Kobayashi Kazuo, Fujikawa Mayu, Kozawa Takahiro
The Institute of Scientific and Industrial Research, Osaka University , Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan.
Biochemistry. 2015 Jan 20;54(2):334-9. doi: 10.1021/bi500931w. Epub 2014 Dec 19.
The [2Fe-2S] transcriptional factor SoxR, a member of the MerR family, functions as a sensor of oxidative stress in Escherichia coli. The transcriptional activity of SoxR is regulated by the reversible oxidation and reduction of [2Fe-2S] clusters. Electrochemistry measurements on DNA-modified electrodes have shown a dramatic shift in the reduction potential of SoxR from -290 to +200 mV with the promoter DNA-bound [ Gorodetsky , A. A. , Dietrich , L. E. P. , Lee , P. E. , Demple , B. , , Newman , D. K. , and Barton , J. K. ( 2008 ) DNA binding shifts the reduction potential of the transcription factor SoxR , Proc. Natl. Acad. Sci. U.S.A. 105 , 3684 - 3689 ]. To determine the change of the SoxR reduction potential using the new condition, the one-electron oxidation-reduction properties of [2Fe-2S] cluster in SoxR were investigated in the absence and presence of the DNA. The [2Fe-2S] cluster of SoxR was completely reduced by nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CRP) in the presence of a NADPH generating system (glucose 6-dehydrogenase and glucose-6 phosphate), indicating that CRP can serve as an NADPH-dependent electron carrier for SoxR. The reduction potential of SoxR was measured from equilibrium data coupled with NADPH and CRP in the presence of electron mediators. The reduction potentials of DNA-bound and DNA-free states of SoxR were -320 and -293 mV versus NHE (normal hydrogen electrode), respectively. These results indicate that DNA binding causes a moderate shift in the reduction potential of SoxR.
[2Fe-2S]转录因子SoxR是MerR家族的成员之一，在大肠杆菌中作为氧化应激的传感器发挥作用。SoxR的转录活性受[2Fe-2S]簇可逆氧化和还原的调节。在DNA修饰电极上进行的电化学测量表明，与启动子DNA结合时，SoxR的还原电位从-290 mV急剧转变为+200 mV [戈罗德茨基，A. A.，迪特里希，L. E. P.，李，P. E.，登普尔，B.，纽曼，D. K.，和巴顿，J. K.（2008年）DNA结合改变转录因子SoxR的还原电位，《美国国家科学院院刊》105，3684 - 3689]。为了在新条件下确定SoxR还原电位的变化，研究了在有无DNA存在的情况下SoxR中[2Fe-2S]簇的单电子氧化还原性质。在存在NADPH生成系统（葡萄糖6-脱氢酶和6-磷酸葡萄糖）的情况下，烟酰胺腺嘌呤二核苷酸磷酸（NADPH）-细胞色素P450还原酶（CRP）可将SoxR的[2Fe-2S]簇完全还原，这表明CRP可作为SoxR的NADPH依赖性电子载体。在存在电子介质的情况下，根据与NADPH和CRP相关的平衡数据测量SoxR的还原电位。与标准氢电极（NHE）相比，SoxR与DNA结合状态和无DNA状态的还原电位分别为-320 mV和-293 mV。这些结果表明，DNA结合导致SoxR的还原电位发生适度变化。
