Nox4: a hydrogen peroxide-generating oxygen sensor.
Nox4: a hydrogen peroxide-generating oxygen sensor.
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DOI：
10.1021/bi500331y
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发表时间：
2014-08-12
期刊：
影响因子：
2.9
通讯作者：
Lambeth JD
中科院分区：
文献类型：
--
作者：
Nisimoto Y;Diebold BA;Cosentino-Gomes D;Lambeth JD
Nox4是NADPH氧化酶（七种从分子氧产生活性氧（ROS）的同工酶）Nox家族成员中的一个奇怪的成员，因为它具有组成性活性。除Nox4外，所有其他Nox酶都需要上游活化剂，要么是钙，要么是组织者/活化剂亚基（p47phox、NOXO1/p67phox和NOXA1）。Nox4也可能是不寻常的，因为据报道它释放过氧化氢（H2O2），而Nox1-Nox3和Nox5释放超氧化物，尽管这一结果存在争议，部分原因是超氧化物可能被膜区隔化，这可能会阻止检测。我们的研究进行了：(1)使用无膜、部分纯化的Nox4制剂来鉴定Nox4 ROS产物；(2)验证Nox4活性不受激活蛋白或钙的强烈调节，而是由细胞pO2调节的假设，使其能够作为O2传感器，其输出信号是H2O2。我们发现大约90%的电子通量通过分离的Nox4产生H2O2, 10%形成超氧化物。H2O2形成的动力学机制符合一个氧分子结合，然后被血红素通过两个单电子还原步骤依次还原，形成一个结合的超氧化物中间体，然后形成H2O2；动力学与先前提出的内部超氧化物失配机制不一致，该机制涉及每个H2O2形成的两个氧结合/还原步骤。关键的是，Nox4具有异常高的氧Km(~ 18%)，与已知的氧感应酶的值相似，而吞噬细胞NADPH氧化酶Nox2的Km为2-3%。这使得Nox4能够产生H2O2作为氧浓度在pO2值的生理范围内的函数，并迅速响应pO2的变化。
Nox4 is an oddity among members of the Nox family of NADPH oxidases [seven isoenzymes that generate reactive oxygen species (ROS) from molecular oxygen] in that it is constitutively active. All other Nox enzymes except for Nox4 require upstream activators, either calcium or organizer/activator subunits (p47phox, NOXO1/p67phox, and NOXA1). Nox4 may also be unusual as it reportedly releases hydrogen peroxide (H2O2) in contrast to Nox1–Nox3 and Nox5, which release superoxide, although this result is controversial in part because of possible membrane compartmentalization of superoxide, which may prevent detection. Our studies were undertaken (1) to identify the Nox4 ROS product using a membrane-free, partially purified preparation of Nox4 and (2) to test the hypothesis that Nox4 activity is acutely regulated not by activator proteins or calcium, but by cellular pO2, allowing it to function as an O2 sensor, the output of which is signaling H2O2. We find that approximately 90% of the electron flux through isolated Nox4 produces H2O2 and 10% forms superoxide. The kinetic mechanism of H2O2 formation is consistent with a mechanism involving binding of one oxygen molecule, which is then sequentially reduced by the heme in two one-electron reduction steps first to form a bound superoxide intermediate and then H2O2; kinetics are not consistent with a previously proposed internal superoxide dismutation mechanism involving two oxygen binding/reduction steps for each H2O2 formed. Critically, Nox4 has an unusually high Km for oxygen (∼18%), similar to the values of known oxygen-sensing enzymes, compared with a Km of 2–3% for Nox2, the phagocyte NADPH oxidase. This allows Nox4 to generate H2O2 as a function of oxygen concentration throughout a physiological range of pO2 values and to respond rapidly to changes in pO2.
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影响因子：
3.3
作者：
Diebold I;Petry A;Hess J;Görlach A
通讯作者：
Görlach A
影响因子：
4.8
作者：
Cheng, Guangjie;Diebold, Becky A.;Lambeth, J. David
通讯作者：
Lambeth, J. David
影响因子：
4.8
作者：
Ambasta, RK;Kumar, P;Brandes, RP
通讯作者：
Brandes, RP
影响因子：
4.8
作者：
Geiszt, M;Witta, J;Leto, TL
通讯作者：
Leto, TL
影响因子：
56.9
作者：
DIEKMANN, D;ABO, A;HALL, A
通讯作者：
HALL, A