The redox state of the [2Fe-2S] clusters in SoxR protein regulates its activity as a transcription factor

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\u27s 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

Redox signal transduction: Mutations shifting [2Fe-2S] centers of the SoxR sensor-regulator to the oxidized form

SoxR is a [2Fe-2S] transcription factor triggered by oxidative stress and activated in vitro by one-electron oxidation or assembly of the iron- sulfur centers. To distinguish which mechanism operates in cells, we studied constitutively active SoxR (SoxR(c)) proteins. Three SoxR(c) proteins contained [2Fe-2S] centers required for in vitro transcription and, like wild-type SoxR, were inactivated by chemical reduction. However, in vivo spectroscopy showed that even without oxidative stress, the three SoxR(c) proteins failed to accumulate with reduced [2Fe-2S] (≤4% compared to ≤40% for wild type). One SoxR(c) protein had a redox potential 65 mV lower than wild type, consistent with its accumulation in the oxidized (activated) form in vivo. These results link in vitro and in vivo approaches showing novel redox regulation that couples an iron-sulfur oxidation state to promoter activation

John B. Little Center Annual Symposium

The Annual Symposium of the John B. Little Center for Radiation Sciences and Environmental Health at the Harvard School of Public Health seeks to educate radiobiologists and biomedical scientists in related areas on the leading research related to the effects of ionizing radiation and related environmental agents in biological systems. This effort seeks to further the training of individuals in this field, and to foment productive interactions and collaborations among scientists at Harvard and with other institutions. The Symposium attracts world-class scientists as speakers, and a broad cross-section of attendees from academic, government, and industrial research centers, as well as editorial staff from leading scientific publications. In order to maintain this quality, funding to support the travel and local expenses of invited speakers is sought, along with funds to allow use of appropriate conference facilities

DNA binding shifts the redox potential of the transcription factor SoxR

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

Accelerated search kinetics mediated by redox reactions of DNA repair enzymes

A Charge Transport (CT) mechanism has been proposed in several papers (e.g., Yavin, et al. PNAS, v102 3546 (2005)) to explain the localization of Base Excision Repair (BER) enzymes to lesions on DNA. The CT mechanism relies on redox reactions of iron-sulfur cofactors that modify the enzyme's binding affinity. These redox reactions are mediated by the DNA strand and involve the exchange of electrons between BER enzymes along DNA. We propose a mathematical model that incorporates enzyme binding/unbinding, electron transport, and enzyme diffusion along DNA. Analysis of our model within a range of parameter values suggests that the redox reactions can increase desorption of BER enzymes not already bound to their targets, allowing the enzymes to be recycled, thus accelerating the overall search process. This acceleration mechanism is most effective when enzyme copy numbers and enzyme diffusivity along the DNA are small. Under such conditions, we find that CT BER enzymes find their targets more quickly than simple "passive" enzymes that simply attach to the DNA without desorbing.Comment: 17 pages, 8 figure