Abstract
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.
Highlights
Fer reactions are well established, but new roles have recently been found for these metallocenters (Beinert, 1990; Johnson, 1994). [4Fe-4S] clusters are involved directly in hydrolytic catalysis by dehydratases such as aconitase (Klausner et al, 1993) and dihydroxy-acid dehydratase. (Flint et al, 1993a)
A recent report proposed that the [4Fe-4S] center of endonuclease III of Escherichia coli is involved in DNA recognition (Thayer et al, 1995)
Regulatory roles for FeS centers have been described for the mammalian iron response protein (IRP; Klausner et al (1993)) and are implicated for two bacterial regulators, Fnr protein (Khoroshilova et al, 1995) and SoxR protein (Hidalgo and Demple (1994); see below)
Summary
Fer reactions are well established, but new roles have recently been found for these metallocenters (Beinert, 1990; Johnson, 1994). [4Fe-4S] clusters are involved directly in hydrolytic catalysis by dehydratases such as aconitase (Klausner et al, 1993) and dihydroxy-acid dehydratase. (Flint et al, 1993a). Regulatory roles for FeS centers have been described for the mammalian iron response protein (IRP; Klausner et al (1993)) and are implicated for two bacterial regulators, Fnr protein (Khoroshilova et al, 1995) and SoxR protein (Hidalgo and Demple (1994); see below). Some tetranuclear [4Fe-4S] clusters appear to be very sensitive to damage via oxidation Both E. coli dihydroxy acid-dehydratase (Flint et al, 1993b) and Bacillus subtilis phosphoribosyl diphosphate 5-amidotransferase (Bernlohr and Switzer, 1981; Grandoni et al, 1989) lose their respective clusters in the presence of hyperbaric oxygen in vivo and in vitro. Cluster assembly/disassembly seems to be employed deliberately as a regulatory mechanism in mammalian IRP In the latter case, iron limitation (Klausner et al, 1993) or oxidative damage (Drapier et al, 1993) lead to formation of the apoprotein in vivo; apo-IRP binds certain mRNAs to effect post-transcriptional control, which is lost when the metalloprotein is regenerated. The SoxR FeS centers are not required for DNA binding, but are essential for promoting open-complex formation by RNA polymerase and triggering expression of the soxS
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