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 superoxide-generating agents or nitric oxide as a powerful transcription activator of the soxS gene, whose product activates approximately 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 of E. 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 K. 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.
Hidalgo
E
E
Department of Molecular and Cellular Toxicology, Harvard University, School of Public Health, Boston, Massachusetts 02115, USA.
Bollinger
J M
JM
Bradley
T M
TM
Walsh
C T
CT
Demple
B
B
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003300:Copper / Q000737:chemistry; D017931:DNA Primers; D004278:DNA, Superhelical; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D010618:Phenanthrolines / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013698:Templates, Genetic; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D017931:DNA Primers; D004278:DNA, Superhelical; D007506:Iron-Sulfur Proteins; D010618:Phenanthrolines; D013438:Sulfhydryl Compounds; D014157:Transcription Factors; C070724:SoxR protein, Bacteria; C090893:5-phenyl-1,10-phenanthroline; D003300:Copper; D007501:Iron