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

The NADPH-dependent oxidase of human neutrophils is a multicomponent s ystem including cytosolic and membrane proteins. Activation requires t ranslocation of cytosolic proteins p47(phox), p67(phox) ,and Rac2 to t he 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. Fr om a random peptide phage display Library, we used biopanning with pur ified flavocytochrome b to select phage peptides that mimicked potenti al p47(phox) binding residues. Using this approach, we identified a re gion of p47(phox) from residue 323 to 342 as a site of interaction wit h flavocytochrome b. Synthetic peptides (315)SRKRLSQD-AYRRNS(328), (32 3)AYRRNSVRFL(332) and (334)QRRRQARPG-PQSPG(347) inhibited superoxide ( O-2(radical anion)) production in the broken cell system with IC, of 1 8, 57, and 15 mu M, respectively. (323)AYRRNSVRFL(332) and its derivat ive peptides inhibited phosphorylation of p47(phox). However, the func tional importance of this peptide was independent of its effects on ph osphorylation, since (323)AYRRNA-VRFL(332) inhibited O-2(radical anion ) production, but had no effect on phosphorylation. None of the peptid es blocked O-2(radical anion) production when added after enzyme activ ation, suggesting that they inhibited the assembly, rather than the ac tivity, 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.