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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 迷迭香洛里亚教授对通过ESR检测原核细胞在体内产生的一氧化氮感兴趣。哺乳动物中的一氧化氮合酶（NOS）是二聚体蛋白，其含有将精氨酸转化为瓜氨酸和NO的N-末端加氧酶结构域和将电子穿梭至加氧酶结构域的血红素的还原酶结构域。NOS的同源物存在于原核细胞中，但这些蛋白质产生NO的直接证据一直缺乏。目前的工作的目的是证明，NOS也产生可扩散的NO。为了证明NO的生产，使用自旋陷阱二硫代羧基肌氨酸（DTCS）。 DTCS结合铁形成活性自旋陷阱Fe（DTCS）2，其缺乏ESR信号。 当NO与Fe（DTCS）2络合物结合后，自旋陷阱产生一个特征性的3峰信号。 Fe（DTCS）2与S.肿胀导致NO-Fe（DTCS）2的特征性3峰信号的出现，其与通过添加人工NO供体在对照实验中产生的信号相同。 在NOS缺失突变培养物的样品中未观察到NO特异性3峰信号。 这些结果证实，NOS依赖的自由扩散的NO释放的thaxtomin生产链霉菌，是第一个数据记录在体内生产NO的原核NOS。 细菌NOS的结构保守性预测了其他革兰氏阳性细菌的NO产生，包括医学和环境重要的生物体，如炭疽芽孢杆菌，金黄色葡萄球菌和耐辐射球菌。 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一个重要部分。

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.