Binding of FAD to cytochrome b558 is facilitated during activation of the phagocyte NADPH oxidase, leading to superoxide production
Binding of FAD to cytochrome b558 is facilitated during activation of the phagocyte NADPH oxidase, leading to superoxide production
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DOI：
10.1074/jbc.m309724200
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发表时间：
2004-06-18
影响因子：
4.8
通讯作者：
Fujii, H
中科院分区：
文献类型：
--
作者：
Hashida, S;Yuzawa, S;Fujii, H
产生超氧化物的吞噬细胞NADPH氧化酶可以在无细胞系统中重组。NADPH氧化酶的活性依赖于FAD，但FAD在该酶中的生理状态尚未完全阐明。为了阐明FAD在NADPH氧化酶中的作用，制备了不含FAD的全长重组蛋白p47(Phox)、p67(Phox)、p40(Phox)和Rac，并将这些蛋白与含有不同量FAD的纯化的细胞色素b(558)(Cytb(558))重组。用纯化的细胞色素b558、三元络合物(p47-p67-p40(Phox))和RAc，获得了高达100mumol/S/mumol血红素以上的酶活性。用FAD滴定纯化的不含FAD的Cytb(558)重组NADPH氧化酶的活性，测得FAD在重组酶Cytb(558)中的解离常数K-d接近1 nm。我们还考察了FAD在重组酶组装过程中的添加情况。当组装过程中存在FAD时，FAD的活性显著提高，与在组装过程中加入FAD时相比，将FAD掺入纯化的Cyt b(558)中的FAD空位的效率增加。厌氧条件下重组酶的吸收光谱表明，FAD掺入Cytb(558)后，恢复了NADPH向血红素的电子流动。根据FAD的K-d值和FAD在重组酶Cytb(558)中的掺入量，结合光谱，我们提出了激活后Cytb(558)中Fad的K-d值是变化的，Fad结合作为开关来调节NADPH氧化酶中的电子传递的模型。
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 mumol/s/mumol 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 b(558), 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.