Nox4 is an oddity among members of the Nox family of NADPH oxidases [seven isoenzymes that generate reactive oxygen species (ROS) from molecular oxygen] in that it is constitutively active. All other Nox enzymes except for Nox4 require upstream activators, either calcium or organizer/activator subunits (p47phox, NOXO1/p67phox, and NOXA1). Nox4 may also be unusual as it reportedly releases hydrogen peroxide (H2O2) in contrast to Nox1–Nox3 and Nox5, which release superoxide, although this result is controversial in part because of possible membrane compartmentalization of superoxide, which may prevent detection. Our studies were undertaken (1) to identify the Nox4 ROS product using a membrane-free, partially purified preparation of Nox4 and (2) to test the hypothesis that Nox4 activity is acutely regulated not by activator proteins or calcium, but by cellular pO2, allowing it to function as an O2 sensor, the output of which is signaling H2O2. We find that approximately 90% of the electron flux through isolated Nox4 produces H2O2 and 10% forms superoxide. The kinetic mechanism of H2O2 formation is consistent with a mechanism involving binding of one oxygen molecule, which is then sequentially reduced by the heme in two one-electron reduction steps first to form a bound superoxide intermediate and then H2O2; kinetics are not consistent with a previously proposed internal superoxide dismutation mechanism involving two oxygen binding/reduction steps for each H2O2 formed. Critically, Nox4 has an unusually high Km for oxygen (∼18%), similar to the values of known oxygen-sensing enzymes, compared with a Km of 2–3% for Nox2, the phagocyte NADPH oxidase. This allows Nox4 to generate H2O2 as a function of oxygen concentration throughout a physiological range of pO2 values and to respond rapidly to changes in pO2.

An activation domain in p67phox (

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.

Activation of the respiratory burst oxidase involves the assembly of the membrane-associated flavocytochrome b 558 with the cytosolic components p47 phox , p67 phox , and the small GTPase Rac. Herein, the interaction between Rac and p67 phox is explored using functional and physical methods.