BINUCLEAR [2FE-2S] CLUSTERS IN THE ESCHERICHIA-COLI SOXR PROTEIN AND ROLE OF THE METAL CENTERS IN TRANSCRIPTION

SoxR protein of Escherichia coli is activated by super oxide-generating agents or nitric oxide as a powerful transcription activator of the soxS gene, whose product activates similar to 10 other promoters, SoxR contains non heme iron essential for abortive initiation of transcription in vitro, Here we show that this metal dependence extends to full-length transcription in vitro, In the presence off. coli sigma(70) RNA polymerase, iron-containing SoxR mediates open complex formation at the soxS promoter, as determined using footprinting with Cu-5-phenyl-1,10-phenanthroline. We investigated the nature of the SoxR iron center by chemical analyses and electron paramagnetic resonance spectroscopy. Dithionite-reduced Fe-SoxR exhibited an almost axial paramagnetic signature with g values of 2.01 and 1.93 observable up to 100 g. These features, together with quantitation of spin, iron, and S2-, and hydrodynamic evidence that SoxR is a homodimer in solution, indicate that (SoxR)(2) contains two [2Fe-2S] clusters. Treatment of Fe-SoxR with high concentrations of dithiothreitol caused subtle changes in the visible absorption spectrum and blocked transcriptional activity without generating reduced [2Fe-2S] centers, but was also associated with the loss of iron from the protein, However, lowering the thiol concentration by dilution allowed spontaneous regeneration of active Fe-SoxR.