Ding H, Hidalgo E, Demple B
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
J Biol Chem. 1996 Dec 27;271(52):33173-5. doi: 10.1074/jbc.271.52.33173.
SoxR protein is a redox-responsive transcription factor that governs a regulon of oxidative stress and antibiotic resistance genes in Escherichia coli. Purified SoxR contains oxidized [2Fe-2S] clusters and stimulates in vitro transcription of its target gene soxS up to 100-fold. SoxR transcriptional activity, but not DNA binding, is completely dependent on the [2Fe-2S] clusters; apo-SoxR prepared in vitro binds the soxS promoter with unchanged affinity but does not have transcription activity. Thus, modulation of the SoxR [2Fe-2S] clusters was proposed to control the protein's function in transcription. Here, we provide evidence that SoxR with reduced [2Fe-2S] clusters is inactive. Redox titration of purified SoxR revealed a midpoint potential of -285 +/- 10 mV (pH 7.6). In vitro transcription assays showed that SoxR was inactivated when the [2Fe-2S] cluster was reduced (-380 mV), and full activity was restored upon reoxidation (+100 mV). The results suggest that one-electron oxidation and reduction of the [2Fe-2S] cluster regulate SoxR transcriptional activity.
SoxR蛋白是一种氧化还原反应敏感的转录因子，它调控大肠杆菌中氧化应激和抗生素抗性基因的一个调控子。纯化的SoxR含有氧化型[2Fe-2S]簇，并能在体外将其靶基因soxS的转录刺激高达100倍。SoxR的转录活性而非DNA结合活性完全依赖于[2Fe-2S]簇；体外制备的脱辅基SoxR以不变的亲和力结合soxS启动子，但没有转录活性。因此，有人提出对SoxR [2Fe-2S]簇的调节来控制该蛋白在转录中的功能。在此，我们提供证据表明具有还原型[2Fe-2S]簇的SoxR是无活性的。纯化的SoxR的氧化还原滴定显示中点电位为-285±10 mV（pH 7.6）。体外转录试验表明，当[2Fe-2S]簇被还原（-380 mV）时SoxR失活，而再氧化（+100 mV）后恢复全部活性。结果表明，[2Fe-2S]簇的单电子氧化和还原调节SoxR的转录活性。
