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

Shukichi Hashida, Satoru Yuzawa, Nobuo N. Suzuki, Yuko Fujioka, Takayuki Takikawa, Hideki Sumimoto, Fuyuhiko Inagaki, Hirotada Fujii

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32 Citations (Scopus)


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, p67phox, p40phox, 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 Kd 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 Kd 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 Kd values of FAD in cyt b558 is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase.

Original languageEnglish
Pages (from-to)26378-26386
Number of pages9
JournalJournal of Biological Chemistry
Issue number25
Publication statusPublished - Jun 18 2004

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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