Nox4：产生过氧化氢的氧气传感器,Biochemistry

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Nox4：产生过氧化氢的氧气传感器
Biochemistry ( IF 2.9 ) Pub Date : 2014-08-01 00:00:00 , DOI: 10.1021/bi500331y
Yukio Nisimoto ₁ , Becky A Diebold , Daniela Cosentino-Gomes , Daniela Constentino-Gomes , J David Lambeth

Affiliation

Nox4 是 NADPH 氧化酶 Nox 家族成员 [从分子氧产生活性氧 (ROS) 的七种同工酶] 中的一个奇怪之处，因为它具有组成型活性。除 Nox4 外的所有其他 Nox 酶都需要上游激活剂，钙或组织者/激活剂亚基（p47 ^phox、NOXO1/p67 ^phox和 NOXA1）。Nox4 也可能不寻常，因为据报道它会释放过氧化氢 (H ₂ O ₂) 与释放超氧化物的 Nox1-Nox3 和 Nox5 形成对比，尽管这一结果存在争议，部分原因是超氧化物可能存在膜区室化，这可能会阻止检测。我们进行了研究 (1) 使用无膜、部分纯化的 Nox4 制剂来鉴定 Nox4 ROS 产物，以及 (2) 测试 Nox4 活性不是由激活蛋白或钙而是由细胞 pO ₂急剧调节的假设，使其能够用作 O ₂传感器，其输出信号为 H ₂ O ₂。我们发现大约 90% 的电子通量通过孤立的 Nox4 产生 H ₂ O ₂并且 10% 形成超氧化物。H的动力学机制₂ O _{2 的}形成与一个氧分子结合的机制一致，然后在两个单电子还原步骤中被血红素依次还原，首先形成结合的超氧化物中间体，然后形成 H ₂ O ₂；动力学与先前提出的内部超氧化物歧化机制不一致，该机制涉及每个形成的H ₂ O _{2 的}两个氧结合/还原步骤。关键的是，Nox4对氧的K _m异常高（~18%），与已知的氧感应酶的值相似，而Nox2（吞噬细胞 NADPH 氧化酶）的K _m为 2-3%。这允许 Nox4 生成 H ₂ø ₂为氧浓度在整个PO的生理范围内的功能₂值，并在PO迅速响应变化₂。

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Nox4: A Hydrogen Peroxide-Generating Oxygen Sensor

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 (p47^phox, NOXO1/p67^phox, 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 K_m for oxygen (∼18%), similar to the values of known oxygen-sensing enzymes, compared with a K_m 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.

更新日期：2014-08-01

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