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Originally published In Press as doi:10.1074/jbc.M603968200 on June 8, 2006

J. Biol. Chem., Vol. 281, Issue 33, 23567-23578, August 18, 2006
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Biochemical Characterization of the Structural Zn2+ Site in the Bacillus subtilis Peroxide Sensor PerR*Formula

Jin-Won Lee and John D. Helmann1

From the Department of Microbiology, Cornell University, Ithaca, New York 14853-8101

Received for publication, April 25, 2006 , and in revised form, June 6, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In Bacillus subtilis most peroxide-inducible oxidative stress genes are regulated by a metal-dependent repressor, PerR. PerR is a dimeric, Zn2+-containing metalloprotein with a regulatory metal-binding site that binds Fe2+ (PerR:Zn,Fe) or Mn2+ (PerR: Zn,Mn). Reaction of PerR:Zn,Fe with low levels of hydrogen peroxide (H2O2) leads to oxidation of two His residues thereby leading to derepression. When bound to Mn2+, the resulting PerR:Zn,Mn is much less sensitive to oxidative inactivation. Here we demonstrate that the structural Zn2+ is coordinated in a highly stable, intrasubunit Cys4:Zn2+ site. Oxidation of this Cys4:Zn2+ site by H2O2 leads to the formation of intrasubunit disulfide bonds. The rate of oxidation is too slow to account for induction of the peroxide stress response by micromolar levels of H2O2 but could contribute to induction under severe oxidative stress conditions. In vivo studies demonstrated that inactivation of PerR:Zn,Mn required 10 mM H2O2, a level at least 1000 times greater than that needed for inactivation of PerR:Zn,Fe. Surprisingly even under these severe oxidation conditions there was little if any detectable oxidation of cysteine residues in vivo: derepression was correlated with oxidation of the regulatory site. Because oxidation at this site required bound Fe2+ in vitro, we suggest that treatment of cells with 10 mM H2O2 released sufficient Fe2+ into the cytosol to effect a transition of PerR from the PerR:Zn,Mn form to the peroxide-sensitive PerR: Zn,Fe form. This model is supported by metal ion affinity measurements demonstrating that PerR bound Fe2+ with higher affinity than Mn2+.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacillus subtilis PerR is a metal-dependent repressor that regulates the peroxide-inducible expression of oxidative stress genes. The PerR regulon includes the major vegetative catalase (KatA), alkyl hydroperoxide reductase (AhpCF), enzymes of heme biosynthesis (HemAXCDBL), a zinc uptake P-type ATPase (ZosA), a Dps-like protein (MrgA), and both the PerR and Fur metalloregulatory proteins. Expression of these genes is elevated in a perR null mutant, and PerR binds directly to operator sites overlapping the promoters for these operons, establishing PerR as a direct repressor of transcription (1–3).

PerR is a member of the Fur family of metal-dependent regulators, and many Fur family proteins contain one Zn2+ ion per monomer in addition to a regulatory metal ion. The x-ray absorption spectroscopy of Escherichia coli Fur (FurEC) revealed Zn2+ bound in a tetrahedral environment composed of two S and two N/O donor ligands (4). Subsequent studies using chemical modification and mass spectroscopy assigned Cys92 and Cys95 as Zn2+ ligands (5), a conclusion supported by site-directed mutagenesis studies (6). To date, the only structure available for a protein from the Fur superfamily is Pseudomonas aeruginosa Fur (FurPA) that was crystallized in the presence of Zn2+ (7). FurPA contains two metal-binding sites assigned as a high affinity Zn2+-binding site and low affinity Fe2+-binding site, respectively, by accompanying spectroscopy. No cysteine residues were involved in metal coordination in FurPA, and there is still considerable uncertainty surrounding the functional role of the metal-binding sites. For example, multiple mutants in the corresponding residues in Bradyrhizobium japonicum Fur failed to abrogate metal sensing (8). Moreover putative Zn2+ ligands in FurPA correspond to residues in PerR required for binding the regulatory metal ion (Fe2+ or Mn2+) but not the structurally important Zn2+ (9).

Most PerR-regulated genes are derepressed when cells are exposed to low levels of H2O2 (<10 µM), by limitation for metal ions or by transition into stationary phase during aerobic growth, which leads to an increase in the endogenous production of H2O2 (2, 10, 11). Physiological studies have revealed that PerR-regulated genes are strongly repressed in medium supplemented with manganese (and lacking added iron) and that, under these growth conditions, the ability of H2O2 to induce expression is greatly reduced. In contrast, in iron-containing growth medium PerR repression is rapidly relieved upon exposure to H2O2 (2).

These results led to a model in which PerR could bind either Mn2+ or Fe2+ as corepressor to generate two forms of PerR that differ in their sensitivity to reactive oxygen species. Biochemical analyses of the reconstituted PerR:Zn,Fe and PerR:Zn,Mn forms of the repressor provide an explanation for this difference: the high sensitivity of PerR:Zn,Fe results from the regio-specific oxidation of two His residues directly coordinated to the activating ferrous ion (9). Thus, in contrast with most peroxide sensors characterized to date (12–14), PerR does not appear to use thiol disulfide chemistry to sense oxidative stress despite the presence of four conserved cysteine residues in the protein sequence. Previous results from homology modeling of PerR on the FurPA structure, combined with site-directed mutagenesis, support a model in which these four residues form a high affinity Cys4:Zn2+ structural site (9).

Although the Cys4:Zn2+ site is not involved in sensing low levels of peroxides, this does not rule out a possible role under conditions of severe oxidative stress. Indeed it has been previously proposed that other PerR homologs sense H2O2 by Cys-based redox reactions as inferred from studies done with 5–10 mM H2O2 (15, 16). Analysis of E. coli HSP33 indicates that cells may contain systems that respond specifically to severe oxidative stress conditions. HSP33 is a redox-regulated protein chaperone that contains a Cys4:Zn2+ site that is only oxidized in the presence of high levels of peroxides (e.g. 4 mM H2O2) and elevated temperatures (e.g. 43 °C) (17).

In the present study, we investigated oxidation of the Cys4:Zn2+ site and its possible role in peroxide sensing in vivo. By monitoring Zn2+ release, we found a rate constant for the in vitro oxidation of this Cys4:Zn2+ site by H2O2 of ~0.05 M–1 s–1. Oxidation led to the formation of intramolecular disulfide bonds between cysteine residues in the two CXXC motifs, and the resulting oxidized species could be resolved by SDS-PAGE. The in vitro sensitivity of the Cys4:Zn2+ site to oxidation suggests that this site could mediate inactivation of the PerR: Zn,Mn form of the repressor under severe oxidative stress conditions. However, even when treated with high levels of H2O2 there was little if any detectable cysteine oxidation in vivo, and derepression was correlated with oxidation of the regulatory site. Because PerR bound Fe2+ with higher affinity than Mn2+, we propose a model in which high levels of H2O2 release sufficient Fe2+ into the cytosol to effect a transition of PerR from the PerR:Zn,Mn form to the peroxide-sensitive PerR:Zn,Fe form.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents—All of the chemicals used were reagent grade. The disodium salt of 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS)2 was purchased from Molecular Probes (Eugene, OR). Anti-FLAG M2-alkaline phosphatase, anti-FLAG from rabbit, anti-FLAG M2 affinity gel, anti-rabbit IgG-alkaline phosphatase antibody produced in goat, proteomics grade trypsin, 4-(2-pyridylazo)resorcinol (PAR), diamide, iodoacetamide, catalase (18,400 units/mg of protein), H2O2 (30%, w/w), and trichloroacetic acid solution were purchased from Sigma. Protease inhibitor mixture tablets (Complete Mini, EDTA-free) were purchased from Roche Diagnostics. Chelex-100 resin and Micro Bio-Spin 6 chromatography columns were purchased from Bio-Rad. (NH4)2Fe(SO4)2 ·6H2O (99.997%), MnCl2 ·4H2O (99.99%), and ZnSO4 ·7H2O (99.999%) were purchased from Aldrich. [{alpha}-32P]dATP and [{gamma}-32P]dATP were purchased from PerkinElmer Life Sciences.

Media and Growth—E. coli and B. subtilis cells were grown at 37 °C in Luria-Bertani (LB) media with appropriate antibiotics. For metal-restricted growth of B. subtilis, 10 µM FeCl3 or 10 µM MnCl2 was added to defined MOPS-buffered minimal medium (MM) containing 40 mM MOPS (adjusted to pH 7.4 with NaOH), 2 mM potassium phosphate buffer (pH 7.0), 20 g/liter glucose, 2 g/liter (NH4)2SO4, 0.2 g/liter MgSO4 ·7H2O, 1 g/liter sodium citrate ·2H2O, 1 g/liter potassium glutamate, 10 mg/liter tryptophan, 3 nM (NH4)6Mo7O24, 400 nM H3BO3, 30 nM CoCl2, 10 nM CuSO4, 10 nM ZnSO4, and 80 nM MnCl2.

Construction of PerR-overexpressing E. coli Strains—The perR open reading frame was PCR-amplified using forward primer 5'-GGTGCATGACCCATGGCTGCACAT-3' and reverse primer 5'-TGACCGTTTCGTGGGATCCGCTTA-3', creating NcoI and BamHI sites, respectively (underlined), with B. subtilis CU1065 chromosomal DNA as template. For the construction of the PerR-FLAG-overexpressing E. coli strain, forward primer 5'-AAGAGAGGTGCATGACCCATGGCTGCA-3' and reverse primer 5'-GATGACCTCGGATCCACCGGAATTAGC-3', creating NcoI and BamHI sites, respectively (underlined), were used with HB9612 genomic DNA (see below) as template. These PCR fragments were cloned into the NcoI and BamHI sites of expression vector pET16b (Novagen) resulting in plasmids named pJL041 and pJL069, respectively. These plasmids were introduced into E. coli BL21 (DE3) pLysS resulting in PerR- and PerR-FLAG-overexpressing E. coli strains named HE9501 and HE9526, respectively.

Purification of PerR after Overexpression in E. coli—E. coli cells were harvested from the 10 ml of overnight culture in LB medium and inoculated into 1 liter of fresh LB medium containing 0.4% glucose, 34 mg/liter chloramphenicol, and 100 mg/liter ampicillin. Isopropyl 1-thio-beta-D-galactopyranoside was added to final concentrations of 1 mM at A600 of ~0.6, and the cells were allowed to grow for an additional 2 h. Cells were harvested by centrifugation, and the cell pellets were resuspended in 50 ml of buffer A (20 mM Tris ·HCl (pH 8.0), 100 mM NaCl, and 5% (v/v) glycerol) containing 10 mM EDTA. After lysis by sonication, cell debris were removed by centrifugation, and the resulting supernatant was loaded onto a 20-ml heparin-Sepharose column. After application of a linear gradient of 0.1–1 M NaCl, PerR-containing fractions were pooled and buffer-exchanged to buffer A containing 10 mM EDTA using a YM-10 ultrafiltration membrane under the pressure of N2 gas. The resultant PerR was applied to a Mono Q column using an FPLC system (GE Healthcare) and separated using a linear gradient of 0.1–1 M NaCl. The PerR-containing factions were further purified on a Superdex 200 HiLoad 16/60 column (GE Healthcare) equilibrated with Chelex-100-treated buffer A without EDTA. Purification of PerR was also carried out as above in the absence of EDTA or in the presence of DTT. Active PerR (purified in the presence of 10 mM EDTA) contained ~1.6 mol of zinc/dimer as judged by inductively coupled plasma MS and could be reconstituted with Mn2+ to give protein with ~60% active molecules in a DNA binding assay. PerR purified in the absence of EDTA was also dimeric and contained stoichiometric zinc (~1.9 mol of zinc and ~0.1 mol of iron/dimer) but was largely inactive due to oxidation of the regulatory metal site as reported previously (9).

Determination of Protein Concentration—The concentration of purified PerR was determined by measuring A277 nm using the calculated value of {epsilon}277 nm = 10,400 M–1 cm–1. The concentration of proteins in the cell crude extract was determined by Bio-Rad protein assay and/or Bio-Rad DC protein assay (for samples containing SDS) using bovine serum albumin as standard.

Construction of FLAG-tagged PerR in B. subtilis—A partial perR gene corresponding to about 350 bp from the 3'-end of perR excluding the stop codon was amplified by PCR using forward primer 5'-GCAGGTACCGCTCTGGAAGGGAAATTTCCTAACATGAGC-3' and reverse primer 5'-GCACGGCCGATGATTTTCTTTTTTCGAACACTCTTGGCAGAC-3' with B. subtilis CU1065 chromosomal DNA as template. These PCR fragments were cloned into the KpnI and EagI sites of epitope protein-tagging integration vector pMUTIN-FLAG (18) resulting in plasmid pJL062. This plasmid was passed through E. coli JM105 (recA), and the resulting plasmids were used for transformation of B. subtilis CU1065. The generation of perR-FLAG, upon integration of pJL062 into perR, was verified by PCR followed by sequencing, and the resulting strain was named HB9612. The perR-FLAG open reading frame was PCR-amplified using forward primer 5'-CCATGTAGCCGAAAAGCTTCAAACCC-3' and reverse primer 5'-GTTTCCACCGGAATTCGCTTGCATGG-3' with HB9612 genomic DNA as template. These PCR fragments were cloned into the HindIII and EcoRI sites of pDG1730 resulting in plasmid pJL070. The ScaI digest of pJL070 was used for transformation of HB2080 (CU1065 perR::kan amyE::cat pheA1) or HB9703 (CU1065 perR::tet) to generate transformants containing perR-FLAG in amyE locus designated HB9620 or HB9735, respectively.

Construction of Reporter Fusion Strains—SPbeta phages from HB1122 (19), HB0618 (20), and HB8010 (21) were used for the transduction of CU1065 to give strains containing reporter fusion for mrgA-cat-lacZ, feuA-cat-lacZ, and yciC-cat-lacZ, respectively. The HB9738 strain was made by transduction of HB9735 with SPbeta phage from HB1122.

SDS-PAGE Sample Preparation and Analysis—For Fig. 1A, purified PerR protein was incubated with 10 mM DTT, 10 mM H2O2, 10 mM diamide, or 10 mM EDTA at 80 °C for 5 min in SDS sample buffer containing 2% SDS and resolved by non-reducing SDS-PAGE using a Tris-Tricine buffer system (22). For Fig. 1B, purified PerR protein was treated with 1 mM DTT, 10 mM H2O2, or 10 mM diamide at room temperature for 30 min in buffer A, and residual reagents were removed by precipitation and washing with 10% trichloroacetic acid. Recovered samples were modified by 50 mM AMS in the presence of 10 mM EDTA and 2% SDS and separated by non-reducing SDS-PAGE. For Fig. 4B, under anaerobic conditions 10 µM PerR (in 20 µl) was treated with 100 µM Fe2+ or Mn2+ for 5 min and then exposed to varying amounts of H2O2 for 10 min. PerR was recovered by precipitation with 20 µl of 20% trichloroacetic acid. After washing with 10% trichloroacetic acid, the pellets were resuspended in buffer A containing 2% SDS, 20 mM EDTA, and 50 mM AMS and incubated for 30 min in the dark. Then the samples were analyzed by non-reducing SDS-PAGE.

On-gel Zn2+ Detection—PerR protein (20 µg) treated with 10 mM DTT or 10 mM H2O2 was resolved on non-reducing SDS-PAGE gels. The SDS-PAGE gel was soaked in buffer A containing 500 µM PAR for 1 min, and subsequently 50 mM H2O2 was added to release Zn2+. Because the color development (light orange) was transient, a photograph of the gel was taken within 10 min.


Figure 1
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  		FIGURE 1.
Electrophoretic mobility of PerR on SDS-PAGE gel. A, purified PerR protein was incubated alone (lane 1), with 10 mM DTT (lane 2), with 10 mM H2O2 (lane 3), with 10 mM diamide (DA; lane 4), with 10 mM EDTA (lane 5), or with 10 mM DTT and 10 mM EDTA (lane 6) at 80 °C for 5 min in SDS sample buffer containing 2% SDS. B, purified PerR was not treated (lanes 1 and 2), treated with 1 mM DTT (lanes 3 and 4), treated with 10 mM H2O2 (lanes 5 and 6), or treated with 10 mM diamide (lanes 7 and 8) for 30 min at room temperature in 20 mM Tris (pH 8.0) containing 5% glycerol and 100 mM NaCl. After precipitation and washing with 10% trichloroacetic acid, samples were incubated with 50 mM AMS in the presence of 2% SDS and 10 mM EDTA to alkylate reduced Cys residues (lanes 2, 4, 6, and 8). Lane M is protein molecular mass standards. C, purified PerR protein (20 µg) was incubated alone, with 10 mM DTT or with 10 mM H2O2 in SDS sample buffer. After non-reducing SDS-PAGE, gels were stained with Coomassie for protein or PAR for Zn2+ ion.

 
Analysis of SDS-PAGE-fractionated Protein by MALDI-TOF MS—To alkylate reduced Cys residues, the SDS-PAGE gels were directly submerged and incubated in buffer A containing 50 mM EDTA and 50 mM iodoacetamide for 30 min with gentle rocking in the dark. Then the gels were stained with Coomassie Brilliant Blue R and subsequently destained. The stained bands were cut and washed three times by incubating with 100 µl of 50% acetonitrile, 50 mM ammonium bicarbonate for 15 min at 37 °C. The washed gel pieces were dried for 20 min in a Speed-Vac and rehydrated with 10–20 µl of trypsin solution (20 ng/µl trypsin in 9% acetonitrile solution containing 40 mM ammonium bicarbonate). The samples were digested for ~4 h (Fig. 2; partial digestion) or ~18 h (Fig. 3; full digestion) at 37 °C. A volume of 0.5 µl of sample was mixed with an equal volume of matrix (saturated solution of {alpha}-cyano-4-hydroxysuccinamic acid in 50% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid) on the target plate and allowed to air dry. MALDI mass spectra were recorded with an Applied Biosystems 4700 mass spectrometer. For the analysis of oxidation of PerR:Zn,Mn (Fig. 5), PerR:Zn,Mn (10 µM purified PerR + 100 µM MnCl2) was incubated with 0, 0.1, 1, 10, or 100 mM H2O2 for 10 min under anaerobic conditions, and the proteins were recovered by precipitation with 10% trichloroacetic acid, modified using 50 mM iodoacetamide, trypsinized, and analyzed using a Bruker BIFLEXIII mass spectrometer.


Figure 2
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  		FIGURE 2.
MALDI-TOF MS analysis of PerR bands with different mobilities during SDS-PAGE. A, tryptic fragments (T1–T16) of PerR. Calculated monoisotopic m/z values are shown in parentheses. T11 and T14 peptides contain 96CXXC99 and 136CXXC139 motifs, respectively. T5 and T11 peptides contain His37 and His91 residues, respectively, which are susceptible to metal-catalyzed oxidation. T5 and T7 contain Met35 and Met54 residues, respectively, which are susceptible to nonspecific oxidation. B, MALDI-TOF MS spectrum of the low mobility band (band 2 in Fig. 1A). T11 (containing 96CXXC99) corresponds to the peak of m/z = 2083.91, T14 (containing 136CXXC139) corresponds to the peak of m/z = 1369.66, and T14 + T15 corresponds to the peak of m/z = 1497.75. Note that each of these peaks is characterized by a loss of 2 m/z units from the calculated m/z value, indicative of disulfide bond formation between Cys residues. C, MALDI-TOF MS spectrum of high mobility band (band 1 in Fig. 1A). T11* (m/z = 2199.93), T14* (m/z = 1485.70), and T14 + T15*(m/z = 1613.79) all display a 114-Da mass increase from their calculated m/z values, indicative of two carbamidomethyl-modified Cys residues. Purified PerR was subjected to SDS-PAGE, and reduced Cys residues were alkylated by treating the whole SDS-PAGE gel with 50 mM iodoacetamide. Then band 1 and band 2 (see Fig. 1A, lane 1) were cut and analyzed by in-gel tryptic digestion followed by MALDI-TOF analysis. Peaks were assigned based on calculated and observed m/z values ({Delta}m/z < 0.1) except for T11, T14, and T14 + T15. Peaks containing an intrapeptide disulfide bond are shown with filled triangles, and peaks containing two carbamidomethyl-modified Cys residues are shown with open triangles and marked with asterisks.

 
Electrospray Mass Spectrometry Analysis—Electrospray ionization MS was performed using a Bruker Esquire-LC ion trap mass spectrometer (Bruker Daltonics, Breman, Germany). The ion spray voltage was set at 4 kV, the orifice voltage was set at 80 V, and the interface temperature was set at 80 °C. PerR (stored in buffer A) was buffer-exchanged with 10 mM ammonium bicarbonate (pH 8.0) by using a Micro Bio-Spin 6 chromatography column. For standard acidic denaturing conditions, 25% (v/v) methanol and 1% (v/v) acetic acid in 5 mM ammonium bicarbonate was used as vector solution. For the detection of Zn2+-bound PerR, 5% (v/v) methanol in 5 mM ammonium bicarbonate was used as vector solvent. Sample volumes of 20 µl were infused into the spectrometer using a syringe pump at a flow rate of 3 µl/min. Mass spectra were analyzed and manipulated using the Bruker Daltonics DataAnalysis program.

Measurement of Zn2+ Release by H2O2 Using PAR—Under our buffer conditions (20 mM Tris (pH 8.0), 100 mM NaCl, and 5% glycerol; 25 °C) the absorption maximum of Zn2+-PAR complex was observed at 494 nm, and the {epsilon}494 nm was measured to be 8.5 ± 0.25 x 104 M–1 cm–1 in the range of 0–6 µM Zn2+ (compared with {epsilon}500 nm = 6.6 ± 0.2 x 104 M–1 cm–1 for (PAR)2-Zn2+ at pH 7 (23)). 5 µM PerR (PerR: Zn) in buffer A (or buffer A containing 7 M urea) was treated with 0, 1, 10, or 100 mM H2O2 in the presence of 100 µM PAR at 25 °C, and released Zn2+ was measured by monitoring the Zn2+-PAR complex at 494 nm every 1 s for 30 min. Because the affinity of PAR to Zn2+ was high and the rate of association of the Zn2+ with PAR was much faster than that of Zn2+ release from PerR (reported value of second order k for forming Zn2+-PAR complex at pH 7 is ~2 x 107 M–1 s–1), all the released Zn2+ ions were assumed to be present as Zn2+-PAR complex. The data were fitted to an effective first order rate equation, A = Ao (1 – e–k[H2O2]t) + B, where A is absorbance at 494 nm, Ao is the amplitude of absorbance, k is the second order rate constant, t is time in seconds, and B is the initial absorbance.

beta-Galactosidase Assay—Overnight cultures of cells in MM containing 10 µM FeCl3 and 10 µM MnCl2 were washed with MM and inoculated by 25x dilution into fresh MM containing 10 µM FeCl3 or 10 µM MnCl2. At an A600 of ~0.6, aliquots of each culture were treated with H2O2 and further incubated for 30 min. Alternatively cells were treated with H2O2 for 10 min, residual H2O2 was removed by centrifugation, and cells were resuspended in fresh MM containing metal ions and further incubated for 30 min. beta-Galactosidase assays were performed as reported previously (2).

Analysis of in Vivo Oxidation Status of Cys Residues by AMS Modification—HB9738 cells grown overnight in MM containing 10 µM FeCl3 and 10 µM MnCl2 were washed twice with MM and diluted 25x in 55 ml of fresh LB medium, MM containing 10 µM FeCl3, or MM containing 10 µM MnCl2. At A600 = 0.6 aliquots of 10-ml culture were mixed with 1.1 ml of trichloroacetic acid at a given time point before and after H2O2 treatment. After centrifugation, pellets were resuspended in 10% trichloroacetic acid, split into two fractions, and washed with acetone. Each fraction was sonicated in 150 µl of 20 mM iodoacetamide or 20 mM AMS solution containing 200 mM Tris (pH 8.0), 5% glycerol, 1% SDS, and 20 mM EDTA. Alkylations were performed at room temperature in the dark for 1 h, and ~10 µl (corresponding to 30 µg of protein as assayed by DC protein assay (Bio-Rad)) of alkylated samples were resolved by SDS-PAGE. After electrophoresis, proteins were blotted to a polyvinylidene difluoride membrane and probed with polyclonal anti-FLAG antibody from rabbit and anti-rabbit antibody conjugated with alkaline phosphatase.

Analysis of in Vivo Oxidation of PerR by MS—HB9738 cells were grown overnight in 25 ml of MM containing 10 µM FeCl3 and 10 µM MnCl2. Cells were washed two times with MM and inoculated into 0.8 liter of MM supplemented with 10 µM MnCl2.Atan A600 of ~0.5, aliquots of 250-ml cell culture were treated with 0, 0.1, or 10 mM H2O2 for 5 min, harvested by centrifugation at 6000 rpm for 10 min, and washed two times with Tris-buffered saline (50 mM Tris (pH 7.4) and 150 mM NaCl) containing 20 mM EDTA (TBS + EDTA). Cells were sonicated in 1.3 ml of TBS + EDTA containing protease inhibitor mixture, 1 mg/ml lysozyme, and 2% Triton X-100. After removal of cell debris by centrifugation at 16,100 x g for 10 min, the resulting supernatants were incubated with 40 µl of anti-FLAG M2 affinity gel for 1 h and then washed with 1 ml of TBS + EDTA three times. Anti-FLAG M2 affinity gel suspensions containing PerR-FLAG were modified with 50 mM iodoacetamide in the presence of 20 mM EDTA and 1% SDS for 30 min in the dark. PerR-FLAG proteins were recovered using SDS-PAGE and analyzed by MALDI-TOF MS after in-gel tryptic digestion (see above).

Fluorescence Anisotropy Experiments—Fluorescence anisotropy experiments were performed as described previously (9). A 6-carboxyfluorescein (6F)-labeled mrgA-PerR box DNA fragment was generated by annealing of 5'-6F-CTAAATTATAATAATTATAATTTAG-3' and its complement (Integrated DNA Technologies). Fluorescence anisotropy measurements ({lambda}ex = 492 nm, slit width = 15 nm; {lambda}em = 520 nm, slit width = 20 nm) were performed anaerobically in 3 ml of 20 mM Tris ·HCl (pH 7.0), 5% glycerol, and 100 mM KCl with 100 nM DNA and 160 nM dimeric PerR:Zn (~100 nM active molecules). Metal ions were dissolved in anaerobic H2O and added through a silicon stopper using a Hamilton syringe (total volume increase was <5% by the end of reaction).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Purified PerR Contains Stably Bound Zn2+—As reported previously, PerR is susceptible to oxidative inactivation during purification, and this process is exacerbated by the presence of thiol-reducing agents and the absence of a metal-chelating agent (9). Consequently PerR was overexpressed in E. coli and purified in the presence of 10 mM EDTA and in the absence of thiol-reducing agents. As purified, PerR contained one stably bound Zn2+ per monomer as judged by both inductively coupled plasma MS metal analysis and the appearance of a protein peak corresponding to a monomeric, Zn2+-bound species in electrospray ionization mass spectroscopy under non-acidic conditions (see supplemental Fig. 1S).

When analyzed by SDS-PAGE, purified PerR routinely displayed two bands (Fig. 1A). The lower mobility band (band 2) migrated as expected given the molecular mass of the PerR monomer (16.3 kDa) and was increased in intensity when samples were treated with thiol-oxidizing agents (H2O2 or diamide) or the metal chelator EDTA. The higher mobility band (band 1) was increased in intensity by DTT (lane 2). These results suggest that band 1 may contain Zn2+ that remains associated with PerR even during electrophoresis in the presence of SDS. Removal of Zn2+ from PerR was observed when EDTA was present in the SDS sample buffer (lanes 5 and 6) but not when native protein was treated with EDTA (e.g. during purification).

We used the cysteine-alkylating agent AMS to determine whether all four Cys residues in PerR were reduced. For this experiment, samples were treated with DTT, H2O2, or diamide for 30 min in the absence of SDS prior to analysis of AMS reactivity (in the presence of SDS and EDTA to remove associated Zn2+). Treatment of purified PerR with AMS resulted in a single band corresponding to the addition of four AMS moieties to PerR (Fig. 1B, lane 2). The appearance of a single modified band despite the presence of two bands in the starting material (Fig. 1A, lane 1) is consistent with the hypothesis that the difference in mobility of bands 1 and 2 is not due to proteolysis but instead reflects the presence or absence of bound Zn2+. Oxidation of Cys residues was apparent after treatment of PerR with 10 mM H2O2 for 30 min (as judged by the reduced susceptibility of the protein to AMS modification; lane 6) but not after exposure to diamide (lane 8). Thus, the ability of diamide to oxidize PerR (Fig. 1A, lane 4) requires the presence of harsher conditions (e.g. SDS and heating) relative to H2O2. Indeed PerR:Zn,Mn retains DNA binding activity even after treatment with 50 mM diamide for 15 min (data not shown).

These results suggest that band 1 corresponds to monomeric PerR containing bound Zn2+, whereas band 2 represents PerR that lacks bound Zn2+. This supposition is supported by direct staining of the SDS-PAGE gels with the Zn2+-binding dye, PAR. Zn2+ was clearly associated with the protein as purified and after DTT treatment but not with the H2O2-treated sample (Fig. 1C). Moreover in the presence of DTT, incubation of PerR with Zn2+, but not other divalent cations, greatly increased the fraction of band 1 (data not shown), whereas all four single Cys -> Ser mutants migrate in the position of band 2 during SDS-PAGE, suggesting that Zn2+ binding has been eliminated (9).

PerR Apoprotein Forms Intrasubunit Disulfide Bonds—We initially speculated that the mobility difference between bands 1 and 2 might originate from an N- or carboxyl-terminal proteolytic cleavage during SDS-PAGE sample preparation. To explore this possibility, we treated an SDS-polyacrylamide gel with iodoacetamide prior to Coomassie Blue staining and destaining (to prevent oxidation of Cys residues) and analyzed the two bands using in-gel tryptic digestion and MALDI-TOF MS analysis. We detected the amino-terminal tryptic peptide (T1) (in Met-excised form) in protein from both bands 1 and 2 implying that there was no difference at the amino terminus (consistent with electrospray ionization MS data of intact protein; see supplemental Fig. 1S). Although the carboxyl-terminal tryptic peptide (T15 and T16 K and ENH) could not be detected under the m/z range of our experiments, we did detect carboxyl-terminal epitope tag in both bands 1 and 2 by immunoblotting analysis of PerR-FLAG (data not shown). These data are consistent with the hypothesis that the primary difference between bands 1 and 2 is the presence of bound Zn2+ in band 1.


Figure 3
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  		FIGURE 3.
Effects of Cys oxidation by H2O2 on the mobility of PerR during SDS-PAGE. A, purified PerR was treated with 0 (lane 1), 10 (lane 2), or 100 mM H2O2 (lane 3) and resolved by SDS-PAGE. Lanes M are protein molecular mass standards. B–D, after treatment of the whole gel with 50 mM iodoacetamide, band 3 (B), band 2(C), and band 4 (D) were analyzed by in-gel tryptic digestion followed by MALDI-TOF MS. Spectra in the upper right insets correspond to the regions of m/z values from 3300 to 3700 with intensities multiplied by 10. E, schematic representations of bands 1, 2, 3, and 4. The dotted line indicates coordination of Zn2+ by Cys thiolate (band 1) or disulfide bonds between Cys residues (bands 2–4). For band 4, alternate disulfide bond(s) formation is also possible.

 
In the spectrum of PerR isolated from band 2, the tryptic peptides containing 96CXXC99 and 136CXXC139 (T11, m/z = 2083.91; T14, m/z = 1369.66; and T14 + T15, m/z = 1497.75) were each detected with a loss of 2 m/z units from the calculated values. This mass change is indicative of an intrafragment disulfide bond formation. No peaks corresponding to interfragment disulfide bond formation were detected in this air-oxidized protein. In contrast, peptides from band 1 contained reduced Cys residues as judged by their quantitative modification by iodoacetamide. The modified peptides (labeled as T11*, m/z = 2199.93; T14*, m/z = 1485.70; and T14 + T15*, m/z = 1613.79) display a 114-Da mass increase (57 Da for each carbamidomethyl-modified Cys residue). Thus, PerR in band 1 is fully reduced as expected for a Cys4:Zn2+ site, whereas PerR in band 2 is oxidized. In this case, air oxidation leading to the appearance of band 2 likely occurs after unfolding of the protein by SDS because AMS modification (Fig. 1B) and iodoacetamide modification followed by electrospray ionization MS (data not shown) indicate that all four Cys residues are fully reduced in the protein as purified.

Oxidation of PerR Apoprotein with H2O2 Leads to Both Disulfide Bond and Sulfonic Acid Formation—To monitor disulfide bond formation during H2O2-mediated protein oxidation, we treated PerR:Zn with either 10 or 100 mM H2O2 and separated the resulting proteins by SDS-PAGE. Under these severe oxidation conditions, two additional protein bands appear (bands 3 and 4; Fig. 3A). Bands 2, 3, and 4 from the protein treated with 100 mM H2O2 were analyzed by MALDI-TOF MS. As before, band 2 corresponds to protein containing two intrasubunit disulfide bonds (Cys96–Cys99 and Cys136–Cys139). Note that even after treatment with 100 mM H2O2, there was no mass increase corresponding to the oxidation of His91 (in peptide T11), consistent with the requirement for Fe2+ for modification of ligands in the high sensitivity peroxide-sensing site (9). Under these conditions, peptides containing Met residues are either partially (T7) or fully (T5) oxidized as judged by a +16-Da mass increase. This presumably corresponds to the formation of methionine sulfoxide as also documented previously for the iron-containing form of the protein (9).


Figure 4
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  		FIGURE 4.
Oxidation of Cys4:Zn2+ site by H2O2 leads to Zn2+ release and Cys oxidation. A, release of Zn2+ from PerR:Zn by H2O2.5 µM PerR (PerR:Zn) was treated with 0, 1, 10, or 100 mM H2O2 in the presence of 100 µM PAR at 25 °C, and the formation of the Zn2+-PAR complex was monitored by measuring absorbance at 494 nm every 1 s for 30 min. Data were fit to an effective first order rate equation, A = Ao (1 – e–k[H2O2]t) + B, where A is absorbance at 494 nm, Ao is the amplitude of the absorbance change, k is the second order rate constant, t is time in seconds, and B is the initial absorbance. The calculated second order rate constant is 0.054 ± 0.009 M–1 s–1. B, oxidation of Cys residues by H2O2. PerR:Zn,Fe or PerR:Zn,Mn was treated with H2O2 under anaerobic conditions, and the remaining reduced Cys residues were monitored by AMS modification.

 
Protein in band 3 contains one disulfide bond (Cys136–Cys139) and two sulfonic acids (at Cys96 and Cys99). There was no evidence for sulfonic acid formation in the peptide containing Cys136 and Cys139. Protein in the faster migrating band 4 contains cross-links between the tryptic peptide containing Cys96 and Cys99 (T11) and that containing Cys136 and Cys139 (T14). This band contains approximately equal levels of two distinct species. The 3452.39-Da peptide corresponds to two disulfide cross-links between T11 and T14 (which may include the parallel or antiparallel doubly cross-linked peptides or both), whereas the 3550.40 peak corresponds in mass to one disulfide bond and two sulfonic acids. There was little if any evidence of the non-cross-linked peptides, T11 and T14.

Together these results indicate that the mobility of PerR during SDS-PAGE is a sensitive indicator of conformational changes elicited by Zn2+ binding and by disulfide bond formation. Oxidation leading to disulfide bond formation between vicinal Cys residues (in the two CXXC motifs) led to a lower mobility species (band 2) that was further retarded under conditions leading to sulfonic acid formation (band 3). In contrast, the more compact conformation of PerR due to the presence of bound Zn2+ (band 1) could be mimicked by one or more disulfide linkages between the two CXXC motifs (band 4). These results are consistent with the previously proposed model of an intra-subunit Cys4:Zn2+ site as supported by homology modeling of PerR on the FurPA structure and by the observation that all four PerR Cys -> Ser mutant proteins migrated at a position corresponding to band 2 during SDS-PAGE analysis (supplemental material in Ref. 9).

Oxidation of PerR Leads to Release of Zn2+ Independent of Metal Binding at the Regulatory Site—Oxidation of the Cys4:Zn2+ site in the stress-regulated E. coli chaperone HSP33 occurs under conditions of severe heat and oxidative stress and activates chaperone activity (24). We reasoned that the Cys4:Zn2+ site in PerR might also serve a regulatory role in vivo. Specifically under conditions in which PerR exists predominantly in the manganese-cofactored form (PerR:Zn,Mn), oxidation of the Zn2+ site might still allow derepression under severe oxidative stress.

To test this hypothesis, we first measured the rate of Zn2+ release from PerR:Zn in the presence of H2O2. Release of Zn2+, as monitored by formation of the colored PAR complex, was dependent on added H2O2 with a second order rate constant of 0.054 ± 0.009 M–1 s–1 (Fig. 4A). This corresponds to a half-time of 21.4 min in the presence of 10 mM H2O2. Even in the presence of 7 M urea, the rate of H2O2-mediated Zn2+ release was very slow (0.119 ± 0.029 M–1 s–1; data not shown). These data are consistent with a structural role for the Cys4:Zn2+ site that helps maintain a locally folded domain even in the presence of protein denaturants such as urea and SDS.

To monitor oxidation of the Cys4:Zn2+ site in the PerR:Zn,Fe and PerR:Zn,Mn forms of the protein we used AMS to monitor the number of reduced cysteines (Fig. 4B). In each case, no significant oxidation of protein was detected by 1 mM H2O2, and ~30% of the protein was fully oxidized by 10 mM H2O2. Complete oxidation of all four Cys residues could be attained with 100 mM H2O2. These results suggest that the rate of oxidation of the Cys4:Zn2+ site is similar in the PerR:Zn (Fig. 1B, lane 6), PerR:Zn,Fe, and PerR:Zn,Mn forms of the repressor (Fig. 4B). The rate of oxidative inactivation measured here also explains the previously reported 50% inactivation of DNA binding activity noted for reconstituted PerR:Zn,Mn by treatment with 10 mM H2O2 for 20 min (9). In contrast, the DNA binding activity of the PerR:Zn,Fe form is highly sensitive to peroxide inactivation (estimated kinact, ~105 M–1 s–1) due to histidine oxidation at the Fe2+-binding site (9).


Figure 5
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  		FIGURE 5.
Oxidation of PerR:Zn,Mn by H2O2 leads to disulfide bond formation between Cys residues without His modification. PerR:Zn,Mn (10 µM purified PerR + 100 µM MnCl2) was incubated with 0 (A), 0.1 (B), 1(C), 10 (D), or 100 (E) mM H2O2 for 10 min under anaerobic conditions. Proteins were recovered by precipitation with 10% trichloroacetic acid, modified using 50 mM iodoacetamide, trypsinized, and analyzed by MALDI-TOF MS. Peptides containing two carbamidomethyl-modified Cys residues are designated with open triangles and marked with asterisks as in Fig. 2. CXXC motif-containing peptides (T11 and T14) are exclusively detected as fully alkylated forms up to 1 mM H2O2 treatment. In samples treated with 100 mM H2O2 (E), CXXC motif-containing peptides are detected without alkylation at Cys residues. Note that T11 shows no increase in oxidation at His91 residue despite the full oxidation at Cys96 and Cys99 residues by 100 mM H2O2 treatment (T11 contains a small amount of oxidation as judged by the small peak with a +16-Da mass shift. This oxidation is present in the purified PerR protein and is not further increased by peroxide treatment of Mn2+-supplemented protein. This corresponds to His91 oxidation and correlates with the observation that the protein, as purified, is ~60% active for DNA binding.) T7 containing Met54 gains +16 m/z units, indicative of methionine sulfoxide formation. By analogy, the gain of +16 m/z units on T5 seems to indicate the oxidation at Met35 rather than His37.

 
We also monitored the effects of high levels of H2O2 on reconstituted PerR:Zn,Mn protein by MALDI-TOF MS studies (Fig. 5). Consistent with the AMS modification studies, iodoacetamide modification was reduced in the 10 mM H2O2-treated samples and eliminated in the samples treated with 100 mM H2O2. There was a near quantitative +16-Da mass shift in the two peptides containing Met residues (T5 and T7) but no evidence of significant oxidation of the peptide containing His91 (T11). Thus, PerR:Zn,Mn is insensitive to oxidation in vitro at the Mn2+-binding site, even under severe conditions, and is instead inactivated by oxidation of the structural Cys4:Zn2+ site.

Inactivation of PerR:Zn,Mn Under Severe Oxidative Stress in Vivo Is Not Mediated by Cys Oxidation—In numerous studies, we have observed that the induction of PerR-regulated genes is highly sensitive to the metal composition of the growth medium. In medium containing iron or iron and manganese, induction is efficient with complete derepression elicited by <100 µM H2O2 or by exposure to NO-generating agents. In contrast, in low iron minimal medium supplemented with manganese, the resulting PerR:Zn,Mn form of the repressor is relatively insensitive to peroxide- and NO-mediated inactivation (2, 10, 25).

To determine whether oxidation of the Cys4:Zn2+ site might contribute to PerR regulation in vivo, we first determined the levels of H2O2 needed to inactivate the PerR:Zn,Mn form of the repressor. Whereas induction in iron-containing minimal medium is readily observed with 0.1 mM H2O2, it took 10 mM H2O2 to induce expression in the manganese-supplemented minimal medium (Fig. 6A). Similar results were observed in a strain expressing PerR-FLAG rather than PerR (data not shown). Although Fur also contains a structural Zn2+ ion, we were unable to detect peroxide-mediated inactivation of Fur (Fig. 6A). Similarly there was no evidence for derepression of a reporter fusion (PyciC-cat-lacZ) regulated by the third Fur paralog in B. subtilis, Zur (data not shown). The high selectivity of peroxide inactivation is consistent with previous transcriptome analyses: treatment of cells with micromolar levels of H2O2 led to derepression of PerR-regulated genes but not genes controlled by Fur or Zur (10).

Based on our in vitro analyses, it seemed likely that the in vivo inactivation of PerR:Zn,Mn by 10 mM H2O2 was due to oxidation of Cys residues associated with the structural Zn2+ site. To test this hypothesis directly, we used AMS modification to trap reduced Cys residues before and after treatment of cells with 10 mM H2O2. Under all conditions tested (Fig. 6B and data not shown), all four Cys residues were fully modified by AMS. In some experiments, there was a faint band corresponding to unmodified PerR after treatment, consistent with formation of either disulfide or sulfonic acid modifications (data not shown). However, this band was always a minor species, and it seemed unlikely that this small amount of oxidation was sufficient to account for the observed derepression (Fig. 6A). These data led us to reject the hypothesis that inactivation of PerR in vivo, in the presence of 10 mM H2O2, is principally mediated by Cys oxidation.


Figure 6
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  		FIGURE 6.
Effects of severe oxidative stress on the derepression of the PerR-regulated mrgA gene. A, wild type B. subtilis strain (CU1065) containing mrgA-cat-lacZ or feuA-cat-lacZ (Fur-regulated) transcriptional fusions (inserted into the SPbeta prophage) were grown in MM containing 10 µM FeCl3 or 10µM MnCl2. Cultures at ~0.6 A600 were split and treated with 0, 0.1, 1, or 10 mM H2O2. After further incubation for 30 min, cells were harvested and assayed for beta-galactosidase activity. The derepression of feuA-cat-lacZ in MM + MnCl2 is consistent with the requirement of iron for the activity of Fur (values in the presence of iron were below 1 unit). Error bars indicate mean ± S.D. (11 independent experiments for mrgA-cat-lacZ fusion and five independent experiments for feuA-cat-lacZ). B, a B. subtilis strain containing PerR-FLAG (HB9738) was grown in LB medium, MM + 10 µM FeCl3, or MM + 10 µM MnCl2. At A600 ~0.6, cultures were harvested before or after 10 mM H2O2 treatment (at times indicated) by 10% trichloroacetic acid precipitation. The pellets were modified with iodoacetamide or AMS, and the mobility of PerR-FLAG was monitored by Western blotting using anti-FLAG antibody. PerR-FLAG purified from E. coli after overexpression (2 ng/lane) was loaded in the last two lanes of the upper panel. The lower mobility bands in the AMS-treated samples correspond to PerR having all four Cys residues modified by AMS.

 
In Vivo Oxidation of PerR:Zn,Mn Is Correlated with Regulatory Site Oxidation—To determine what modification(s) might be associated with PerR:Zn,Mn inactivation in vivo, we immunoprecipitated FLAG epitope-tagged PerR from B. subtilis cells after treatment with either 0.1 or 10 mM H2O2 (Fig. 7A). MALDI-TOF analysis of in situ trypsin-digested PerR was consistent with the presence of four reduced Cys residues under all conditions: in each case the T11 and T14 tryptic peptides were fully alkylated by iodoacetamide. In the sample treated with 10 mM H2O2, the T11 peptide appeared as a doublet with ~30–40% oxidation corresponding to a species previously shown to represent His91 oxidation (9). The other histidine residue previously shown to be oxidized by Fe2+-mediated hydroxyl radical modification is in peptide T5. This peptide appeared as a doublet (with a 16-Da mass shift) in all three samples, likely due to methionine sulfoxide formation during sample preparation and analysis (Met oxidation is also consistent with the doublet noted for T7, the other Met-containing peptide). With this background oxidation and the poor recovery of highly oxidized T5 peptides noted during matrix desorption, it is difficult to assess the level of His37 oxidation. Nevertheless these results indicate that the in vivo oxidation of PerR:Zn,Mn leads to oxidation of His91 in the regulatory metal-binding site but not to significant Cys oxidation.


Figure 7
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  		FIGURE 7.
In vivo oxidation of the regulatory metal-binding site by 10 mM H2O2. A, PerR-FLAG proteins prepared from B. subtilis by immunoprecipitation after treatment with 0 (lane 1), 0.1 (lane 2), or 10 (lane 3) mM H2O2 for 5 min. Samples were treated with 50 mM iodoacetamide prior to electrophoresis. B–D, MALDI-TOF MS analysis of PerR-FLAG (after in-gel tryptic digestion) from cells treated with no H2O2 (B), 0.1 mM H2O2 (C), or 10 mM H2O2 (D). In samples treated with 10 mM H2O2 (D), the decrease of intensity of T11* and increase of intensity corresponding to T11* + T16 are indicative of His91 oxidation.

 
Treatment of reconstituted PerR:Zn,Mn (in the absence of Fe2+) with 10–100 mM H2O2 oxidized the Cys4:Zn2+ site but did not lead to appreciable His oxidation (Fig. 5). In contrast, in vivo oxidation in cells containing PerR:Zn,Mn led to His91 oxidation with no significant Cys oxidation. Because this pattern of oxidation is characteristic of PerR:Zn,Fe (9), we suggest that treatment of cells with 10 mM H2O2 led to the release of sufficient intracellular Fe2+ to allow a shift in the active species of PerR from PerR:Zn,Mn to PerR:Zn,Fe.

PerR:Zn Binds Fe2+ with Higher Affinity than Mn2+—We next determined the affinity of PerR:Zn for Fe2+ and Mn2+ using a fluorescence anisotropy-based DNA binding assay (Fig. 8). The apparent Kd for activation by Fe2+ (~0.1 µM) is significantly greater than that for Mn2+ (~2.8 µM). Although lower absolute affinities were observed in several replicate experiments (particularly for Fe2+, perhaps due to trace oxygen contamination), the data are generally consistent with a significant preference for Fe2+ relative to Mn2+. Indeed we previously demonstrated that 10 µM Mn2+ is unable to protect PerR:Zn against oxidative inactivation in the presence of 10 µM Fe2+, and only partial protection is afforded by 100 µM Mn2+ (9). We also estimated the affinity for Mn2+ using electrophoretic mobility shift assay experiments. By inclusion of Mn2+ in the binding and running buffers as well as in the polyacrylamide gel, we could measure the apparent DNA binding affinity of PerR as a function of Mn2+ concentration (supplemental Fig. S2). Under these assay conditions, DNA binding affinity increased 40-fold as the Mn2+ concentration was increased from 1 to 100 µM. Together these studies indicate that PerR:Zn is activated for DNA binding by Mn2+ in the low micromolar range and by Fe2+ with even higher affinity.


Figure 8
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  		FIGURE 8.
Measurement of metal-dependent binding of PerR to DNA by fluorescence anisotropy. The anisotropy was monitored in samples with various amounts of metal ions (filled circle for Fe2+ and open circle for Mn2+). Samples contained 100 nM 25-bp mrgA-PerR box and 170 nM PerR dimer (corresponding to 100 nM active PerR dimer) in 20 mM Tris (pH 7.0) with 5% glycerol and 100 mM NaCl. Because the Kd of PerR (e.g. dimeric PerR:Zn,Mn) for DNA is < 1 nM and the amount of PerR (active dimer) and DNA used is 100 nM each, the assumption was made to let [PerR:Zn,Mn or PerR:Zn,Fe] ~ [PerR-DNA complex], and the data were fit to a simple 1:1 interaction between metal ions and PerR:Zn. The Kd values were determined to be 0.1 and 2.8 µM for Fe2+ and Mn2+, respectively. Data shown are from the experiments yielding the highest measured affinity of five independent experiments for each metal ion. Somewhat lower affinities for Fe2+ seen in some experiments likely reflect trace oxygen contamination in the sample (see text).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Cells respond to oxidative stress by the inducible synthesis of a variety of protective enzymes and proteins. Most peroxide sensors characterized to date use cysteine thiolates to detect peroxides, often leading to the formation of either intra- or intermolecular disulfides (12–14). In contrast, B. subtilis PerR has been shown recently to detect low levels (<100 µM) of H2O2 by iron-catalyzed oxidation of either of two His residues, His37 and His91 (9). These two His residues are required for Fe2+ binding and are predicted, by homology modeling, to be direct ligands to the regulatory metal ion. PerR also contains a tightly bound Zn2+ ion (1) proposed to exist as part of a Cys4:Zn2+ site (9). In the present study we investigated the redox sensitivity of this Cys4:Zn2+ site and assessed the role of this site in sensing high levels of H2O2 both in vitro and in vivo.

PerR retained tightly bound Zn2+ even after purification in the presence of EDTA. In the course of our studies, we noted a correlation between conditions favoring Zn2+ binding and the appearance of a more rapidly migrating form of PerR during SDS-PAGE (band 1). Using MALDI-TOF analysis of in situ digested protein samples, we demonstrated that non-reducing SDS-PAGE provides a sensitive assay for monitoring the status of the Cys4:Zn2+ site. When this site was intact, PerR migrated as a 14.5-kDa protein, whereas when Zn2+ was lost the resulting protein migrated at the expected position of 16.4 kDa. Loss of Zn2+ was accompanied by oxidation of PerR to a disulfide-bonded form. When treated with high levels of H2O2 (in the presence of protein denaturant and heat) two additional bands appeared including a slowly migrating band in which PerR contains two sulfonic acid residues and one disulfide bond (band 3) and a more rapidly migrating band containing one or two long range disulfide bonds (band 4).

Because many Fur homologs are known or predicted to contain a structural Zn2+ site, our results may provide an explanation for the observation that many Fur homologs migrate as a doublet during SDS-PAGE. Examples include B. subtilis and Bacillus cereus Fur (26, 27), Streptomyces reticuli FurS (15), and Streptomyces coelicolor CatR (16). However, analysis of E. coli Fur by amino-terminal sequencing and by electrospray ionization MS revealed that the fast migrating band is a proteolytic product of intact protein lacking the amino-terminal nine amino acids (28, 29). Recently it has been noted that Zn2+ binding is required for stable folding of the carboxyl-terminal domain of E. coli Fur and for protein dimerization (30).

Previously we (1) and others (15, 16) had suggested that PerR and its homologs might sense H2O2 by Cys-based redox reactions. However, each of these studies used high levels of H2O2 (5–10 mM), and the physiological role, if any, of this Cys-based oxidation has not been established. In the present study we demonstrated that the structural Cys4:Zn2+ site in PerR was remarkably resistant to H2O2 with an observed second order rate constant for Zn2+ release of ~0.054 M–1 s–1 (corresponding to a half-time for inactivation of ~20 min by 10 mM H2O2). A similar oxidation rate was determined by several assays including Zn2+ release studies (using PerR:Zn; Fig. 4A), monitoring of Cys status by AMS modification of PerR:Zn,Fe and PerR:Zn,Mn (Fig. 4B), and monitoring of Cys status by iodoacetamide modification of PerR:Zn,Mn followed by MALDI-TOF MS (Fig. 5) and by measuring oxidative inactivation of DNA binding activity for PerR:Zn,Mn (9). Significantly the Cys4: Zn2+ site in PerR was even less reactive than free cysteine (2–20 M–1 s–1; Refs. 31–33), the major low molecular weight thiol in B. subtilis (34). Thus, these Cys residues are actually protected against peroxidative attack by Zn2+ coordination. Comparison of the rate reported here for oxidation of the Cys4:Zn2+ site (kinact ~ 0.05 M–1 s–1) with the rate of peroxide-mediated inactivation of PerR:Zn,Fe (kinact ~ 105 M–1 s–1) highlights the fact that the structural Zn2+ and regulatory Fe2+ sites have vastly different sensitivities toward oxidation.

The oxidative modification of thiolates coordinated to Zn2+ has been implicated in several other redox-sensitive proteins. The S. coelicolor RsrA anti-{sigma} factor is regulated by disulfide stress. Treatment with diamide, a thiol-specific oxidant, leads to Zn2+ release and inactivation of the anti-{sigma}. This results in activation of {sigma}R and expression of thiol reductants such as thioredoxin and thioredoxin reductase (35, 36). E. coli HSP33 is a redox-regulated protein chaperone that contains a high affinity Cys4:Zn2+ site (17). Oxidation of this Cys4:Zn2+ site, accompanied by Zn2+ release, activates this protein chaperone, which can functionally replace the redox-sensitive DnaK protein under severe oxidative stress conditions (24). Activation of HSP33 requires harsh conditions including high levels of peroxides (e.g. 4 mM H2O2) and elevated temperatures (e.g. 43 °C). B. subtilis also contains an HSP33 ortholog (37), but the redox regulation of this protein has not yet been characterized.

Because the Cys4:Zn2+ site in PerR is structurally similar to the regulatory site in HSP33, we speculated that oxidation at this metal center might provide a backup mechanism for induction under severe oxidative stress conditions for iron-starved cells (in which PerR would be present in the PerR:Zn,Mn form). Indeed in vitro oxidation of reconstituted PerR:Zn,Mn by 10 or 100 mM H2O2 was correlated with Cys oxidation and not with oxidation of His residues in the regulatory metal-binding site (Fig. 5). In contrast, in vivo treatment of manganese-supplemented cells with 10 mM H2O2 (a level sufficient to induce a PerR-regulated reporter; Fig. 6) did not lead to PerR Cys oxidation (Fig. 7). The lack of Cys oxidation in vivo may be explained by the lower effective concentration of H2O2 in vivo due to the presence of catalase, peroxidases, and competition from low molecular mass thiols such as cysteine. Instead induction was correlated with oxidation of at least one of the two His residues previously implicated in peroxide sensing (9). Oxidation of these His residues is diagnostic for PerR:Zn,Fe. It is likely that exposure of cells to high levels of peroxide releases sufficient Fe2+ to allow PerR to switch from the relatively insensitive Per-R:Zn,Mn form to the thermodynamically preferred and much more peroxide-sensitive PerR:Zn,Fe form.

To evaluate this model it is necessary to determine (i) the relative affinity of PerR:Zn for Fe2+ and Mn2+ and (ii) the levels of free metal ions in the cytosol under different growth conditions. PerR:Zn binds Fe2+ with higher affinity than Mn2+ as judged by both metal-dependent DNA binding measurement (Fig. 8) and the ability of Mn2+ to protect against Fe2+-catalyzed protein inactivation (9). This is consistent with the observation that PerR:Zn,Mn only forms in cells grown under iron-limiting conditions. Although it is difficult to measure the levels of free metal ions in the cell, it is possible to make reasonable estimates. For example MntR, the B. subtilis protein responsible for regulating Mn2+ levels in the cell binds two Mn2+ per monomer with apparent dissociation constants (at pH 8.0) of 0.2–2.0 and 5–13 µM (38). Somewhat lower affinities were reported based on EPR measurements of Mn2+ binding by the Bacillus anthracis MntR ortholog with average dissociation constants in the range of ~50 µM (39). Because MntR represses Mn2+ uptake when in its Mn2+-bound state, it is reasonable to assume that intracellular levels of free Mn2+ are normally maintained at levels sufficient to saturate PerR (to generate the PerR:Zn,Mn form of the repressor). The levels of exchangeable Fe2+ in the cytosol are difficult to measure directly, but in E. coli chelatable iron levels of 10–30 µM have been measured by EPR in the presence of desferrioxamine (40, 41). If similar values pertain to B. subtilis, this would explain why, in the presence of both iron and manganese in the growth medium, PerR is predominantly in the peroxide-sensing PerR:Zn,Fe form. Apparently in cells grown in manganese-supplemented minimal medium (with no added iron), the intracellular level of iron is lowered sufficiently to allow the PerR:Zn,Mn form of the repressor to predominate (2, 25). Yet because of the relatively higher affinity for Fe2+, a small increase in intracellular free Fe2+ could shift the repressor from the PerR:Zn,Mn to the Per-R:Zn,Fe form. Treatment of cells with 10 mM H2O2 is likely to damage iron-sulfur clusters (42) and perhaps other non-heme iron proteins, leading to elevation of free Fe2+ in the cell.


   FOOTNOTES
 
* This work was supported by National Science Foundation Grant MCB-0235255. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. Back

1 To whom correspondence should be addressed: Dept. of Microbiology, 370 Wing Hall, Cornell University, Ithaca, NY 14853. Tel.: 607-255-6570; Fax: 607-255-3904; E-mail: jdh9{at}cornell.edu.

2 The abbreviations used are: AMS, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; PAR, 4-(2-pyridylazo)resorcinol; MM, MOPS-buffered minimal medium; MOPS, 4-morpholinepropanesulfonic acid; DTT, dithiothreitol; MS, mass spectrometry; Tricine, N-[2-hydroxy-1,1-bis(hydroxy-methyl)ethyl]glycine; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Mayuree Fuangthong for initial observations on the effects of oxidants on PerR mobility and R. Sherwood for assistance with mass spectrometry.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Herbig, A. F., and Helmann, J. D. (2001) Mol. Microbiol. 41, 849–859[CrossRef][Medline] [Order article via Infotrieve]
  2. Fuangthong, M., Herbig, A. F., Bsat, N., and Helmann, J. D. (2002) J. Bacteriol. 184, 3276–3286[Abstract/Free Full Text]
  3. Bsat, N., Herbig, A., Casillas-Martinez, L., Setlow, P., and Helmann, J. D. (1998) Mol. Microbiol. 29, 189–198[CrossRef][Medline] [Order article via Infotrieve]
  4. Jacquamet, L., Aberdam, D., Adrait, A., Hazemann, J. L., Latour, J. M., and Michaud-Soret, I. (1998) Biochemistry 37, 2564–2571[CrossRef][Medline] [Order article via Infotrieve]
  5. Gonzalez de Peredo, A., Saint-Pierre, C., Adrait, A., Jacquamet, L., Latour, J. M., Michaud-Soret, I., and Forest, E. (1999) Biochemistry 38, 8582–8589[CrossRef][Medline] [Order article via Infotrieve]
  6. Coy, M., Doyle, C., Besser, J., and Neilands, J. B. (1994) Biometals 7, 292–298[Medline] [Order article via Infotrieve]
  7. Pohl, E., Haller, J. C., Mijovilovich, A., Meyer-Klaucke, W., Garman, E., and Vasil, M. L. (2003) Mol. Microbiol. 47, 903–915[CrossRef][Medline] [Order article via Infotrieve]
  8. Friedman, Y. E., and O'Brian, M. R. (2004) J. Biol. Chem. 279, 32100–32105[Abstract/Free Full Text]
  9. Lee, J. W., and Helmann, J. D. (2006) Nature 440, 363–367[CrossRef][Medline] [Order article via Infotrieve]
  10. Helmann, J. D., Wu, M. F., Gaballa, A., Kobel, P. A., Morshedi, M. M., Fawcett, P., and Paddon, C. (2003) J. Bacteriol. 185, 243–253[Abstract/Free Full Text]
  11. Chen, L., James, L. P., and Helmann, J. D. (1993) J. Bacteriol. 175, 5428–5437[Abstract/Free Full Text]
  12. Kiley, P. J., and Storz, G. (2004) PLoS Biol. 2, e400[CrossRef][Medline] [Order article via Infotrieve]
  13. Green, J., and Paget, M. S. (2004) Nat. Rev. Microbiol. 2, 954–966[CrossRef][Medline] [Order article via Infotrieve]
  14. Paget, M. S., and Buttner, M. J. (2003) Annu. Rev. Genet. 37, 91–121[CrossRef][Medline] [Order article via Infotrieve]
  15. Ortiz de Orue Lucana, D., and Schrempf, H. (2000) Mol. Gen. Genet. 264, 341–353[CrossRef][Medline] [Order article via Infotrieve]
  16. Hahn, J. S., Oh, S. Y., Chater, K. F., Cho, Y. H., and Roe, J. H. (2000) J. Biol. Chem. 275, 38254–38260[Abstract/Free Full Text]
  17. Jakob, U., Muse, W., Eser, M., and Bardwell, J. C. (1999) Cell 96, 341–352[CrossRef][Medline] [Order article via Infotrieve]
  18. Kaltwasser, M., Wiegert, T., and Schumann, W. (2002) Appl. Environ. Microbiol. 68, 2624–2628[Abstract/Free Full Text]
  19. Chen, L., Keramati, L., and Helmann, J. D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8190–8194[Abstract/Free Full Text]
  20. Fuangthong, M., and Helmann, J. D. (2003) J. Bacteriol. 185, 6348–6357[Abstract/Free Full Text]
  21. Gaballa, A., and Helmann, J. D. (1998) J. Bacteriol. 180, 5815–5821[Abstract/Free Full Text]
  22. Schagger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368–379[CrossRef][Medline] [Order article via Infotrieve]
  23. Hunt, J. B., Neece, S. H., and Ginsburg, A. (1985) Anal. Biochem. 146, 150–157[CrossRef][Medline] [Order article via Infotrieve]
  24. Winter, J., Linke, K., Jatzek, A., and Jakob, U. (2005) Mol. Cell 17, 381–392[CrossRef][Medline] [Order article via Infotrieve]
  25. Moore, C. M., Nakano, M. M., Wang, T., Ye, R. W., and Helmann, J. D. (2004) J. Bacteriol. 186, 4655–4664[Abstract/Free Full Text]
  26. Bsat, N., and Helmann, J. D. (1999) J. Bacteriol. 181, 4299–4307[Abstract/Free Full Text]
  27. Harvie, D. R., and Ellar, D. J. (2005) Curr. Microbiol. 50, 246–250[CrossRef][Medline] [Order article via Infotrieve]
  28. Michaud-Soret, I., Adrait, A., Jaquinod, M., Forest, E., Touati, D., and Latour, J. M. (1997) FEBS Lett. 413, 473–476[CrossRef][Medline] [Order article via Infotrieve]
  29. Coy, M., and Neilands, J. B. (1991) Biochemistry 30, 8201–8210[CrossRef][Medline] [Order article via Infotrieve]
  30. Pecqueur, L., D'Autreaux, B., Dupuy, J., Nicolet, Y., Jacquamet, L., Brutscher, B., Michaud-Soret, I., and Bersch, B. (May 11, 2006) J. Biol. Chem. 10.1074/jbc.M601278200[Abstract/Free Full Text]
  31. Stone, J. R. (2004) Arch. Biochem. Biophys. 422, 119–124[CrossRef][Medline] [Order article via Infotrieve]
  32. Winterbourn, C. C., and Metodiewa, D. (1999) Free Radic. Biol. Med. 27, 322–328[CrossRef][Medline] [Order article via Infotrieve]
  33. Imlay, J. A. (2003) Annu. Rev. Microbiol. 57, 395–418[CrossRef][Medline] [Order article via Infotrieve]
  34. Newton, G. L., Arnold, K., Price, M. S., Sherrill, C., Delcardayre, S. B., Aharonowitz, Y., Cohen, G., Davies, J., Fahey, R. C., and Davis, C. (1996) J. Bacteriol. 178, 1990–1995[Abstract/Free Full Text]
  35. Kang, J. G., Paget, M. S., Seok, Y. J., Hahn, M. Y., Bae, J. B., Hahn, J. S., Kleanthous, C., Buttner, M. J., and Roe, J. H. (1999) EMBO J. 18, 4292–4298[CrossRef][Medline] [Order article via Infotrieve]
  36. Bae, J. B., Park, J. H., Hahn, M. Y., Kim, M. S., and Roe, J. H. (2004) J. Mol. Biol. 335, 425–435[CrossRef][Medline] [Order article via Infotrieve]
  37. Janda, I., Devedjiev, Y., Derewenda, U., Dauter, Z., Bielnicki, J., Cooper, D. R., Graf, P. C., Joachimiak, A., Jakob, U., and Derewenda, Z. S. (2004) Structure (Lond.) 12, 1901–1907[Medline] [Order article via Infotrieve]
  38. Kliegman, J. I., Griner, S. L., Helmann, J. D., Brennan, R. G., and Glasfeld, A. (2006) Biochemistry 45, 3493–3505[CrossRef][Medline] [Order article via Infotrieve]
  39. Sen, K. I., Sienkiewicz, A., Love, J. F., Vanderspek, J. C., Fajer, P. G., and Logan, T. M. (2006) Biochemistry 45, 4295–4303[CrossRef][Medline] [Order article via Infotrieve]
  40. Keyer, K., and Imlay, J. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13635–13640[Abstract/Free Full Text]
  41. Woodmansee, A. N., and Imlay, J. A. (2002) Methods Enzymol. 349, 3–9[Medline] [Order article via Infotrieve]
  42. Imlay, J. A. (2006) Mol. Microbiol. 59, 1073–1082[CrossRef][Medline] [Order article via Infotrieve]

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