NADPH dehydrogenase activity of p67PHOX, a cytosolic subunit of the leukocyte NADPH oxidase

The leukocyte NADPH oxidase catalyzes the one-electron reduction of oxygen to O-2(-) at the expense of NADPH. It is a multicomponent enzyme comprising a membrane-bound flavocytochrome (cytochrome b(558)) and at least four cytosolic components: p47(PHOX), p67(PHOX), p40(PHOX), and Rac, a small GTPase. All the oxidase components except p40(PHOX) are required for enzyme activity. Many aspects of their function, however, are unclear. Using the electron acceptor ferricyanide, we found that recombinant p67(PHOX) 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 p67(PHOX), not by a trace contaminant in the p67(PHOX) preparation. The NADPH dehydrogenase activity of p67(PHOX) was proportional to enzyme concentration, and showed saturation kinetics with NADPH (K-m 92 +/- 5 mu M), but was inhibited at high concentrations of ferricyanide. NADH was also used as a substrate by p67(PHOX) (K-m 123 +/- 38 mu M). Taken together, these results show that p67(PHOX) is able to mediate pyridine nucleotide dehydrogenation. These findings raise the possibility that p67(PHOX) might participate directly in electron transfer between NADPH and the oxidase flavin.