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 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.
Dang
P M
PM
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Babior
B M
BM
Smith
R M
RM
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation; D005292:Ferricyanides / Q000737:chemistry / Q000378:metabolism; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D010084:Oxidation-Reduction; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics
D005292:Ferricyanides; D010750:Phosphoproteins; D011994:Recombinant Proteins; C079789:neutrophil cytosol factor 67K; C007931:hexacyanoferrate III; D009249:NADP; D009252:NADPH Dehydrogenase