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 b(558) (cyt b(558)) with different amounts of FAD. A remarkably high activity, over 100 micromol/s/micromol heme, was obtained in the oxidase with purified cyt b(558), ternary complex (p47-p67-p40(phox)), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b, the dissociation constant K(d) of FAD in cyt b(558) 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 b(558) recovered electron flow from NADPH to heme. From both K(d) values of FAD and the amount of incorporated FAD in cyt b(558) of reconstituted oxidase, in combination with spectra, we propose the model in which the K(d) values of FAD in cyt b(558) is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase.
Hashida
Shukichi
S
Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812.
Yuzawa
Satoru
S
Suzuki
Nobuo N
NN
Fujioka
Yuko
Y
Takikawa
Takayuki
T
Sumimoto
Hideki
H
Inagaki
Fuyuhiko
F
Fujii
Hirotada
H
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D002850:Chromatography, Gel; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004579:Electron Transport; D004583:Electrons; D004789:Enzyme Activation; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D019255:NADPH Oxidases / Q000737:chemistry* / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010587:Phagocytosis; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D013481:Superoxides / Q000378:metabolism; D013552:Swine; D020744:rac GTP-Binding Proteins / Q000378:metabolism
D003573:Cytochrome b Group; D010750:Phosphoproteins; D011994:Recombinant Proteins; C085072:neutrophil cytosol factor 40K; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D005182:Flavin-Adenine Dinucleotide; C036888:cytochrome b558; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D020744:rac GTP-Binding Proteins; D010100:Oxygen