C-terminal truncation of Noxa1 greatly enhances its ability to activate Nox2 in a pure reconstitution system

Noxa1 activates Nox2 together with Noxo1 and Rac in a pure reconstitution system, but the resulting activity is considerably lower than that induced by p67phox and p47phox. In this study, we found that C-terminal-truncated forms of Noxa1 exhibited higher activities than full-length Noxa1. Of the truncations examined, Noxa1(1-225) showed the highest ability for activation. Kinetic studies revealed that Noxa1(1-225) had a threefold higher Vmax value than full-length Noxa1 with a similar EC50 value. The affinities of Noxo1 and RacQ61L were not much altered by the truncation. Conversely, the affinity of FAD for the Nox2 complex was enhanced after the truncation. In the absence of Noxo1, Noxa1(1-225) showed much higher activity with a lower EC50 than full-length Noxa1. Noxa1(1-225) showed comparable activity to that of p67phox with either Noxo1 or p47phox, although the stability was lower than that with p67phox and p47phox. These findings indicate that the role of the C-terminal half of Noxa1 is autoinhibition. The data suggest a two-step autoinhibition mechanism, comprising self-masking to interrupt the binding to the oxidase, and holding of the activation domain in a suboptimal position to the oxidase. This study reveals that when both types of inhibition are released, Noxa1 achieves high-level superoxide production.