ESR DETECTION OF NITRIC OXIDE PRODUCED IN VIVO BY PROKARYOTIC CELLS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 罗斯玛丽·洛里亚教授对通过电子自旋共振检测原核细胞体内产生的一氧化氮感兴趣。哺乳动物中的一氧化氮合酶(NOSS)是一种二聚体蛋白质，含有N-末端的加氧酶结构域，将精氨酸转化为瓜氨酸和NO，以及一个还原酶结构域，将电子穿梭到加氧酶结构域的血红素上。NOSS的同源物存在于原核生物中，但缺乏这些蛋白质产生NO的直接证据。目前工作的目的是证明一氧化氮合酶也能产生可扩散的一氧化氮。为了证明没有生成，使用了自旋陷阱二硫代羧酸肌氨酸(DTCS)。DTCS与铁结合形成缺少ESR信号的活性自旋陷阱Fe(DTCS)2。在NO与Fe(DTCS)2络合物结合后，自旋陷阱产生特征的3峰信号。将Fe(DTCS)_2与膨大链霉菌野生型培养物孵育后，NO-Fe(DTCS)_2出现与添加人工NO供体的对照实验中相同的特征3峰信号。在一氧化氮合酶缺失突变培养的样品中没有观察到这种NO特异性的3峰信号。这些结果证实了产泰素的链霉菌释放了依赖于一氧化氮合酶的自由扩散的NO，这是首次报道了原核生物NOSS体内产生NO的数据。细菌NOSS的结构保守预测其他革兰氏阳性细菌不会产生，包括医学和环境重要的生物体，如炭疽杆菌、金黄色葡萄球菌和耐辐射葡萄球菌。一篇关于这项工作的论文已提交给《化学与生物学》。这项工作也代表了洛里亚教授的研究生埃文·约翰逊博士论文的重要部分。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Prof Rosemary Loria is interested in detection, via ESR, of nitric oxide produced in vivo by prokaryotic cells. Nitric oxide synthases (NOSs) in mammals are dimeric proteins containing N-terminal oxygenase domains that convert arginine into citrulline and NO, and a reductase domain that shuttles electrons to the heme of the oxygenase domain. Homologs to NOSs exist in prokaryots, but direct evidence for NO production by these proteins has been lacking. The aim of current work was to demonstrate that NOS also produces diffusible NO. To demonstrate NO production, the spin trap dithiocarboxy-sarcosine (DTCS) was used. DTCS binds iron to form the active spin trap Fe(DTCS)2 that lacks an ESR signal. After NO binds to the Fe(DTCS)2 complex, the spin trap produces a characteristic 3-peak signal. Incubation of Fe(DTCS)2 with the wild type culture of S. turgidiscabies resulted in the appearance of the characteristic 3-peak signal of NO-Fe(DTCS)2 identical to that produced in control experiments by addition of artificial NO-donor. This NO-specific 3-peak signal was not observed in samples from NOS-deletion mutant cultures. These results confirmed that NOS-dependent free-diffusing NO is released by thaxtomin-producing streptomycetes and were the first data to document in vivo production of NO by prokaryotic NOSs. Structural conservation of bacterial NOSs predicts NO production by other Gram-positive bacteria including medically and environmentally important organisms such as Bacillus anthracis, Staphylococcus aureus, and Deinococcus radiodurans. A paper on this work was submitted to Chemistry & Biology. This work also represents a substantial part of the PhD thesis of a graduate student of Prof. Loria: Evan Johnson.