Ding H, Demple B
Department of Cancer Cell Biology, School of Public Health, Harvard University, Boston, Massachusetts 02115-6021, USA.
Biochemistry. 1998 Dec 8;37(49):17280-6. doi: 10.1021/bi980532g.
SoxR, a transcription factor containing [2Fe-2S] clusters, governs the cellular response to oxidative stress in Escherichia coli. The oxidation state of the iron-sulfur clusters regulates the SoxR transcriptional activity. When the reduced iron-sulfur clusters become oxidized ([2Fe-2S]2+ state), SoxR is activated to stimulate transcription of the soxS gene, whose product in turn switches on a group of genes encoding various proteins that defend against oxidative stress and antibiotics. A previous study showed that the oxidized [2Fe-2S] clusters of SoxR are destroyed by a free-radical-dependent process in vitro during aerobic exposure to the biological thiol glutathione. Here, we show that different thiols have differing effects on the SoxR [2Fe-2S] clusters. Like reduced glutathione, N-acetyl-L-cysteine, L-cysteine methyl ester, and L-cysteine ethyl ester disrupted the SoxR [2Fe-2S] clusters in aerobic solution. This disruption was blocked by L-cysteine, which was effective at concentrations 100-fold lower (1-10 microM) than the disrupting thiols (1 mM). In view of a previous observation that superoxide dismutase and catalase block the disruption process, this result suggests that L-cysteine may quench reactive SoxR or thiol intermediates involved in the cluster disruption reaction, the detailed mechanism of which remains unknown. In contrast, bifunctional thiols such as dithiothreitol or dithioerythritol promoted the aerobic assembly of the functional [2Fe-2S] clusters into apo-SoxR in the presence of Fe2+ and inorganic sulfide. The dithiol protein thioredoxin-A of E. coli acted catalytically in vitro in the presence of thioredoxin reductase and NADPH to promote [2Fe-2S] cluster assembly into apo-SoxR. The regulatory activity of SoxR in vivo, assessed by monitoring the paraquat-mediated induction of a soxS'::lacZ reporter fusion, was significantly lower in a strain lacking both thioredoxin-A and glutathione reductase, which maintains reduced glutaredoxins. Thus, cellular monothiols and dithiol proteins may contribute to SoxR regulation by affecting the disassembly and reassembly of the [2Fe-2S] clusters.
SoxR是一种含有[2Fe-2S]簇的转录因子，它调控大肠杆菌对氧化应激的细胞反应。铁硫簇的氧化状态调节SoxR的转录活性。当还原态的铁硫簇被氧化（[2Fe-2S]2+状态）时，SoxR被激活，从而刺激soxS基因的转录，其产物进而开启一组编码各种抵御氧化应激和抗生素的蛋白质的基因。先前的一项研究表明，在有氧条件下暴露于生物硫醇谷胱甘肽时，体外SoxR的氧化态[2Fe-2S]簇会通过自由基依赖的过程被破坏。在此，我们表明不同的硫醇对SoxR的[2Fe-2S]簇有不同的影响。与还原型谷胱甘肽一样，N-乙酰-L-半胱氨酸、L-半胱氨酸甲酯和L-半胱氨酸乙酯在有氧溶液中会破坏SoxR的[2Fe-2S]簇。这种破坏被L-半胱氨酸阻断，L-半胱氨酸发挥作用的浓度（1-10 microM）比破坏硫醇（1 mM）低100倍。鉴于之前观察到超氧化物歧化酶和过氧化氢酶可阻断破坏过程，该结果表明L-半胱氨酸可能淬灭参与簇破坏反应的活性SoxR或硫醇中间体，其详细机制仍不清楚。相反，双功能硫醇如二硫苏糖醇或二硫赤藓糖醇在Fe2+和无机硫化物存在的情况下，可促进功能性[2Fe-2S]簇在有氧条件下组装到脱辅基SoxR中。在硫氧还蛋白还原酶和NADPH存在的情况下，大肠杆菌的双硫醇蛋白硫氧还蛋白-A在体外具有催化作用，可促进[2Fe-2S]簇组装到脱辅基SoxR中。通过监测百草枯介导的soxS'::lacZ报告基因融合的诱导来评估SoxR在体内的调节活性，在同时缺乏硫氧还蛋白-A和谷胱甘肽还原酶（维持还原型谷氧还蛋白）的菌株中，该活性显著降低。因此，细胞中的单硫醇和双硫醇蛋白可能通过影响[2Fe-2S]簇的拆卸和重新组装来促进SoxR的调节。
