Gorren A C, Bec N, Schrammel A, Werner E R, Lange R, Mayer B
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, A-8010 Graz, Austria.
Biochemistry. 2000 Sep 26;39(38):11763-70. doi: 10.1021/bi0007775.
To investigate the role of tetrahydrobiopterin (BH4) in the catalytic mechanism of nitric oxide synthase (NOS), we analyzed the spectral changes following addition of oxygen to the reduced oxygenase domain of endothelial nitric oxide synthase (NOS) in the presence of different pteridines at -30 degrees C. In the presence of N(G)-hydroxy-L-arginine (NOHLA) and BH4 or 5-methyl-BH4, both of which support NO synthesis, the first observable species were mixtures of high-spin ferric NOS (395 nm), ferric NO-heme (439 nm), and the oxyferrous complex (417 nm). With Arg, no clear intermediates could be observed under the same conditions. In the presence of the BH4-competitive inhibitor 7,8-dihydrobiopterin (BH2), intermediates with maxima at 417 and 425 nm were formed in the presence of Arg and NOHLA, respectively. In the presence of 4-amino-BH4, the maxima of the intermediates with Arg and NOHLA were at 431 and 423 nm, respectively. We ascribe all four spectra to oxyferrous heme complexes. The intermediates observed in this study slowly decayed to the high-spin ferric state at -30 degrees C, except for those formed in the presence of 4-amino-BH4, which required warming to room temperature for regeneration of high-spin ferric NOS; with Arg, regeneration remained incomplete. From these observations, we draw several conclusions. (1) BH4 is required for reductive oxygen activation, probably as a transient one-electron donor, not only in the reaction with Arg but also with NOHLA; (2) in the absence of redox-active pterins, reductive oxygen activation does not occur, which results in accumulation of the oxyferrous complex; (3) the spectral properties of the oxyferrous complex are affected by the presence and identity of the substrate; (4) the slow and incomplete formation of high-spin ferric heme with 4-amino-BH4 suggests a structural cause for inhibition of NOS activity by this pteridine.
为了研究四氢生物蝶呤（BH4）在一氧化氮合酶（NOS）催化机制中的作用，我们在-30℃下，分析了在不同蝶啶存在的情况下，向内皮型一氧化氮合酶（NOS）的还原加氧酶结构域中加入氧气后的光谱变化。在存在N(G)-羟基-L-精氨酸（NOHLA）和支持NO合成的BH4或5-甲基-BH4的情况下，首先观察到的物种是高自旋铁型NOS（395nm）、铁-NO-血红素（439nm）和氧合亚铁复合物（417nm）的混合物。使用精氨酸时，在相同条件下未观察到明显的中间体。在BH4竞争性抑制剂7,8-二氢生物蝶呤（BH2）存在的情况下，分别在精氨酸和NOHLA存在时形成了最大吸收峰位于417和425nm的中间体。在4-氨基-BH4存在的情况下，与精氨酸和NOHLA反应的中间体的最大吸收峰分别位于431和423nm。我们将这四种光谱都归因于氧合亚铁血红素复合物。在本研究中观察到的中间体在-30℃下缓慢衰减至高自旋铁状态，但在4-氨基-BH4存在时形成的中间体除外，后者需要升温至室温才能使高自旋铁型NOS再生；使用精氨酸时，再生仍不完全。基于这些观察结果，我们得出了几个结论。（1）BH4是还原氧激活所必需的，可能作为瞬态单电子供体，不仅在与精氨酸的反应中，而且在与NOHLA的反应中也是如此；（2）在没有氧化还原活性蝶呤的情况下，不会发生还原氧激活，这会导致氧合亚铁复合物的积累；（3）氧合亚铁复合物的光谱特性受底物的存在和特性影响；（4）4-氨基-BH4导致高自旋铁血红素形成缓慢且不完全，这表明该蝶啶抑制NOS活性存在结构上的原因。
