Superoxide generation by endothelial nitric oxide synthase:: The influence of cofactors
Superoxide generation by endothelial nitric oxide synthase:: The influence of cofactors
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DOI：
10.1073/pnas.95.16.9220
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发表时间：
1998-08-04
影响因子：
11.1
通讯作者：
Pritchard, KA
中科院分区：
文献类型：
--
作者：
Vásquez-Vivar, J;Kalyanaraman, B;Pritchard, KA
采用电子自旋共振自旋捕获技术，以5-二氧基磷酸-5-甲基-1-吡咯啉n-氧化物为原料研究了内皮型一氧化氮合酶（eNOS）产生超氧化物的机理。在缺乏钙/钙调素的情况下，eNOS产生少量的超氧化物。当钙/钙调素结合激活eNOS电子转移反应时，超氧化物的生成增加，血红素-铁配体、氰化物、咪唑和苯（二氮）基自由基抑制超氧化物的生成，在活化的eNOS中加入l -精氨酸、n - g -羟基- l -精氨酸、l -硫氨酸和l - ng -单甲基精氨酸后，无抑制作用。这些结果表明，超氧化物是通过铁-二氧络合物的解离从加氧酶结构域产生的，而l -精氨酸结合位点的占据并不会抑制这一过程，然而，l -精氨酸和四氢生物蝶呤（BH4）的同时添加消除了eNOS产生的超氧化物，在这些条件下，l -瓜氨酸的产量接近最大值。我们的数据表明BH4将l -精氨酸氧化与NADPH消耗完全耦合，并阻止铁-二氧络合物的解离，在这些条件下，eNOS不产生超氧化物。黄素的存在，在NOS测定系统中通常采用的浓度，增强了还原酶域的超氧化物生成。我们的数据表明BH4浓度的调节可以调节eNOS产生的超氧化物与一氧化氮的比例。
The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium/calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium/calmodulin binding, superoxide formation is increased, Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation, No inhibition is observed after addition of L-arginine, N-G-hydroxy-L-arginine, L-thiocitrulline, and L-NG-monomethyl arginine to activated eNOS, These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous-dioxygen complex and that occupation of the L-arginine binding site does Plot inhibit this process, However, the concomitant addition of L-arginine and tetrahydrobiopterin (BH4) abolishes superoxide generation by eNOS, Under these conditions, L-citrulline production is close to maximal. Our data indicate that BH4 fully couples L-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous-dioxygen complex, Under these conditions, eNOS does not generate superoxide. The presence of flavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.