Low-temperature stabilization and spectroscopic characterization of the dioxygen complex of the ferrous neuronal nitric oxide synthase oxygenase domain.
Low-temperature stabilization and spectroscopic characterization of the dioxygen complex of the ferrous neuronal nitric oxide synthase oxygenase domain.
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亚铁神经元一氧化氮合酶加氧酶结构域双氧复合物的低温稳定性和光谱表征。
DOI：
10.1021/bi990619h
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Sono,M
中科院分区：
文献类型：
--
作者：
Ledbetter,AP;McMillan,K;Roman,LJ;Masters,BS;Dawson,JH;Sono,M
一氧化氮（NO）是一种细胞间信使和免疫细胞毒性剂，由一氧化氮合酶（NOS）家族合成，NOS是巯基连接的含血红素单加氧酶，使用NADPH作为电子供体，以四氢生物蝶呤（BH 4）依赖性方式转化1-Arg tol-瓜氨酸和NO。亚铁酶的双氧复合物被认为是NOS催化循环的关键中间体。在这项研究中，我们已经产生了一个稳定的亚铁-O2复合物的加氧酶结构域的大鼠神经元NOS（nNOS）通过鼓泡O2通过溶液中的连二亚硫酸盐还原酶在-30 °C在低温溶剂含有50%乙二醇。最稳定的双氧复合物是使用加氧酶结构域获得的，该加氧酶结构域已在4 °C下与BH 4/二硫苏糖醇和底物类似物抑制剂NG-甲基-L-精氨酸预孵育延长的时间。由此制备的nNOS加氧酶结构域的O2复合物表现出UV-可见吸收（在419和553 nm处最大，在585 nm处肩峰）和磁性圆二色谱，与亚铁-O2细胞色素P450-CAM的光谱几乎相同。对于nNOS加氧酶结构域，使用停流快速扫描光谱法，我们的光谱数据与在10 °C下观察到的短寿命瞬态物质（λmax= 427 nm）相比明显蓝移[Abu-Soud，H. M.，Gachhui河，Raushel，F. M.，Stuehr，D. J.（1997）J.Biol.Chem.272，17349]，但有点类似于在-30 °C下产生的无-Arg全长nNOS（λmax= 415 - 416.5 nm）的相对稳定的O2加合物[Bec，N.，Gorren，A. C. F.、Voelder，C.，Mayer，B.，和Lange，R.（1998）J.Biol.Chem.273，13502]。然而，与亚铁− O2 P450-CAM相比，nNOS加氧酶结构域的亚铁− O2加合物更容易自氧化，O2−CO交换反应明显较慢。如本文所述，nNOS加氧酶结构域的稳定亚铁− O2加合物的产生将促进对该重要中间体的进一步机理和光谱研究。
Nitric oxide (NO), an intercellular messenger and an immuno-cytotoxic agent, is synthesized by the family of nitric oxide synthases (NOS), which are thiolate-ligated, heme-containing monooxygenases that convertl-Arg tol-citrulline and NO in a tetrahydrobiopterin (BH4)-dependent manner, using NADPH as the electron donor. The dioxygen complex of the ferrous enzyme has been proposed to be a key intermediate in the NOS catalytic cycle. In this study, we have generated a stable ferrous−O2complex of the oxygenase domain of rat neuronal NOS (nNOS) by bubbling O2through a solution of the dithionite-reduced enzyme at −30 °C in a cryogenic solvent containing 50% ethylene glycol. The most stable dioxygen complex is obtained using the oxygenase domain which has been preincubated for an extended length of time at 4 °C with BH4/dithiothreitol andNG-methyl-l-arginine, a substrate analogue inhibitor. The O2complex of the nNOS oxygenase domain thus prepared exhibits UV−visible absorption (maxima at 419 and 553 nm, shoulder at ∼585 nm) and magnetic circular dichroism spectra that are nearly identical to those of ferrous−O2cytochrome P450-CAM. Our spectral data are noticeably blue-shifted from those seen at 10 °C for a short-lived transient species (λmax= 427 nm) for the nNOS oxygenase domain using stopped-flow rapid-scanning spectroscopy [Abu-Soud, H. M., Gachhui, R., Raushel, F. M., and Stuehr, D. J. (1997)J. Biol. Chem.272, 17349], but somewhat similar to those of a relatively stable O2adduct ofl-Arg-free full-length nNOS (λmax= 415−416.5 nm) generated at −30 °C [Bec, N., Gorren, A. C. F., Voelder, C., Mayer, B., and Lange, R. (1998)J. Biol. Chem.273, 13502]. Compared with ferrous−O2P450-CAM, however, the ferrous−O2adduct of the nNOS oxygenase domain is considerably more autoxidizable and the O2−CO exchange reaction is noticeably slower. The generation of a stable ferrous−O2adduct of the nNOS oxygenase domain, as described herein, will facilitate further mechanistic and spectroscopic investigations of this important intermediate.