NADPH oxidase 4 function as a hydrogen peroxide sensor

Nox4, a member of the NADPH- and oxygen-dependent oxidoreductases that generate reactive oxygen species (ROS), is widely expressed and constitutively active. To understand better its function and regulation, specific mutations in the Nox4 dehydrogenase (DH) domain were examined for effects on Nox4 oxidase activity. Transfection of His₆-tagged Nox4 increased the amount of p22^phox subunit in HEK293 cells, and a higher level of the heterodimer was observed in the nucleus- enriched fraction (NEF). NEF from Nox4-expressing HEK293 cells exhibited oxygen and H₂O₂ concentration-dependent NADPH oxidation rate. In Nox4-expressing cells, NEF and its partially purified form, the Nox4(P437H) mutant almost completely lost its oxidase activity, while Nox4(C546S), Nox4(C546L) or/and (C547L) had a significantly decreased rate of ROS production. The NADPH-dependent reduction of cytochrome c or cytochrome b₅ by purified Nox4 DH domain was found regulated by the H₂O₂ concentration, and C546L and C547L mutants showed lower rates of the hemeprotein reduction. These conserved Cys residues in the DH domain respond to the cytosolic H₂O₂ concentration to regulate Nox4 activity.