A domain of p477^phox that interacts with human neutrophil flavocytochrome b₅₅₈

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 fiavocytoehrome b. Synthetic peptides ³¹⁵SRKRLSQD-AYRRNS³²⁸, ³²³AYRRNSVRFL³³², and ³³⁴QRRRQARPG-PQSPG³⁴⁷ inhibited superoxide (O^-.₂) production in the broken cell system with IC₅₀ of 18, 57, and 15 μM, respectively. ³²³AYRRNSVRFL³³² and its derivative peptides inhibited phosphorylation of p47^phox. However, the functional importance of this peptide was independent of its effects on phosphorylation, since ³²³SAYRRNA-VRFL³³² inhibited O^-.₂ production, but had no effect on phosphorylation. None of the peptides blocked O^-.₂ 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 in the broken cell translocation assay and O^-.₂ production by electropermeabilized neutrophils, thereby supporting the interpretation that this region of p47^phox interacts with fiavoeytochrome b.