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首页> 外文期刊>Biochemistry >Essential thiol requirement to restore pterin- or substrate-binding capability and to regenerate native enzyme-type high-spin heme spectra in the Escherichia coli-expressed tetrahydrobiopterin-free oxygenase domain of neuronal nitric oxide synthase.
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Essential thiol requirement to restore pterin- or substrate-binding capability and to regenerate native enzyme-type high-spin heme spectra in the Escherichia coli-expressed tetrahydrobiopterin-free oxygenase domain of neuronal nitric oxide synthase.

机译：基本的硫醇需求，以恢复蝶呤或底物的结合能力，并在大肠杆菌表达的神经元一氧化氮合酶的无四氢生物蝶呤加氧酶域中再生天然酶型高自旋血红素光谱。

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摘要

Nitric oxide (NO) synthases (NOS) are thiolate-ligated heme-, tetrahydrobiopterin (BH(4))-, and flavin-containing monooxygenases which catalyze the NADPH-dependent conversion of L-arginine (L-Arg) to NO AND citrulline. NOS consists of two domains: an N-terminal oxygenase (heme- and BH(4)-bound) domain and a C-terminal reductase (FMN- and FAD-bound) domain. In this study, we have spectroscopically examined the binding of L-Agr and BH(4) to the dimeric, BH(4)-free ferric neuronal NOS (NNOS) oxygenase domain expressed in Escherichia coli separately from the reductase domain. Addition of L-Arg or its analogue inhibitors (N(G)()-methyl-L-Arg, N(G)()-nitro-L-Arg) and BH(4), together with dithiothreitol (DTT), to the pterin-free ferric low-spin oxygenase domain (gamma(MAX): 419, 538, 568 NM) and incubation for 2-3 days at 4 degrees C converted the domain to a native enzyme-type, predominantly high-spin state (gamma(MAX): approximately 395, approximately 512, approximately 650 NM). 7,8-Dihydrobiopterin and other thiols (E.G., beta-mercaptoethanol, cysteine, and glutathione, with less effectiveness) can replace BH(4) and DTT, respectively. the UV-visible absorption spectrum of L-Arg-bound ferric full length NNOS, which exhibits a relatively intense band at approximately 650 NM (epsilon equals 7.5-8 MM(-)(1) CM(-)(1)) due to the presence of a neutral flavin semiquinone, can then be quantitatively reconstructed by combining the spectra of equimolar amounts of the oxygenase and reductase domains. Of particular note, the heme spin-state conversion does not occur in the absence of a thiol even after prolonged (35-48 H) incubation of the oxygenase domain with BH(4) and/or L-Arg under anaerobic conditions. Thus, DTT (or other thiols) plays a significant role(s) beyond keeping BH(4) in its reduced form, In restoring the pterin- and/or substrate-binding capability of the E. coli-expressed, BH(4) free, dimeric NNOS oxygenase domain. Our results in combination with recently available X-ray crystallography and site-directed mutagenesis data suggest that the observed DTT effects arise from the involvement of an intersubunit disulfide bond or its rearrangement in the NOS dimer.
机译：一氧化氮（NO）合酶（NOS）是硫醇盐连接的血红素，四氢生物蝶呤（BH（4））和含黄素的单加氧酶，它们催化L-精氨酸（L-Arg）的NADPH依赖性转化为NO和瓜氨酸。 。 NOS由两个域组成：N末端加氧酶（血红素和BH（4）结合）域和C末端还原酶（FMN和FAD结合）域。在这项研究中，我们已经光谱学地检查了L-Agr和BH（4）与在大肠杆菌中与还原酶结构域分开表达的二聚体，无BH（4）的铁神经元NOS（NNOS）加氧酶结构域的结合。将L-Arg或其类似物抑制剂（N（G）（）-甲基-L-Arg，N（G）（）-硝基-L-Arg）和BH（4）与二硫苏糖醇（DTT）一起添加到不含蝶呤的铁低旋加氧酶结构域（γ（MAX）：419、538、568 NM）并在4摄氏度下温育2-3天，将结构域转变为天然酶型，主要是高旋态（ γ（MAX）：大约395，大约512，大约650海里）。 7,8-Dihydrobiopterin和其他硫醇（例如，β-巯基乙醇，半胱氨酸和谷胱甘肽，效果较差）可以分别代替BH（4）和DTT。 L-Arg结合的铁全长NNOS的紫外可见吸收光谱，由于大约650 NM（ε等于7.5-8 MM（-）（1）CM（-）（1））表现出相对较强的谱带通过结合等摩尔量的加氧酶和还原酶结构域的光谱，可以定量地重建中性黄素半醌的存在。特别要注意的是，即使在无氧条件下将加氧酶结构域与BH（4）和/或L-Arg长时间（35-48 H）孵育后，在没有硫醇的情况下也不会发生血红素自旋态转化。因此，DTT（或其他硫醇）在将BH（4）保持其还原形式方面起着重要作用。在恢复大肠杆菌表达的BH（4）的蝶呤和/或底物结合能力方面游离的二聚体NNOS加氧酶结构域。我们的结果与最近可获得的X射线晶体学和定点诱变数据相结合，表明观察到的DTT效应是由于亚单位间二硫键的参与或其在NOS二聚体中的重排而引起的。

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