Institute of Microbiology and Infection and School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, S10 2TN, UK.
Microbiology (Reading). 2018 Apr;164(4):563-575. doi: 10.1099/mic.0.000629. Epub 2018 Mar 1.
Synthesis of the Escherichia coli YtfE protein, also known as RIC, for the repair of damaged iron centres, is highly induced during anaerobic growth under conditions of nitrosative stress. How YtfE repairs nitrosative damage remains unclear. Contrary to previous reports, we show that strains defective in YtfE that lack the high-affinity NO reductase activity of the hybrid cluster protein (Hcp) are less sensitive to nitrosative stress than isogenic ytfE strains, which are extremely sensitive. Evidence that this sensitivity is due to YtfE-dependent release of NO into the cytoplasm includes: relief of growth inhibition by PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide), which degrades NO; relief of nitrosative stress by deletion of narG encoding the nitrate reductase that is the major source of NO from nitrite; partial suppression of nitrosative stress due to loss of Hcp function by a further mutation in ytfE; YtfE-dependent loss of aconitase and fumarase activities in the absence of Hcp; and YtfE-dependent relief of NsrR repression of the hcp promoter in response to cytoplasmic NO. We suggest that a major role for YtfE is to reverse nitrosative damage by releasing, directly or indirectly, NO from nitrosylated proteins into the cytoplasm where the high-affinity NO reductase activity of Hcp ensures its reduction to N2O. If so, the concerted action of YtfE and Hcp would not only maintain the cytoplasmic concentration of NO in the low nM range, but also provide a rationalization for the coordinate regulation of Hcp and YtfE synthesis by NsrR.
大肠杆菌 YtfE 蛋白（也称为 RIC）的合成，用于修复受损的铁中心，在厌氧条件下生长并受到亚硝化应激时会被高度诱导。YtfE 如何修复亚硝化损伤尚不清楚。与之前的报道相反，我们表明，缺乏杂种簇蛋白（Hcp）高亲和力 NO 还原酶活性的 ytfE 缺陷菌株在亚硝化应激下比具有相同遗传背景的 ytfE 菌株更不敏感，后者对亚硝化应激极其敏感。表明这种敏感性是由于 YtfE 依赖性将 NO 释放到细胞质中的证据包括：PTIO（2-苯基-4,4,5,5-四甲基咪唑啉-1-氧自由基 3-氧化物）降解 NO 可缓解生长抑制；硝酸盐还原酶编码基因 narG 的缺失可缓解亚硝化应激，硝酸盐还原酶是亚硝酸盐产生 NO 的主要来源；由于 ytfE 中的进一步突变导致 Hcp 功能丧失，部分缓解了亚硝化应激；在没有 Hcp 的情况下，依赖于 YtfE 的 aconitase 和延胡索酸酶活性丧失；以及 YtfE 依赖性缓解 NsrR 对 hcp 启动子的抑制作用，以响应细胞质中的 NO。我们认为，YtfE 的主要作用是通过直接或间接地将来自亚硝化蛋白的 NO 释放到细胞质中，从而逆转亚硝化损伤，Hcp 的高亲和力 NO 还原酶活性可确保其还原为 N2O。如果是这样，YtfE 和 Hcp 的协同作用不仅可以将细胞质中 NO 的浓度维持在低 nM 范围内，还可以为 NsrR 对 Hcp 和 YtfE 合成的协调调节提供合理化解释。
