Department of Pathology and Laboratory Medicine, Emory University Medical School, Atlanta, Georgia 30322, USA.
Biochemistry. 2010 Mar 23;49(11):2433-42. doi: 10.1021/bi9022285.
NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47(phox) and p67(phox) and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K(m) for NADPH of 55 +/- 10 microM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of approximately 200 mol of H(2)O(2) min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H(2)O(2) production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide (470 min(-1)), and other electron acceptors (artificial dyes and cytochrome b(5)). Rates were similar to those observed for H(2)O(2) production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.
NADPH 氧化酶 4（Nox4）是组成性激活的，而 Nox2 需要细胞质调节亚基 p47（phox）和 p67（phox）以及激活的 Rac，并通过佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）激活。本研究旨在确定赋予组成性活性的 Nox4 结构域。来自 Nox4 表达细胞的裂解物表现出组成型 NADPH-但不是 NADH 依赖性过氧化氢产生，NADPH 的 K(m)为 55 +/- 10 microM。使用 Western blot 估计细胞裂解物中的 Nox4 浓度，并允许计算大约 200 mol 的 H(2)O(2) min(-1)（mol 的 Nox4)(-1)的周转率。由 Nox2 跨膜（TM）结构域和 Nox4 脱氢酶（DH）结构域组成的嵌合蛋白（Nox2/4）在没有细胞质调节亚基的情况下表现出 H(2)O(2)产生。相比之下，由 Nox4 TM 和 Nox2 DH 结构域组成的嵌合体 Nox4/2 表现出依赖 PMA 的激活，需要共表达调节亚基。几种 Nox 同工型的 Nox DH 结构域被纯化并评估其电子转移酶活性。Nox1 DH、Nox2 DH 和 Nox5 DH 结构域对人工电子受体几乎没有检测到活性，而 Nox4 DH 结构域表现出细胞色素 c（160 min(-1)，主要与超氧化物歧化酶无关）、铁氰化物（470 min(-1)）和其他电子受体（人工染料和细胞色素 b(5)）的显著还原速率。速率与细胞裂解物中 Nox4 全酶产生 H(2)O(2)的速率相似。该活性需要添加 FAD，并且可以与 NADPH 但不是 NADH 一起观察到。这些结果表明，Nox4 DH 结构域处于固有激活状态，并且从 NADPH 到 FAD 的电子转移可能是全酶 Nox4 依赖 NADPH 的氧还原的限速步骤。
