Binding of FAD to cytochrome b₅₅₈ is facilitated during activation of the phagocyte NADPH oxidase, leading to superoxide production

The superoxide-producing phagocyte NADPH oxidase can be reconstituted in a cell-free system. The activity of NADPH oxidase is dependent on FAD, but the physiological status of FAD in the oxidase is not fully elucidated. To clarify the role of FAD in NADPH oxidase, FAD-free full-length recombinant p47 ^phox, p67^phox, p40^phox, and Rac were prepared, and the activity was reconstituted with these proteins and purified cytochrome b₅₅₈ (cyt b₅₅₈) with different amounts of FAD. A remarkably high activity, over 100 μmol/s/μmol heme, was obtained in the oxidase with purified cyt b₅₅₈, ternary complex (p47-p67-p40 ^phox), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b₅₅₈, the dissociation constant K_d of FAD in cyt b₅₅₈ of reconstituted oxidase was estimated as nearly 1 nM. We also examined addition of FAD on the assembly process in reconstituted oxidase. The activity was remarkably enhanced when FAD was present during assembly process, and the efficacy of incorporating FAD into the vacant FAD site in purified cyt b ₅₅₈ increased, compared when FAD was added after assembly processes. The absorption spectra of reconstituted oxidase under anaerobiosis showed that incorporation of FAD into cyt b₅₅₈ recovered electron flow from NADPH to heme. From both K_d values of FAD and the amount of incorporated FAD in cyt b₅₅₈ of reconstituted oxidase, in combination with spectra, we propose the model in which the K_d values of FAD in cyt b₅₅₈ is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase.