Oxygen-induced radical intermediates in the nNOS oxygenase domain regulated by L-arginine, tetrahydrobiopterin, and thiol
Oxygen-induced radical intermediates in the nNOS oxygenase domain regulated by L-arginine, tetrahydrobiopterin, and thiol
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DOI：
10.1021/bi701677r
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发表时间：
2008-01-08
期刊：
影响因子：
2.9
通讯作者：
Tsai, Ah-Lim
中科院分区：
文献类型：
--
作者：
Berka, Vladimir;Wang, Lee-Ho;Tsai, Ah-Lim
全偶联一氧化氮合酶（NOS）催化L-精氨酸、NADPH和氧气形成一氧化氮（NO）、L-瓜氨酸、NADP（+）和水。未偶联或部分偶联的NOS催化活性氧物质如超氧化物、过氧化氢和过氧亚硝酸盐的合成，这取决于催化过程中辅因子四氢生物蝶呤（BH 4）和L-精氨酸的可用性。我们在有或没有BH 4和/或L-精氨酸的亚铁内皮NOS加氧酶结构域（eNOS（ox））中鉴定了三种不同的氧诱导自由基中间体[Berka，V.，吴，G.，是的，H。C.的方法，Palmer，G.，和Tsai，A.- L.（2004）J.Biol.Chem.279，32243-32251]。BH 4和L-精氨酸对分离的神经元NOS加氧酶结构域（nNOS（ox））中氧诱导的自由基中间体的影响已经类似地研究了在存在或不存在二硫苏糖醇（DTT）的情况下，通过单周转停流和快速冷冻淬灭EPR动力学测量。与eNOS（ox）一样，我们在亚铁nNOS（ox）与氧的反应中发现了不同的自由基中间体。(1)nNOS（Ox）（不含BH 4或L-Arg）在DTT存在或不存在下产生超氧化物。(2)nNOS（ox）（与BH 4和L-Arg）以独立于DTT的方式产生典型的BH 4自由基。(3)nNOS（ox）（有BH_4而无L-Arg）产生一个新的自由基。在没有DTT的情况下，EPR显示出超氧化物和生物蝶呤自由基的混合物。（4）nNOS（ox）（不含BH_4但含L-Arg）中仅存在L-精氨酸可导致约70%的超氧自由基转化为新的自由基，这解释了L-精氨酸如何降低nNOS（ox）（不含BH_4但含L-Arg）中超氧自由基的产生水平。因此，L-精氨酸在nNOS中的调节作用与eNOS中的调节作用非常不同，eNOS中的底物仅降低超氧化物的形成速率而不是自由基的总量。DTT的作用也不同。DTT阻止两种亚型中BH 4的氧化，但在nNOS中，DTT还抑制nNOS中两个关键半胱氨酸（ox）的氧化，以防止底物结合的丧失。仅在nNOS中发现的巯基的这种新作用在神经退行性疾病中可能是重要的。
Fully coupled nitric oxide synthase (NOS) catalyzes formation of nitric oxide (NO), L-citrulline, NADP(+), and water from L-arginine, NADPH, and oxygen. Uncoupled or partially coupled NOS catalyzes the synthesis of reactive oxygen species such as superoxide, hydrogen peroxide, and peroxynitrite, depending on the availability of cofactor tetrahydrobiopterin (BH4) and L-arginine during catalysis. We identified three distinct oxygen-induced radical intermediates in the ferrous endothelial NOS oxygenase domain (eNOS(ox)) with or without BH4 and/or L-arginine [Berka, V., Wu, G., Yeh, H. C., Palmer, G., and Tsai, A.-L. (2004) J. Biol. Chem. 279, 32243-32251]. The effects of BH4 and L-arginine on the oxygen-induced radical intermediates in the isolated neuronal NOS oxygenase domain (nNOS(ox)) have been similarly investigated by single-turnover stopped-flow and rapid-freeze quench EPR kinetic measurements in the presence or absence of dithiothreitol (DTT). Like for eNOS(ox), we found different radical intermediates in the reaction of ferrous nNOS(ox) with oxygen. (1) nNOS(Ox) (without BH4 or L-Arg) produces superoxide in the presence or absence of DTT. (2) nNOS(ox) (with BH4 and L-Arg) yields a typical BH4 radical in a manner independent of DTT. (3) nNOS(ox) (with BH4 and without L-Arg) yields a new radical. Without DTT, EPR showed a mixture of superoxide and biopterin radicals. With DTT, a new similar to 75 G wide radical EPR was observed, different from the radical formed by eNOS(ox), (4) The presence of only L-arginine in nNOS(ox) (without BH4 but with L-Arg) caused conversion of similar to 70% of superoxide radical to a novel radical, explaining how L-arginine decreases the level of superoxide production in nNOS(ox) (without BH4 but with L-Arg). The regulatory role of L-arginine in nNOS is thus very different from that in eNOS where substrate was only to decrease the rate of formation of superoxide but not the total amount of radical. The role of DTT is also different. DTT prevents oxidation of BH4 in both isoforms, but in nNOS, DTT also inhibits oxidation of two key cysteines in nNOS(ox) to prevent the loss of substrate binding. This new role of thiol found only for nNOS may be significant in neurodegenerative diseases.