A DOMAIN OF P47(PHOX) THAT INTERACTS WITH HUMAN NEUTROPHIL FLAVOCYTOCHROME B(558)

The NADPH-dependent oxidase of human neutrophils is a multicomponent system including cytosolic and membrane proteins. Activation requires translocation of cytosolic proteins p47(phox), p67(phox) ,and Rac2 to the plasma membrane and association with the membrane flavocytochrome b to assemble a functioning oxidase. We report the location of a region in p47(phox) that mediates its interaction with flavocytochrome b. From a random peptide phage display Library, we used biopanning with purified flavocytochrome b to select phage peptides that mimicked potential p47(phox) binding residues. Using this approach, we identified a region of p47(phox) from residue 323 to 342 as a site of interaction with flavocytochrome b. Synthetic peptides (315)SRKRLSQD-AYRRNS(328), (323)AYRRNSVRFL(332) and (334)QRRRQARPG-PQSPG(347) inhibited superoxide (O-2(radical anion)) production in the broken cell system with IC, of 18, 57, and 15 mu M, respectively. (323)AYRRNSVRFL(332) and its derivative peptides inhibited phosphorylation of p47(phox). However, the functional importance of this peptide was independent of its effects on phosphorylation, since (323)AYRRNA-VRFL(332) inhibited O-2(radical anion) production, but had no effect on phosphorylation. None of the peptides blocked O-2(radical anion) production when added after enzyme activation, suggesting that they inhibited the assembly, rather than the activity, of the oxidase. Furthermore these peptides inhibited membrane association of p47(phox), the broken cell translocation assay and O-2(radical anion) production by electropermeabilized neutrophils, thereby supporting the interpretation that this region of p47(phox) interacts with flavocytochrome b.