consisted of recombinant Noxa1, Noxo1, Rac, and purified cytochrome b558. ► Using the system, we clarified the kinetic properties of Noxa1 in Nox2 activation. ► Noxa1 was a less potent activator of Nox2 than p67phox either with Noxo1 or p47phox. ► Noxa1 has remarkably lower affinity for the oxidase complex than p67phox.