Binding of p67 ^phox to Nox2 is stabilized by disulfide bonds between cysteines in the ³⁶⁹ Cys-Gly-Cys ³⁷¹ triad in Nox2 and in p67 ^phox

A central event in the activation of the phagocyte NADPH oxidase involves binding of p67 ^phox to the dehydrogenase region of Nox2. The identity of the binding site in Nox2 is unknown. By measuring binding of p67 ^phox to synthetic Nox2 peptides, we previously identified a sequence corresponding to Nox2 residues 357–383, as a potential binding site. A key role was attributed to a ³⁶⁹ Cys-Gly-Cys ³⁷¹ triad, shared by peptides 357–371 (peptide 24) and 369–383 (peptide 28). In this study, we show that (1) oxidation of cysteines in peptides 24 and 28 by a variety of oxidants markedly enhances the binding of p67 ^phox ; (2) replacing cysteines by arginine abolishes the response to oxidants and the enhanced binding of p67 ^phox ; (3) oxidants act by generating an intramolecular disulfide bond linking cysteines 369 and 371, generating such bond during peptide synthesis reproduces the effect of oxidants; (4) for the disulfide bond to lead to enhanced binding, cysteines must be separated by an intervening residue; bonds joining adjacent cysteines, or cysteines located on two peptides, do not enhance binding; (5) dissociating disulfide bonds by reducing agents abolishes enhanced binding; (6) treating p67 ^phox with the alkylating agent N-ethylmaleimide suppresses binding; and (7) mutating all nine cysteines in p67 ^phox to serines abolishes binding and diminishes the ability of p67 ^phox to support NADPH oxidase activation in vitro. Results show that the primary interaction of p67 ^phox with Nox2 is followed by a stabilizing step, based on the establishment of disulfide bonds between cysteine(s) in the ³⁶⁹ Cys-Gly-Cys ³⁷¹ triad and cysteine(s) in p67 ^phox .