NADPH Dehydrogenase Activity of p67^PHOX, a Cytosolic Subunit of the Leukocyte NADPH Oxidase

Abstract

The leukocyte NADPH oxidase catalyzes the one-electron reduction of oxygen to O2- at the expense of NADPH. It is a multicomponent enzyme comprising a membrane-bound flavocytochrome (cytochrome b558) and at least four cytosolic components: p47PHOX, p67PHOX, p40PHOX, and Rac, a small GTPase. All the oxidase components except p40PHOX are required for enzyme activity. Many aspects of their function, however, are unclear. Using the electron acceptor ferricyanide, we found that recombinant p67PHOX from baculovirus-infected Sf9 cells could mediate the dehydrogenation of NADPH. NADPH dehydrogenation was not dependent on FAD and was insensitive to superoxide dismutase. Several control experiments showed that NADPH dehydrogenation was accomplished by p67PHOX, not by a trace contaminant in the p67PHOX preparation. The NADPH dehydrogenase activity of p67PHOX was proportional to enzyme concentration, and showed saturation kinetics with NADPH (Km 92 +/- 5 microM), but was inhibited at high concentrations of ferricyanide. NADH was also used as a substrate by p67PHOX (Km 123 +/- 38 microM). Taken together, these results show that p67PHOX is able to mediate pyridine nucleotide dehydrogenation. These findings raise the possibility that p67PHOX might participate directly in electron transfer between NADPH and the oxidase flavin.

Functional Epitope on Human Neutrophil Flavocytochrome b558

“…6), as previously suggested (35,39). According to this view, the kinetic parameters influenced by NL7 correspond to the low affinity (ϳ26 M) NADPH binding site, presumably on gp91 phox (Fig.…”

Phosphatidylinositol 3-Phosphate-dependent and -independent Functions of p40phox in Activation of the Neutrophil NADPH Oxidase

“…Immunodepletion of p40 phox from this system resulted in a profound decrease in PMA-stimulated intracellular ROS production that could be rescued by complementation with recombinant wild-type p40 phox but not by p40 phox containing mutations in either the PX, PB1, or SH3 domains, illustrating the p40 phox dependence of this system and indicating that all three domains are required for its biochemical activity. p40 phox is constitutively bound to p67 phox through PB1-PB1 domain-mediated interactions, and p67 phox has been shown to both bind to NADPH (40) and to facilitate electron flow from NADPH to FAD in the cytochrome (6,10). As such, we investigated whether p40 phox might function as a molecular "chaperone" for p67 phox to facilitate interactions of NADPH with the oxidase.…”

“…Upon stimulation, the cytosolic components translocate to and activate the cytochrome (7). p47 phox and p67 phox have relatively well established roles in activation as follows: p47 phox is essential for localization of the cytosolic phox components to the cytochrome (8); p67 phox has been implicated as the cytosolic NADPH-binding protein (9,10) and facilitates electron transport by the cytochrome (11). Mutations in either of these proteins results in CGD.…”

Role of Putative Second Transmembrane Region of Nox2 Protein in the Structural Stability and Electron Transfer of the Phagocytic NADPH Oxidase

“…In Taylor's model of the "dehydrogenase" domain of Nox2 (11), there is an open space above the NADPH-and FAD-binding sites. This void could provide a region for other cytoplasmic residues to be involved in the organization of the NADPH/FAD-binding site so as to control electron transfer (11,14,28,37). Candidates include the C-terminal aromatic residue Phe-570, a conserved and essential residue in the whole ferredoxin-NADP ϩ reductase family, and/or the region centered on Cys-369, which is a docking site for p67 phox (28).…”