Thiol-mediated disassembly and reassembly of [2Fe-2S] clusters in the redox-regulated transcription factor SoxR.
SoxR, a transcription factor containing [2Fe-2S] clusters, governs the cellular response to oxidative stress in Escherichia coli. The oxidation state of the iron-sulfur clusters regulates the SoxR transcriptional activity. When the reduced iron-sulfur clusters become oxidized ([2Fe-2S]2+ state), SoxR is activated to stimulate transcription of the soxS gene, whose product in turn switches on a group of genes encoding various proteins that defend against oxidative stress and antibiotics. A previous study showed that the oxidized [2Fe-2S] clusters of SoxR are destroyed by a free-radical-dependent process in vitro during aerobic exposure to the biological thiol glutathione. Here, we show that different thiols have differing effects on the SoxR [2Fe-2S] clusters. Like reduced glutathione, N-acetyl-L-cysteine, L-cysteine methyl ester, and L-cysteine ethyl ester disrupted the SoxR [2Fe-2S] clusters in aerobic solution. This disruption was blocked by L-cysteine, which was effective at concentrations 100-fold lower (1-10 microM) than the disrupting thiols (1 mM). In view of a previous observation that superoxide dismutase and catalase block the disruption process, this result suggests that L-cysteine may quench reactive SoxR or thiol intermediates involved in the cluster disruption reaction, the detailed mechanism of which remains unknown. In contrast, bifunctional thiols such as dithiothreitol or dithioerythritol promoted the aerobic assembly of the functional [2Fe-2S] clusters into apo-SoxR in the presence of Fe2+ and inorganic sulfide. The dithiol protein thioredoxin-A of E. coli acted catalytically in vitro in the presence of thioredoxin reductase and NADPH to promote [2Fe-2S] cluster assembly into apo-SoxR. The regulatory activity of SoxR in vivo, assessed by monitoring the paraquat-mediated induction of a soxS'::lacZ reporter fusion, was significantly lower in a strain lacking both thioredoxin-A and glutathione reductase, which maintains reduced glutaredoxins. Thus, cellular monothiols and dithiol proteins may contribute to SoxR regulation by affecting the disassembly and reassembly of the [2Fe-2S] clusters.
Ding
H
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Department of Cancer Cell Biology, School of Public Health, Harvard University, Boston, Massachusetts 02115-6021, USA.
Demple
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B
D001059:Apoproteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D003545:Cysteine / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D005978:Glutathione / Q000494:pharmacology; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D013879:Thioredoxins / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*
D001059:Apoproteins; D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D013438:Sulfhydryl Compounds; D014157:Transcription Factors; C070724:SoxR protein, Bacteria; D013879:Thioredoxins; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol