The transcriptional activation of SoxR in Escherichia coli occurs through direct modification of the [2Fe-2S] by nitric oxide (NO) to form a dinitrosyl iron complex (DNIC). Here, we investigated the reactions of NO with [2Fe-2S] clusters of SoxR; the results were compared with those obtained for spinach ferredoxin (Fd). UV-visible absorption and ESR spectra of SoxR upon treatment with an NO donor showed the formation of DNIC of SoxR efficiently, whereas those of Fd exhibited small changes. Upon pulse radiolysis of a deaerated solution of SoxR in the presence of sodium nitrite, a biphasic change in absorption, consisting of a faster phase and a slower phase, was observed. The slower phase fraction was increased with increases in the [NO]/[SoxR] molar ratio, reaching a plateau at ∼2 equivalents of NO. On the basis of these results, we propose that the faster phase corresponds to the reaction of the first NO molecule with [2Fe-2S] of SoxR, followed by the reaction of the second NO molecule. In the reaction of NO with Fd, no slower phase was observed. These results suggest that the reaction of the second equivalent of NO is an important process for the formation of DNIC.

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The [2Fe-2S] transcription factor, SoxR, a member of the MerR family, functions as a bacterial sensor of oxidative stress in Escherichia coli. SoxR is activated by reversible one-electron oxidation of the [2Fe-2S] cluster and enhances the production of various antioxidant proteins through the SoxRS regulon. Ultraviolet resonance Raman (UVRR) spectroscopic analysis of SoxR revealed conformational changes upon reduction of the [2Fe-2S] cluster in the absence and presence of promoter oligonucleotide. UVRR spectra reflected the environmental or structural changes of Trp following reduction. Notably, the environment around Trp91 contacting the [2Fe-2S] cluster was altered to become more hydrophilic, whereas that around Trp98 exhibited a small change to become more hydrophobic. In addition, changes in cation-π interactions between the [2Fe-2S] cluster and Trp91 were suggested. On the other hand, the environment around Tyr was barely affected by the [2Fe-2S] reduction. Binding of the promoter oligonucleotide triggered changes in Tyr located in the DNA-binding domain, but not Trp. Furthermore, conformational changes induced upon reduction of DNA-bound SoxR were not significantly different from those of DNA-free SoxR.