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Query: KEGG:D02011 (
FAD
)
5,530
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
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.
...
PMID:Binding of FAD to cytochrome b558 is facilitated during activation of the phagocyte NADPH oxidase, leading to superoxide production. 1510 59
NADPH oxidase is a crucial element of phagocytes involved in microbicidal mechanisms. It becomes active when membrane-bound cytochrome b(558), the redox core, is assembled with cytosolic p47(phox), p67(phox),
p40
(phox), and rac proteins to produce superoxide, the precursor for generation of toxic reactive oxygen species. In a previous study, we demonstrated that the potential second intracellular loop of Nox2 was essential to maintaining NADPH oxidase activity by controlling electron transfer from
FAD
to O(2). Moreover, replacement of this loop by the Nox4-D-loop (D-loop(Nox4)-Nox2) in PLB-985 cells induced superoxide overproduction. In the present investigation, we demonstrated that both soluble and particulate stimuli were able to induce this superoxide overproduction. Superoxide overproduction was also observed after phosphatidic acid activation in a purified cell-free-system assay. The highest oxidase activity was obtained after ionomycin and fMLF stimulation. In addition, enhanced sensitivity to Ca(2+) influx was shown by thapsigargin, EDTA, or BTP2 treatment before fMLF activation. Mutated cytochrome b(558) was less dependent on phosphorylation triggered by ERK1/2 during fMLF or PMA stimulation and by PI3K during OpZ stimulation. The superoxide overproduction of the D-loop(Nox4)-Nox2 mutant may come from a change of responsiveness to intracellular Ca(2+) level and to phosphorylation events during oxidase activation. Finally the D-loop(Nox4)-Nox2-PLB-985 cells were more effective against an attenuated strain of Pseudomonas aeruginosa compared to WT-Nox2 cells. The killing mechanism was biphasic, an early step of ROS production that was directly bactericidal, and a second oxidase-independent step related to the amount of ROS produced in the first step.
...
PMID:Characterization of superoxide overproduction by the D-Loop(Nox4)-Nox2 cytochrome b(558) in phagocytes-Differential sensitivity to calcium and phosphorylation events. 2070 98
