Inactivation of neutrophil NADPH oxidase upon dilution and its prevention by cross-link and fusion of phox proteins

Activation of the phagocyte NADPH oxidase involves assembly of p47 ^phox, p67 ^phox, Rac, and flavocytochrome b ₅₅₈, and the activation can be triggered in a cell-free system with an anionic amphiphile. We find that the activated oxidase in a pure cell-free system was rapidly inactivated upon dilution. When the activated oxidase was treated with a chemical cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the half-life of the oxidase in dilution was extended from 1 min to 4 h at 25°C. The cross-linked oxidase was resistant to inhibition by inactive flavin analogs, indicating that cross-linking prevents flavin exchange. When a fusion protein p67N-p47N plus RacQ61L was added, flavocytochrome b ₅₅₈ became spontaneously active. Cross-linking of this mixture produced an oxidase that was extremely stable to dilution (t _1/2 = 6.6 h). Western blotting analysis showed the presence of a cross-link between p67N-p47N and RacQ61L. These results suggest that covalently linked phox components prevents FAD loss and stabilizes the longevity of the stoichiometric complex, extending the lifespan of the active oxidase.