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 p47phox, p67phox, Rac, and flavocytochrome b558, 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 b558 became spontaneously active. Cross-linking of this mixture produced an oxidase that was extremely stable to dilution (t1/2 = 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.