Noxa1 as a moderate activator of Nox2-based NADPH oxidase

Noxa1 was discovered as an activating factor for Nox1, an O2 - generating enzyme. Subsequent studies have shown that Noxa1 is colocalized with Nox2 in several cell types, including vascular cells. Nox2 activation by Noxa1 has been examined in reconstituted model cells. However, little is known about the kinetic properties of Noxa1 in Nox2 activation. In the present study, we used purified cyt.b ₅₅₈ (Nox2 plus p22 ^phox), Rac(Q61L), and Noxo1 to examine the ability of Noxa1 to activate Nox2. In the pure reconstitution system, Noxa1 activated Nox2 with lower efficiency than p67 ^phox, a canonical activator of Nox2. The EC ₅₀ value of Noxa1 was considerably higher than that of p67 ^phox. The V _max value with Noxa1 and Noxo1 was one-third of that with p67 ^phox and p47 ^phox. The EC ₅₀ value of Noxo1 or Rac(Q61L) was also higher when Noxa1 was used. The affinity of FAD for the oxidase and the stability of the active complex were remarkably low when Noxa1 and Noxo1 were used compared with p67 ^phox and p47 ^phox. The stability was not improved by fusion of Noxa1 with Rac(Q61L). These findings show that Noxa1 has quite different kinetic properties from p67 ^phox and suggest that Noxa1 may function as a moderate activator of Nox2.