Gorodetsky Alon A, Dietrich Lars E P, Lee Paul E, Demple Bruce, Newman Dianne K, Barton Jacqueline K
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3684-9. doi: 10.1073/pnas.0800093105. Epub 2008 Mar 3.
Electrochemistry measurements on DNA-modified electrodes are used to probe the effects of binding to DNA on the redox potential of SoxR, a transcription factor that contains a [2Fe-2S] cluster and is activated through oxidation. A DNA-bound potential of +200 mV versus NHE (normal hydrogen electrode) is found for SoxR isolated from Escherichia coli and Pseudomonas aeruginosa. This potential value corresponds to a dramatic shift of +490 mV versus values found in the absence of DNA. Using Redmond red as a covalently bound redox reporter affixed above the SoxR binding site, we also see, associated with SoxR binding, an attenuation in the Redmond red signal compared with that for Redmond red attached below the SoxR binding site. This observation is consistent with a SoxR-binding-induced structural distortion in the DNA base stack that inhibits DNA-mediated charge transport to the Redmond red probe. The dramatic shift in potential for DNA-bound SoxR compared with the free form is thus reconciled based on a high-energy conformational change in the SoxR-DNA complex. The substantial positive shift in potential for DNA-bound SoxR furthermore indicates that, in the reducing intracellular environment, DNA-bound SoxR is primarily in the reduced form; the activation of DNA-bound SoxR would then be limited to strong oxidants, making SoxR an effective sensor for oxidative stress. These results more generally underscore the importance of using DNA electrochemistry to determine DNA-bound potentials for redox-sensitive transcription factors because such binding can dramatically affect this key protein property.
对DNA修饰电极进行电化学测量，以探究与DNA结合对SoxR氧化还原电位的影响。SoxR是一种转录因子，含有一个[2Fe-2S]簇，通过氧化被激活。从大肠杆菌和铜绿假单胞菌中分离出的SoxR，其与DNA结合的电位相对于标准氢电极（NHE）为 +200 mV。该电位值相对于在无DNA情况下测得的值有 +490 mV的显著偏移。使用Redmond红作为共价结合的氧化还原报告分子，固定在SoxR结合位点上方，我们还观察到，与SoxR结合相关的是，与固定在SoxR结合位点下方的Redmond红相比，Redmond红信号减弱。这一观察结果与SoxR结合诱导的DNA碱基堆积结构畸变一致，该畸变抑制了DNA介导的电荷传输至Redmond红探针。因此，基于SoxR-DNA复合物中的高能构象变化，解释了与游离形式相比，结合DNA的SoxR电位的显著偏移。此外，结合DNA的SoxR电位的大幅正向偏移表明，在还原性的细胞内环境中，结合DNA的SoxR主要处于还原形式；结合DNA的SoxR的激活将仅限于强氧化剂，这使得SoxR成为氧化应激的有效传感器。这些结果更普遍地强调了利用DNA电化学来确定对氧化还原敏感的转录因子的DNA结合电位的重要性，因为这种结合会显著影响这一关键的蛋白质特性。
