Binding of Promoter DNA to SoxR Protein Decreases the Reduction Potential of the [2Fe–2S] Cluster

Abstract

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.

NADPH‐related processes studied with a SoxR‐based biosensor in Escherichia coli

“…The RsxABCDGE complex belongs to the family of Rnf complexes, enzymes that drive the endergonic reduction of ferredoxin ( E 0 ′ = −420 mV) with NAD(P)H ( E 0 ′ = −320 mV) by the proton‐ or sodium‐motive force via import of H + or Na + , or, in the reverse reaction, the exergonic reduction of NAD(P) + with reduced ferredoxin coupled to the export of H + or Na + (Biegel, Schmidt, Gonzalez, & Müller, ). The redox potential of SoxR in its DNA‐free and its DNA‐bound state was reported to be −293 and −320 mV (Kobayashi, Fujikawa, & Kozawa, ), respectively, that is, in the same range as the one of NAD(P)H. Consumption of proton‐ or sodium‐motive force via the Rsx complex to drive reduction of SoxR by NADPH allows the cell to keep most SoxR in the reduced state in the absence of inducing conditions. In fact, it was reported that in wild‐type cells overproducing SoxR almost all of the protein was present in the reduced state, but in rsxC and rseC mutants only about 60% and 56% (Koo et al, ).…”

“…The RsxABCDGE complex belongs to the family of Rnf complexes, enzymes that drive the endergonic reduction of ferredoxin (E 0 ′ = −420 mV) with NAD(P)H (E 0 ′ = −320 mV) by the proton-or sodium-motive force via import of H + or Na + , or, in the reverse reaction, the exergonic reduction of NAD(P) + with reduced ferredoxin coupled to the export of H + or Na + (Biegel, Schmidt, Gonzalez, & Müller, 2011). The redox potential of SoxR in its DNA-free and its DNA-bound state was reported to be −293 and −320 mV (Kobayashi, Fujikawa, & Kozawa, 2015), respectively, that is, in the same range as the one of NAD(P)H. Consumption of the protein was present in the reduced state, but in rsxC and rseC mutants only about 60% and 56% (Koo et al, 2003). The final steps of electron transfer to SoxR are unknown at present.…”

Transcription Factors That Defend Bacteria Against Reactive Oxygen Species

“…Indeed, some genes within the E. coli SoxR regulon provide mechanisms to restore NADPH pools (58, 79). However, measurements indicate that the shift in NADPH redox status [to −.314 V, calculated from (57)] is not enough to explain the near-total oxidation of SoxR [Eo’ = −.293 V (53)], and mutations that impair cellular NADPH formation do not activate SoxR (34). Thus although NADPH depletion might augment the inducing effects of redox agents, by itself it is probably not sufficient to turn on the system.…”

13. Reactivity of iron-sulfur clusters with nitric oxide

“…Whether DNA-binding has an important effect on the redox properties of the cluster is controversial. Electrochemical measurements on DNA-modified electrodes revealed a dramatic shift in the reduction potential to +200 mV for SoxR bound to its cognate DNA [193], while more recent solution equilibrium experiments indicated a small decrease in the potential for the DNA-bound form compared to free in solution [194].…”