CNRS, Laboratoire Stress Oxydant et Détoxication, Gif-sur-Yvette, France.
Biophys J. 2012 Jul 3;103(1):109-17. doi: 10.1016/j.bpj.2012.05.032.
H(4)B is an essential catalytic cofactor of the mNOSs. It acts as an electron donor and activates the ferrous heme-oxygen complex intermediate during Arg oxidation (first step) and NOHA oxidation (second step) leading to nitric oxide and citrulline as final products. However, its role as a proton donor is still debated. Furthermore, its exact involvement has never been explored for other NOSs such as NOS-like proteins from bacteria. This article proposes a comparative study of the role of H(4)B between iNOS and bsNOS. In this work, we have used freeze-quench to stop the arginine and NOHA oxidation reactions and trap reaction intermediates. We have characterized these intermediates using multifrequency electron paramagnetic resonance. For the first time, to our knowledge, we report a radical formation for a nonmammalian NOS. The results indicate that bsNOS, like iNOS, has the capacity to generate a pterin radical during Arg oxidation. Our current electron paramagnetic resonance data suggest that this radical is protonated indicating that H(4)B may not transfer any proton. In the 2nd step, the radical trapped for iNOS is also suggested to be protonated as in the 1st step, whereas it was not possible to trap a radical for the bsNOS 2nd step. Our data highlight potential differences for the catalytic mechanism of NOHA oxidation between mammalian and bacterial NOSs.
H（4）B 是 mNOSs 的必需催化辅助因子。它作为电子供体，在 Arg 氧化（第一步）和 NOHA 氧化（第二步）过程中激活亚铁血红素-氧配合物中间物，从而生成一氧化氮和瓜氨酸作为最终产物。然而，其作为质子供体的作用仍存在争议。此外，其确切作用在其他 NOS 中，例如细菌中的 NOS 样蛋白，从未被探索过。本文提出了 iNOS 和 bsNOS 之间 H（4）B 作用的比较研究。在这项工作中，我们使用冷冻猝灭来停止精氨酸和 NOHA 氧化反应并捕获反应中间物。我们使用多频电子顺磁共振对这些中间物进行了表征。据我们所知，这是首次报道非哺乳动物 NOS 中自由基的形成。结果表明，bsNOS 像 iNOS 一样，在 Arg 氧化过程中具有生成蝶呤自由基的能力。我们当前的电子顺磁共振数据表明，该自由基被质子化，表明 H（4）B 可能不传递任何质子。在第二步中，也表明 iNOS 中捕获的自由基被质子化，就像在第一步中一样，而对于 bsNOS 的第二步，则无法捕获自由基。我们的数据突出了哺乳动物和细菌 NOSs 之间 NOHA 氧化催化机制的潜在差异。
