Characterization of the flavoprotein domain of gp91phox which has NADPH diaphorase activity

A series of truncated forms of gp91phox were expressed in Escherichia coli in which the N-terminal hydrophobic transmembrane region was replaced with a portion of the highly soluble bacterial protein thioredoxin. TRX-gp91phox (306-569), which contains the putative FAD and NADPH binding sites, showed weak NADPH-dependent NET (nitroblue tetrazolium) reductase activity, where as TRX-gp91phox (304-423) and TRX-gp91phox (424-569) were inactive, Activit y saturated at about a 1:1 molar ratio of FAD to TRX-gp91phox (366-669), an d showed the same K, for NADPH as that for superoxide generating activity b y the intact enzyme, Activity was not inhibited by superoxide dismutase, in dicating that it was not mediated by superoxide, but was blocked by an inhi bitor of the respiratory burst oxidase, diphenylene iodonium, In the presen ce of Rad, the cytosolic regulatory protein p67phox stimulated the NET redu ctase activity, but p47phox had no effect. Truncated p67phox containing the activation domain (residues 199-210) [C.-H, Dan, J.R. Freeman, T. Lee, S.A . Motalebi, and J.D. Lambeth (1998) J. Biol. Chem. 273, 16663-16668] stimul ated activity approximately a-fold, whereas forms mutated or lacking this r egion failed to stimulate the activity. Our data indicate that: (i) TRX-gp9 1phox (306-569) contains binding sites for both pyridine and flavin nucleot ides; (ii) this flavoprotein domain shows weak diaphorase activity; and (ii i) the flavin-binding domain of gp91phox is the target of regulation by the activation domain of p67phox.