Magnetic circular dichroism spectroscopic characterization of the NOS-like protein from Geobacillus stearothermophilus (gsNOS).
Magnetic circular dichroism spectroscopic characterization of the NOS-like protein from Geobacillus stearothermophilus (gsNOS).
复制标题
嗜热脂肪地芽孢杆菌 (gsNOS) 的 NOS 样蛋白的磁圆二色光谱表征。
DOI：
10.1016/j.jinorgbio.2009.12.014
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发表时间：
2010
影响因子：
3.9
通讯作者：
Dawson,JohnH
中科院分区：
文献类型：
--
作者：
Kinloch,RyanD;Sono,Masanori;Sudhamsu,Jawahar;Crane,BrianR;Dawson,JohnH
一氧化氮合酶(NOS)催化依赖NADPH和O2的L-精氨酸(L-Arg)通过NG-羟基-L-精氨酸中间体氧化成一氧化氮(NO)和瓜氨酸。哺乳动物一氧化氮合酶的研究已经相当广泛；其他真核生物和一些原核生物似乎表达的一氧化氮合酶样蛋白类似于哺乳动物一氧化氮合酶的氧合酶结构域。本研究利用磁性圆二色谱(MCD)和紫外可见吸收光谱技术，对耐热细菌Gebacillusstearthermophilus(GsNOS)的重组类NOS蛋白进行了表征。底物结合的(L-精氨酸或nHA)gsNOS的配体配合物(与O2、NO和CO)的光谱比较表明，gsNOS和哺乳动物NOS的光谱基本相似，其中包括在−50°C低温混合溶剂中研究的关键含氧亚铁配合物。然而，更详细的光谱比较反映了gsNOS和哺乳动物NOS之间的细微结构差异。这可能是由于四氢生物蝶呤(BH4)结合位点不完整和BH4结合亲和力低所致，在gsNOS中存在冷冻剂时，亲和力可能会变得更低。虽然bH4结合可能会发生改变，但gsNOS似乎需要蝶呤才能产生NO，因为只有当在−35°C或−50°C下的单一翻转反应中，O2被冒泡到二亚硫酸盐还原的nHA结合的蛋白质溶液中时，才能观察到稳定的铁-NO产物复合体的形成。
Nitric oxide synthase (NOS) catalyzes the NADPH- and O2-dependent oxidation of l-arginine (l-Arg) to nitric oxide (NO) and citrulline via an NG-hydroxy-l-arginine (NHA) intermediate. Mammalian NOSs have been studied quite extensively; other eukaryotes and some prokaryotes appear to express NOS-like proteins comparable to the oxygenase domain of mammalian NOSs. In this study, a recombinant NOS-like protein from the thermostable bacterium Geobacillus stearothermophilus (gsNOS) has been characterized using magnetic circular dichroism (MCD) and UV–Vis absorption spectroscopic techniques. Spectral comparisons of ligand complexes (with O2, NO and CO) of substrate-bound (l-Arg or NHA) gsNOS, including the key oxyferrous complex studied at −50°C in cryogenic mixed solvents, with analogous mammalian NOS complexes indicate overall spectroscopic similarities between gsNOS and mammalian NOSs. However, more detailed spectral comparisons reflect subtle structural differences between gsNOS and mammalian NOSs. This may be due to an incomplete tetrahydrobiopterin (BH4)-binding site and low BH4-binding affinity, which may become even lower in the presence of cryosolvent in gsNOS. Although BH4-binding may be altered, gsNOS appears to require the pterin for NO production since formation of the stable ferric-NO product complex was only observed when excess BH4(>150μM) over gsNOS was present upon single turnover reaction in which O2was bubbled into dithionite-reduced NHA-bound protein solution at −35°C or −50°C.