Activation of SoxR-dependent transcription in vitro by noncatalytic or NifS-mediated assembly of [2Fe-2S] clusters into apo-SoxR.
SoxR is a transcriptional activator that senses superoxide and nitric oxide stress in Escherichia coli. The active protein isolated from E. coli contains a pair of [2Fe-2S] clusters per SoxR dimer. We previously demonstrated that the iron-free protein (apo-SoxR), isolated during purification in thiol-containing buffers, binds soxS promoter DNA with an affinity equal to that of the metalloprotein (Fe-SoxR), but lacks significant ability to activate transcription in vitro. Here we demonstrate the reversibility of this process: the full transcriptional activity of SoxR can be restored by in vitro assembly of iron-sulfur clusters into the apoprotein. Two methods were used to synthesize the metallocenters of SoxR: (i) nonenzymatic, in which apo-SoxR, incubated in the presence of iron, inorganic sulfide, and a reducing agent, regained full transcriptional activity in 5-6 h; (ii) enzymatic, in which NifS protein of Azotobacter vinelandii regenerated active Fe-SoxR in as little as 2 min. Analysis by electron paramagnetic resonance spectroscopy indicated that binuclear [2Fe-2S] clusters were restored by both the enzymatic and nonenzymatic reconstitutions. A mutant SoxR protein missing one of its four cysteine residues failed to undergo either transcriptional activation or the formation of [2Fe-2S] centers, even in the presence of NifS. Thus, only the presence of an iron-sulfur center is required to restore transcriptional activity to apo-SoxR. Moreover, the catalytic generation of [2Fe-2S] centers extends the known specificity of this enzyme beyond that already shown for [4Fe-4S] centers. Catalytic generation of [2Fe-2S]-containing SoxR could allow for rapid activation of this transcription factor in vivo.
Hidalgo
E
E
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Demple
B
B
D001059:Apoproteins / Q000378:metabolism*; D016948:Azotobacter vinelandii / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D007501:Iron / Q000032:analysis; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis / Q000378:metabolism*; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation; D011401:Promoter Regions, Genetic; D013455:Sulfur / Q000032:analysis; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D001059:Apoproteins; D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C080001:nifS protein, Bacteria; C070724:SoxR protein, Bacteria; C070725:SoxS protein, E coli; D013455:Sulfur; D007501:Iron