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 (SoxRc) proteins. Three SoxRc 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 SoxRc proteins failed to accumulate with reduced [2Fe-2S] (< or = 4% compared to > or = 40% for wild type). One SoxRc 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.