Adak S, Wang Q, Stuehr D J
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
J Biol Chem. 2000 Oct 27;275(43):33554-61. doi: 10.1074/jbc.M004337200.
We studied catalysis by tetrahydrobiopterin (H4B)-free neuronal nitric-oxide synthase (nNOS) to understand how heme and H4B participate in nitric oxide (NO) synthesis. H4B-free nNOS catalyzed Arg oxidation to N(omega)-hydroxy-l-Arg (NOHA) and citrulline in both NADPH- and H(2)O(2)-driven reactions. Citrulline formation was time- and enzyme concentration-dependent but was uncoupled relative to NADPH oxidation, and generated nitrite and nitrate without forming NO. Similar results were observed when NOHA served as substrate. Steady-state and stopped-flow spectroscopy with the H4B-free enzyme revealed that a ferrous heme-NO complex built up after initiating catalysis in both NADPH- and H(2)O(2)-driven reactions, consistent with formation of nitroxyl as an immediate product. This differed from the H4B-replete enzyme, which formed a ferric heme-NO complex as an immediate product that could then release NO. We make the following conclusions. 1) H4B is not essential for Arg oxidation by nNOS, although it helps couple NADPH oxidation to product formation in both steps of NO synthesis. Thus, the NADPH- or H(2)O(2)-driven reactions form common heme-oxy species that can react with substrate in the presence or absence of H4B. 2) The sole essential role of H4B is to enable nNOS to generate NO instead of nitroxyl. On this basis we propose a new unified model for heme-dependent oxygen activation and H4B function in both steps of NO synthesis.
我们研究了无四氢生物蝶呤（H4B）的神经元型一氧化氮合酶（nNOS）的催化作用，以了解血红素和H4B如何参与一氧化氮（NO）的合成。在由NADPH和H₂O₂驱动的反应中，无H4B的nNOS均催化精氨酸氧化生成N（ω）-羟基-L-精氨酸（NOHA）和瓜氨酸。瓜氨酸的形成具有时间和酶浓度依赖性，但相对于NADPH氧化是解偶联的，并且生成亚硝酸盐和硝酸盐而不形成NO。当NOHA作为底物时也观察到类似结果。对无H4B的酶进行稳态和停流光谱分析表明，在NADPH和H₂O₂驱动的反应中启动催化后，均会形成亚铁血红素-NO复合物，这与作为直接产物的硝酰基的形成一致。这与富含H4B的酶不同，后者形成的三价铁血红素-NO复合物作为直接产物，然后可以释放NO。我们得出以下结论。1）H4B对于nNOS氧化精氨酸不是必需的，尽管它有助于在NO合成的两个步骤中将NADPH氧化与产物形成偶联。因此，由NADPH或H₂O₂驱动的反应形成常见的血红素-氧物种，该物种在有或没有H4B的情况下都可以与底物反应。2）H4B的唯一重要作用是使nNOS能够生成NO而不是硝酰基。在此基础上，我们提出了一个新的统一模型，用于解释NO合成两个步骤中血红素依赖性氧激活和H4B的功能。
