Fe - SoxR GSH 1 ( min ) C 0 10 30 m bla 11111 _ 1 2 3 4 5

SoxR is a transcription factor that governs a global defense against the oxidative stress caused by nitric oxide or excess superoxide in Escherichia coli. SoxR is a homodimer containing a pair of [2Fe-2S] clusters essential for its transcriptional activity, and changes in the stability of these metal centers could contribute to the activation or inactivation of SoxR in vivo. Herein we show that reduced glutathione (GSH) in aerobic solution disrupts the SoxR [2Fe-2S] clusters, releasing Fe from the protein and eliminating SoxR transcriptional activity. This disassembly process evidently involves oxygen-derived free radicals. The loss of [2Fe-2S] clusters does not occur in anaerobic solution and is blocked in aerobic solution by the addition of superoxide dismutase and catalase. Although H202 or xanthine oxidase and hypoxanthine (to generate superoxide) were insufficient on their own to cause [2Fe-2S] cluster loss, they did accelerate the rate of disassembly after GSH addition. Oxidized GSH alone was ineffective in disrupting the clusters, but the rate of [2Fe-2S] cluster disassembly was maximal when reduced and oxidized GSH were present at a ratio of -1:3, which suggests the critical involvement of a GSH-based free radical in the disassembly process. Such a reaction might occur in vivo: we found that the induction by paraquat of SoxR-dependent soxS transcription was much higher in a GSH-deficient E. coli strain than in its GSH-containing parent. The results imply that GSH may play a significant role during the deactivation process of SoxR in vivo. Ironically, superoxide production seems both to activate SoxR and, in the GSH-dependent disassembly process, to switch off this transcription factor. Most known proteins that contain iron-sulfur clusters employ these metal centers for electron-transfer, dehydratase reactions, or for structural purposes (1). The gene-regulatory functions of iron-sulfur proteins have been described only recently (2). One clear example for such a regulatory role occurs in the Escherichia coli protein SoxR (3), which is a homodimer of 17-kDa subunits containing a pair of [2Fe-2S] clusters (4, 5). The metal centers appear to be anchored by a cluster of four cysteine residues located near the C terminus of the SoxR polypeptide (T. Bradley, E. Hidalgo, H.D., and B.D., unpublished data). SoxR in vivo governs the induction of more than 12 genes in response to superoxide stress or nitric oxide (6). Preexisting SoxR protein is rapidly converted (<10 min) by these agents into a potent transcriptional activator of the soxS gene (7, 8), whose protein product then elevates transcription of many antioxidant and other defense genes (9-12). After withdrawal of the activating signal, induced soxS transcription decays with a half-life of c40 min (13). In vitro, SoxR specifically binds to the soxS promoter region and activates transcription by or70-containing RNA polymerase (3-6). However, apo-SoxR (lacking the [2Fe-2S] clusters) binds the soxS promoter with unchanged affinity but does not stimulate transcription (3, 4). Footprinting studies indicated specific structural distortions caused by Fe-SoxR but not by apo-SoxR (3, 4), which suggests that SoxR might stimulate transcription by an allosteric mechanism analogous to that proposed for the homologous MerR protein (14, 15). The foregoing suggests that the SoxR [2Fe-2S] clusters may constitute the actual sensor of free-radical-induced stress (3-6). We have therefore investigated the assembly and disassembly processes of the [2Fe-2S] clusters in SoxR under physiological conditions. The [2Fe-2S] clusters can assemble spontaneously in SoxR, but this process is strongly accelerated by the Azotobacter vinelandii NifS protein to produce fully active Fe-SoxR (16). To our knowledge, the factors affecting disassembly of the SoxR [2Fe-2S] clusters have not been studied systematically. However, apo-SoxR is obtained when 2-mercaptoethanol is included in the protein purification buffers (3). In examining the effects of other thiols, we have now found unexpectedly that reduced glutathione (GSH) in aerobic solution effectively disrupts the [2Fe-2S] clusters to inactivate SoxR in vitro. Our studies suggest a role for GSHbased free radicals in this disassembly process, and we show that GSH could also play such an inactivating role in vivo. MATERIALS AND METHODS SoxR Protein. Purification of SoxR protein from E. coli containing the expression plasmid pKOXR was performed as described (3), except that the heparin-agarose columns (Life Technologies, Grand Island, NY) loaded with SoxR protein were extensively washed with 350 mM NaCl/50 mM Hepes-NaOH, pH 7.6, prior to elution of SoxR. The purity of SoxR protein in these experiments was >90%, as judged by staining of SDS/polyacrylamide gels. Transcriptional Activity of SoxR. SoxR activity was assayed by in vitro transcription using plasmid pBD100 as the template. The SoxR-dependent soxS transcript and the SoxRindependent bla transcript were quantified by primerextension analysis (4). SoxR activity in vivo was monitored using a soxS'::lacZ operon fusion present in single copy. The A)D (soxS'::lacZ) fusion was introduced into strains JTG10 (GSH-deficient) and AB1157 (parent of JTG10) (17) by isolating A lysogens of these strains (6). The expression of soxS'::lacZ was measured by assaying ,B-galactosidase activity (7). Spectroscopy. A UV/visible spectrophotometer (PerkinElmer Lambda 3A) was used to measure the absorbance spectrum of SoxR protein and to monitor the absorbance change as a function of time. The X-band EPR spectra of SoxR were obtained using a Bruker model ESP-300 equipped with an Oxford Instruments 910 continuous flow cryostat (courtesy of J. Stubbe's laboratory, Massachusetts Institute of TechnolAbbreviations: Apo-SoxR, SoxR protein without [2Fe-2S] clusters; Fe-SoxR, SoxR protein that contains [2Fe-2S] clusters; GSH, reduced glutathione; GSSG, oxidized glutathione; SOD, superoxide dismutase. *To whom reprint requests should be addressed. e-mail: demple@ mbcrr.harvard.edu. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.