Three different oxygen-induced radical species in endothelial nitric-oxide synthase oxygenase domain under regulation by L-arginine and tetrahydrobiopterin
Three different oxygen-induced radical species in endothelial nitric-oxide synthase oxygenase domain under regulation by L-arginine and tetrahydrobiopterin
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DOI：
10.1074/jbc.m404044200
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发表时间：
2004-07-30
影响因子：
4.8
通讯作者：
Tsai, AL
中科院分区：
文献类型：
--
作者：
Berka, V;Wu, G;Tsai, AL
内皮型一氧化氮合酶（eNOS）在血管生理和稳态中起重要作用。eNOS是否催化一氧化氮生物合成或活性氧物质如超氧化物、过氧化氢和过氧亚硝酸盐的合成取决于eNOS催化过程中四氢生物蝶呤（BH 4）和L-精氨酸的生物利用度。利用eNOS加氧酶结构域（eNOS（ox））的单翻转快速冷冻淬灭和EPR谱研究了BH_4和L-精氨酸对氧诱导自由基中间体的影响。在亚铁eNOS（ox）和氧之间的反应期间，观察到对应于>50%的血红素的三种不同的自由基中间体。在没有L-精氨酸的情况下，无BH 4的eNOS（ox）非常有效地产生超氧自由基。L-精氨酸使超氧阴离子的形成速率降低一个数量级，但不降低其最终水平或EPR线形。对于含BH 4的eNOS（ox），在L-精氨酸存在下仅产生化学计量量的BH 4自由基，但在其不存在下获得新的自由基。这个新的自由基可能是BH_4的过氧自由基，也可能是血红素附近的氨基酸自由基。这种新自由基的形成非常迅速，>150 s（-1），随后转化为BH 4自由基。在BH 4（-）eNOS（ox）反应中，细胞色素c捕获超氧自由基的极限速率为15 s（-1），这是超氧自由基离开血红素口袋到达蛋白质表面的时间;这揭示了常规自旋捕获法在确定自由基形成动力学中的一个普遍问题。细胞色素c未能捕获新的自由基种类。与其他EPR特征一起，我们的数据强烈支持这一结论，即这种新的自由基不是超氧自由基或超氧自由基和生物蝶呤自由基的混合物。我们的研究指出了BH 4和L-精氨酸在调节eNOS自由基中间体中的不同作用。BH 4阻止超氧化物的形成的Fe（II）O-2中间体的化学转化，和L-精氨酸延迟超氧化物的形成通过电子相互作用与血红素结合的氧。
Endothelial nitric-oxide synthase ( eNOS) plays important roles in vascular physiology and homeostasis. Whether eNOS catalyzes nitric oxide biosynthesis or the synthesis of reactive oxygen species such as superoxide, hydrogen peroxide, and peroxynitrite is dictated by the bioavailability of tetrahydrobiopterin (BH4) and L-arginine during eNOS catalysis. The effect of BH4 and L-arginine on oxygen-induced radical intermediates has been investigated by single turnover rapid-freeze quench and EPR spectroscopy using the isolated eNOS oxygenase domain (eNOS(ox)). Three distinct radical intermediates corresponding to >50% of the heme were observed during the reaction between ferrous eNOS(ox) and oxygen. BH4-free eNOS(ox) produced the superoxide radical very efficiently in the absence of L-arginine. L-Arginine decreased the formation rate of superoxide by an order of magnitude but not its final level or EPR line shape. For BH4-containing eNOS(ox), only a stoichiometric amount of BH4 radical was produced in the presence of L-arginine, but in its absence a new radical was obtained. This new radical could be either a peroxyl radical of BH4 or an amino acid radical was in the vicinity of the heme. Formation of this new radical is very rapid, >150 s(-1), and it was subsequently converted to a BH4 radical. The trapping of the superoxide radical by cytochrome c in the reaction of BH4(-) eNOS(ox) exhibited a limiting rate of similar to15 s(-1), the time for the superoxide radical to leave the heme pocket and reach the protein surface; this reveals a general problem of the regular spin-trapping method in determining radical formation kinetics. Cytochrome c failed to trap the new radical species. Together with other EPR characteristics, our data strongly support the conclusion that this new radical is not a superoxide radical or a mixture of superoxide and biopterin radicals. Our study points out distinct roles of BH4 and L-arginine in regulating eNOS radical intermediates. BH4 prevented superoxide formation by chemical conversions of the Fe(II)O-2 intermediate, and L-arginine delayed superoxide formation by electronic interaction with the heme-bound oxygen.