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

Abstract

NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47phox and p67phox and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a Km for NADPH of 55 ± 10 μM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of ∼200 mol of H2O2 min−1 (mol of Nox4)−1. A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H2O2 production in the absence of cytosolic regulatory subunits. In 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 electron transferase activities. Nox1 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 (470 min−1), and other electron acceptors (artificial dyes and cytochrome b5). 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 an 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.

Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

“…Using two different methods of protein synthesis, we demonstrate that the cytosolic domain of Nox4 is responsible for Nox4 constitutive activity. Other authors using a similar portion of the C-terminal part of Nox4 have made the same observations [26,27].…”

NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis

“…Second, several recent independent reports indicate that Nox4 generates predominantly H 2 O 2 (as further confirmed in the current study) whereas Nox2 primarily generates O 2 − (refs. [17][18][19][20]. These differences in regulation, activation mechanism, subcellular location, and ROS generation translate into isoform-specific actions in isolated cellular models (9,19,22,33).…”

“…Nox4 differs from Nox2 and other Nox enzymes in that it is regulated mainly by its expression level and does not require agonist stimulation or association with regulatory subunits for activation (16)(17)(18). Recent studies also suggest that it generates predominantly H 2 O 2 rather than O 2 − (17)(18)(19)(20). Previous work showed that Nox4 is expressed at a low level in the adult mammalian heart and that its abundance increases during pressure overload (11), but its pathophysiological functions in vivo are unknown.…”

Quinone compounds regulate the level of ROS production by the NADPH oxidase Nox4

“…However, two partners of Nox4 have been described recently: the protein disulfide isomerase (PDI) and the polymerase DNA directed-delta-interacting protein (Poldip2). Nox4 is unique among other Nox isoenzymes in that its activity is constitutive and may depend on a specific conformation of the dehydrogenase DH domain that should allow a spontaneous transfer of electrons from NADPH to FAD and to the hemes [24,25]. Data from various studies indicated clearly that Nox4 activity is regulated at the mRNA level, implying that an increase or decrease of ROS production by Nox4 is correlated to an up regulation [9,16,[26][27][28][29][30][31][32][33] or to a decline [34] of Nox4 transcripts.…”