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 NBT (nitroblue tetrazolium) reductase activity, whereas TRX-gp91phox (304-423) and TRX-gp91phox (424-569) were inactive. Activity saturated at about a 1:1 molar ratio of FAD to TRX-gp91phox (306-569), and showed the same K(m) for NADPH as that for superoxide generating activity by the intact enzyme. Activity was not inhibited by superoxide dismutase, indicating that it was not mediated by superoxide, but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium. In the presence of Rac1, the cytosolic regulatory protein p67phox stimulated the NBT reductase activity, but p47phox had no effect. Truncated p67phox containing the activation domain (residues 199-210) [C.-H. Han, J.R. Freeman, T. Lee, S.A. Motalebi, and J.D. Lambeth (1998) J. Biol. Chem. 273, 16663-16668] stimulated activity approximately 2-fold, whereas forms mutated or lacking this region failed to stimulate the activity. Our data indicate that: (i) TRX-gp91phox (306-569) contains binding sites for both pyridine and flavin nucleotides; (ii) this flavoprotein domain shows weak diaphorase activity; and (iii) the flavin-binding domain of gp91phox is the target of regulation by the activation domain of p67phox.
Han
C H
CH
Department of Biochemistry, Emory Medical School, Atlanta, Georgia 30322, USA.
Nisimoto
Y
Y
Lee
S H
SH
Kim
E T
ET
Lambeth
J D
JD
D001665:Binding Sites; D001685:Biological Factors / Q000494:pharmacology; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism / Q000494:pharmacology; D005420:Flavoproteins / Q000378:metabolism*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D009252:NADPH Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D009861:Onium Compounds / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013482:Superoxide Dismutase / Q000378:metabolism; D013816:Thermodynamics; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
D001685:Biological Factors; D004791:Enzyme Inhibitors; D005420:Flavoproteins; D008562:Membrane Glycoproteins; D009861:Onium Compounds; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C079789:neutrophil cytosol factor 67K; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; C007517:diphenyleneiodonium; D013482:Superoxide Dismutase; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D009252:NADPH Dehydrogenase; D020830:rac1 GTP-Binding Protein