Nitric oxide formation by Escherichia coli -: Dependence on nitrite reductase, the NO-sensing regulator FNR, and flavohemoglobin Hmp
Nitric oxide formation by Escherichia coli -: Dependence on nitrite reductase, the NO-sensing regulator FNR, and flavohemoglobin Hmp
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DOI：
10.1074/jbc.m303282200
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发表时间：
2003-08-22
影响因子：
4.8
通讯作者：
Poole, RK
中科院分区：
文献类型：
--
作者：
Corker, H;Poole, RK
一氧化氮（NO）是生物系统中重要的信号分子和防御分子。例如，由巨噬细胞产生的NO的杀菌作用被各种细菌NO解毒酶抵抗，最好理解的是以大肠杆菌Hmp为例的黄血红蛋白。然而，许多细菌，包括E.大肠杆菌，据报道，产生NO的过程是独立的反硝化，其中NO是一个强制性的中间体。我们证明使用NO特异性电极，E。以硝酸盐为末端电子受体的大肠杆菌厌氧培养细胞在加入亚硝酸盐时产生大量NO。通过比较Nrf（+）和Nrf（-）突变体，表明周质细胞色素c亚硝酸还原酶（Nrf）在很大程度上负责NO的产生。令人惊讶的是，hmp突变体没有积累更多的NO，而是不能产生可检测的NO。hmp突变体的厌氧生长不受硝酸盐刺激，并且突变体不能产生周质细胞色素c，从而导致以下假设：在不存在Hmp的情况下积累NO通过与[4Fe-4S]（2+）簇反应而使全局厌氧调节剂Fnr失活（Cruz-Ramos，H.，克拉克，J。，吴，G.，休斯，M。N.，斯科特，C.，汤姆森，A. J.，绿色，J.，和Poole，R. K.等（2002）EMBO J. 21，3235-3244）。因此Fnr不能上调亚硝酸还原酶。该模型得到了支持的fnr突变体不能产生NO和恢复NO积累的hmp突变体后，引入质粒编码Fnr*（D154 A）已知赋予活性的存在下的氧气。细胞色素bd缺陷突变体保留NO生成活性。本研究揭示了一个关键的平衡之间的NO生成和解毒活动在厌氧生长。
Nitric oxide (NO) is a key signaling and defense molecule in biological systems. The bactericidal effects of NO produced, for example, by macrophages are resisted by various bacterial NO-detoxifying enzymes, the best understood being the flavohemoglobins exemplified by Escherichia coli Hmp. However, many bacteria, including E. coli, are reported to produce NO by processes that are independent of denitrification in which NO is an obligatory intermediate. We demonstrate using an NO-specific electrode that E. coli cells, grown anaerobically with nitrate as terminal electron acceptor, generate significant NO on adding nitrite. The periplasmic cytochrome c nitrite reductase (Nrf) is shown, by comparing Nrf(+) and Nrf(-) mutants, to be largely responsible for NO generation. Surprisingly, an hmp mutant did not accumulate more NO but, rather, failed to produce detectable NO. Anaerobic growth of the hmp mutant was not stimulated by nitrate, and the mutant failed to produce periplasmic cytochrome(s) c, leading to the hypothesis that accumulating NO in the absence of Hmp inactivates the global anaerobic regulator Fnr by reaction with the [4Fe-4S](2+) cluster (Cruz-Ramos, H., Crack, J., Wu, G., Hughes, M. N., Scott, C., Thomson, A. J., Green, J., and Poole, R. K. (2002) EMBO J. 21, 3235-3244). Fnr thus failed to up-regulate nitrite reductase. The model is supported by the inability of an fnr mutant to generate NO and by the restoration of NO accumulation to hmp mutants upon introducing a plasmid encoding Fnr* (D154A) known to confer activity in the presence of oxygen. A cytochrome bd-deficient mutant retained NO-generating activity. The present study reveals a critical balance between NO-generating and -detoxifying activities during anaerobic growth.