The molecular basis of Rac‐GTP action—promoting binding of p67phox to Nox2 by disengaging the β hairpin from downstream residues

p67phox fulfils a key role in the assembly/activation of the NADPH oxidase by direct interaction with Nox2. We proposed that Rac‐GTP serves both as a carrier of p67phox to the membrane and an inducer of a conformational change enhancing its affinity for Nox2. This study provides evidence for the latter function: (i) oxidase activation was inhibited by p67phox peptides (106–120) and (181–195), corresponding to the β hairpin and to a downstream region engaged in intramolecular bonds with the β hairpin, respectively; (ii) deletion of residues 181–193 and point mutations Q115R or K181E resulted in selective binding of p67phox to Nox2 peptide (369–383); (iii) both deletion and point mutations led to a change in p67phox, expressed in increased apparent molecular weights; (iv) p67phox was bound to p67phox peptide (181–195) and to a cluster of peptides (residues 97–117), supporting the participation of selected residues within these sequences in intramolecular bonds; (v) p67phox failed to bind to Nox2 peptide (369–383), following interaction with Rac1‐GTP, but a (p67phox‐Rac1‐GTP) chimera exhibited marked binding to the peptide, similar to that of p67phox deletion and point mutants; and (vi) size exclusion chromatography of the chimera revealed its partition in monomeric and polymeric forms, with binding to Nox2 peptide (369–383) restricted to polymers. The molecular basis of Rac‐GTP action entails unmasking of a previously hidden Nox2‐binding site in p67phox, following disengagement of the β hairpin from more C‐terminal residues. The domain in Nox2 binding the “modified” p67phox comprises residues within the 369–383 sequence in the cytosolic dehydrogenase region. Graphical Deletion of residues 181‐193 or severing the bond between Q115 and K181 enhances binding of p67phox to Nox2 residues 369‐383, mimicking the effect of Rac‐GTP