A study of NO trafficking from dinitrosyl–iron complexes to the recombinant E. coli transcriptional factor SoxR
A study of NO trafficking from dinitrosyl–iron complexes to the recombinant E. coli transcriptional factor SoxR
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NO 从二亚硝基铁复合物转运至重组大肠杆菌转录因子 SoxR 的研究
DOI：
10.1007/s00775-008-0383-6
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
S. S. Yu
中科院分区：
文献类型：
--
作者：
Feng;Chang;Chien‐Ming Lee;Ming;Tsai;W. Liaw;S. S. Yu
SoxR是大肠杆菌中的一种转录因子，在氧化应激反应中诱导SoxS的表达，启动酶的产生。除了超氧化物外，SoxR对细胞NO也很敏感，在gav = 2.03时产生具有特征电子顺磁共振（EPR）信号的蛋白结合二硝基铁复合物（DNIC）。为了开发一种基于SoxR这一特性的NO传感策略，我们过表达并纯化了重组his标记的SoxR蛋白。在厌氧条件下用(1)NO溶液、(2)s -亚硝基硫醇（RSNO）和(3)化学合成的低分子量dnic （lw - dnic）处理纯化后的蛋白质，我们观察到蛋白质结合的dnic在gav = 2.03时的EPR信号随着时间的推移而增强，这反映了NO从NO溶液、RSNO和lw - dnic向SoxR的再分配。我们利用这种NO交换来研究各种lw - dics向大肠杆菌细胞中表达的异丙基-β-d-硫代半乳糖苷依赖性SoxR释放和递送NO的动力学和机制。这些实验表明，来自RSNO和lw -DNIC的NO可以穿过生物膜进入细胞质，形成SoxR蛋白结合的DNIC复合物。为了比较，我们还研究了缓冲液中lw - dnic向SoxR蛋白的直接NO转移。一氧化氮的转移是迅速的。从动力学数据中，我们发现具有双齿硫酸盐配体的lw - dics在水溶液中表现出更大的稳定性，但在整个细胞中表现出更容易将NO传递到细胞质SoxR。
SoxR is a transcriptional factor in Escherichia coli that induces the expression of SoxS to initiate the production of enzymes in response to oxidative stress. In addition to superoxide, SoxR is also sensitive to cellular NO to produce a protein-bound dinitrosyl–iron complex (DNIC) with a characteristic electron paramagnetic resonance (EPR) signal at gav = 2.03. Toward developing a strategy for NO sensing based on this property of SoxR, we have overexpressed and purified the recombinant His-tagged SoxR protein. Upon treatment of the purified protein under anaerobic conditions with (1) NO solution, (2) S-nitrosothiol (RSNO), and (3) chemically synthesized low molecular weight DNICs (LMW-DNICs), we have observed enhancement of the EPR signal at gav = 2.03 from the protein-bound DNICs over time, reflecting the redistribution of NO from the NO solution, RSNO and LMW-DNICs to the SoxR. We have exploited this NO exchange to investigate the kinetics and mechanisms of release and delivery of NO from various LMW-DNICs to an isopropyl-β-d-thiogalactopyranoside-dependent SoxR expressed in E. coli cells. These experiments revealed that the NO from RSNO and LMW-DNICs could cross the biological membrane and enter the cytoplasm of the cell to form the SoxR protein-bound DNIC complex. For comparison, we have also studied the direct NO transfer from the LMW-DNICs to the SoxR protein in buffer. The NO transfer was found to be rapid. From the kinetic data derived, we showed that LMW-DNICs with bidentate thiolate ligands displayed greater stability in aqueous solution but exhibited more facile NO delivery to cytoplasmic SoxR in whole cells.
一氧化氮对维氏固氮菌固氮酶各个成分的失活作用的动力学和光谱分析。
影响因子：
2.9
作者：
Hyman,MR;Seefeldt,LC;Morgan,TV;Arp,DJ;Mortenson,LE
通讯作者：
Mortenson,LE
DOI：
10.1073/pnas.84.24.9265
发表时间：
1987-12-01
影响因子：
11.1
作者：
IGNARRO, LJ;BUGA, GM;CHAUDHURI, G
通讯作者：
CHAUDHURI, G
DOI：
10.1073/pnas.97.10.5146
发表时间：
2000-05-09
影响因子：
11.1
作者：
Ding, HG;Demple, B
通讯作者：
Demple, B