Constitutive NADPH-Dependent Electron Transferase Activity of the Nox4 Dehydrogenase Domain

NADPH oxidase 4 (Nox4) is constitutively active, While Nox2 requires the cytosolic regulatory Subunits p47(phox) and p67(phox) and activated Rac With activation by phorbol 12-myristate 13-acetate (PMA). This Study Was undertaken to identify the domain oil Nox4 that confers constitutive activity Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production With a K-m For NADPH of 55 +/- 10 mu M. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of similar to 200 mol of H2O2 min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox-2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H2O2 production in the absence Of cytosolic Subunits. Ill contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression Of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electrom transferase activities. Nox I DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction Of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide(470min(-1)), and other electron acceptors (artificial dyes and cytochrom b(5)). Rates were similar to those observed for H2O2 production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and Was seen with NADPH but not NADH. These results Indicate that the Nox4 DH domain exists in all Intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.