(Received for publication, December 18, 1995; and in revised form, February 5, 1996)
From the
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.
The functions of iron-sulfur (FeS) ()clusters in
electron transfer 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).
Some tetranuclear [4Fe-4S] clusters appear to be very sensitive to damage via oxidation. For example, 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. The amidotransferase apoprotein is immediately degraded upon cluster disassembly (Bernlohr and Switzer, 1981; Grandoni et al., 1989), while the dehydratase apoprotein is stable and, when oxygen levels return to normal, reactivates by the reinsertion of iron and inorganic sulfide (Flint et al., 1993b). 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 specifically binds certain mRNAs to effect post-transcriptional control, which is lost when the metalloprotein is regenerated.
Little is known about the biochemical mechanisms of FeS cluster disassembly or assembly, either into newly synthesized proteins or in the cases of reversible processes cited above. The Azotobacter vinelandii nifS gene was identified as essential for the in vivo formation of active nitrogenase, which contains oxygen-sensitive [4Fe-4S] centers (Kennedy and Dean, 1992). NifS protein catalyzes the formation of these FeS centers in nitrogenase (Zheng et al., 1993), probably by mobilization of inorganic sulfide from L-cysteine (Zheng et al., 1994). In vitro NifS-mediated reconstitution of [4Fe-4S] clusters into the E. coli Fnr regulatory protein has also been described recently (Khoroshilova et al., 1995). Dean and collaborators have proposed a general role for NifS-type proteins in FeS cluster assembly (Zheng and Dean, 1994), although the reconstitution of other types of clusters has not been reported.
SoxR protein is a transcriptional activator that triggers a defense response against excess superoxide or nitric oxide in E. coli (Hidalgo and Demple, 1995). SoxR is post-translationally activated to stimulate transcription of a second regulatory gene, soxS (Nunoshiba et al., 1992; Wu and Weiss, 1992). The newly synthesized SoxS protein then triggers expression of >10 genes involved in defenses against oxidative damage (Amábile-Cuevas and Demple, 1991; Li and Demple, 1994; Wu and Weiss, 1992) and antibiotic resistance (Chou et al., 1993; Ma et al., 1996).
SoxR is a dimeric FeS protein that contains two [2Fe-2S] centers (Hidalgo and Demple, 1994; Wu et al., 1995; Hidalgo et al., 1995). 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 gene (Hidalgo and Demple, 1994; Hidalgo et al., 1995). The biochemical mechanism by which SoxR senses oxidative stress and activates transcription of soxS is unknown, but almost certainly relates to the protein's [2Fe-2S] clusters (Hidalgo and Demple, 1994). Two possible mechanisms of SoxR activation have been considered: (i) redox reactions involving pre-existing, reduced [2Fe-2S] clusters and (ii) assembly of the [2Fe-2S] clusters into pre-existing apoprotein. Here we demonstrate that purified apo-SoxR, which is unable to activate soxS transcription, can be fully activated for this function by in vitro reconstitution of its [2Fe-2S] centers, and that this process is catalyzed by A. vinelandii NifS protein.
Reconstitution reactions were carried out by anaerobic incubation of
diluted apo-SoxR (final concentration 1 µM) with iron
salts and Na
S in the presence of the reducing reagent
2-mercaptoethanol. Samples were removed at various times to assess
their in vitro transcriptional activity specific for the soxS promoter. As shown in Fig. 1, a slow
reconstitution process was observed that was essentially complete by
6 h. There was no activating effect of SoxR on bla transcription under any circumstances (Fig. 1). We
confirmed the presence of [2Fe-2S] clusters in this
spontaneously reconstituted SoxR by concentrating samples of the
reconstitution reactions, reduction with dithionite, and EPR
spectroscopy.
Although clear signatures of
[2Fe-2S] clusters were observed after a 6-h reconstitution,
the yield measured by EPR was lowered than expected. It seems likely
that the instability of the newly synthesized FeS clusters in the
presence of thiols and oxygen prevented observation of the full
complement of [2Fe-2S] centers in reactivated SoxR.
Figure 1: Spontaneous formation of active SoxR with iron and inorganic sulfide. Apo-SoxR (1 µM) was incubated anaerobically at room temperature in the presence of ferrous salts, sodium sulfide, and the reducing agent 2-mercaptoethanol (see ``Materials and Methods''). At the times indicated, 50- to 100-µl aliquots were removed using a syringe and diluted in 0.1 M NaCl, 50 mM HEPES-NaOH, pH 7.6. The transcriptional activity of RNA polymerase (RNAP) alone (lane 1) or in the presence of 10 ng of purified Fe-SoxR (lane 2), untreated apo-SoxR (lane 3) or treated apo-SoxR (lanes 4-7) was determined as described under ``Materials and Methods.'' Similar results were obtained in four independent experiments. The primer extension products (soxS, bla) are indicated by arrows.
Figure 2:
NifS-mediated reactivation of SoxR
transcriptional activity. A, 4.1 µM sample of
apo-SoxR was incubated inside an anaerobic chamber with 0.1 µM NifS protein in the presence of ferrous salts, the sulfide donor L-cysteine, and DTT. The reconstitution reaction was incubated
at 30 °C for 10 min. The sample was immediately removed from the
chamber, and SoxR was analyzed for in vitro transcription
activity (lane 4). Control reactions show the primer extension
products for RNA polymerase (RNAP) alone (lane 1),
purified Fe-SoxR (lane 2), or untreated apo-SoxR (lane
3). Although the amount of soxS transcription relative to bla appears higher in lane 4 than in lane 2,
this effect was not seen in two other experiments in which the
activities (measured as the soxS/bla transcription ratio) of
NifS-reconstituted SoxR and Fe-SoxR were equal. B, as for A, but using SoxR-C117A (cysteine 117 substituted by an
alanine) instead of apo-SoxR in the reconstitution
reaction; the mixture was incubated for 60 min at 30 °C before
transcriptional activity assay. The primer extension products (soxS, bla) are indicated by arrows.
The amount of SoxR in the NifS-mediated reconstitution reaction was 4 µM, high enough for direct EPR analysis without additional concentration. We compared the EPR spectrum of a reduced Fe-SoxR sample (8 µM; Fig. 3A) with those of untreated apo-SoxR or NifS-reconstituted apo-SoxR after dithionite treatment (Fig. 3, B and C, respectively). Comparison of the signals in A and C indicated that essentially complete reconstitution of the [2Fe-2S] centers had been achieved (4 µM estimated from EPR signal versus 4 µM SoxR protein in Fig. 3C).
Figure 3: EPR analysis of NifS-reconstituted Fe-SoxR. Apo-SoxR was incubated anaerobically with NifS as for Fig. 2A, then reduced and subjected to EPR spectroscopy. A 400-µl sample was analyzed at 20 K, with a microwave frequency of 9.42 GHz, modulation frequency of 100 kHz, microwave power of 1 milliwatt, modulation amplitude of 1.29 mT, time constant of 40.96 ms, and magnetic field from 310 to 370 mT. A, purified Fe-SoxR (printing scale 15); B, untreated apo-SoxR (printing scale 14); C, reconstituted Fe-SoxR (printing scale 14).
The results presented here demonstrate two novel points.
First, reassembly of the SoxR iron-sulfur center is sufficient to
restore full transcriptional activity to the protein. No other physical
difference between apo-SoxR and the transcriptionally active protein (e.g. oxidation or reduction of an amino acid side chain)
needs to be postulated to account for the difference in activity.
Second, the reassembly of [2Fe-2S] clusters into SoxR is
efficiently catalyzed by NifS, extending the known specificity of this
enzyme. Since the NifS activity generates S (Zheng et al., 1993), this result implies that apo-SoxR does not
contain sufficient residual inorganic sulfide for the reassembly
process to occur.
SoxR is a transcriptional activator that responds to superoxide and nitric oxide stress. We have been exploring the role of the SoxR-FeS centers in the oxidative stress-sensing and transcription-activating functions of this unusual protein. The mechanism by which SoxR is activated by intracellular superoxide or nitric oxide has not been established, in part because the resting (inactive) state of SoxR in vivo remains unknown. The experiments presented here demonstrate that such activation can occur in vitro by the reconstitution of SoxR's [2Fe-2S] centers, and that the process mediated by NifS protein is sufficiently rapid to mediate in vivo activation. SoxR activation occurs within 10 min in response to the superoxide-generating agent paraquat, as determined using a single-copy soxS`-lacZ operon fusion (Nunoshiba and Demple, 1993).
The
sluggishness of spontaneous SoxR reactivation by FeS cluster assembly
indicates that catalysis would be involved if in vivo activation occurs by this process. The only E. coli activity isolated so far that is able to favor FeS cluster
assembly seems to be related to NifS. ()It will be important
to determine whether this activity also restores [2Fe-2S]
centers and transcriptional activity to apo-SoxR.
The mechanisms by which FeS centers are incorporated into proteins have not been established and could differ for different cluster types. It has been proposed that clusters are preassembled from iron and sulfide in a process mediated by NifS-type enzymes, followed by integration of the clusters into apoproteins (Zheng and Dean, 1994). If so, suitable forms are generated by NifS for both [4Fe-4S] (Zheng and Dean, 1994; Khoroshilova et al., 1995) and [2Fe-2S] proteins, as shown by the catalysis of Fe-SoxR reassembly. Although genetic evidence implicates A. vinelandii NifS in the formation of active nitrogenase (Kennedy and Dean, 1992), the generality of this role has not been established for other FeS proteins. It is also unknown whether NifS-mediated reconstitution of FeS centers applies to both newly synthesized and pre-existing apoproteins. Reconstitution of FeS centers into proteins that have lost them would lead to inactivation of regulatory activity in some cases (e.g. IRP) and activation in others (Fnr, SoxR).
Several
interesting possibilities exist that relate the formation of FeS
centers to the activation of SoxR in vivo. If the resting
state is apo-SoxR, oxidative stress might generate available Fe from
oxidant-sensitive [4Fe-4S] proteins (Gardner and Fridovich,
1991; Liochev and Fridovich, 1992; Flint et al., 1993c) to
serve in the formation of Fe-SoxR. However, we have consistently
detected EPR signatures in vivo in untreated E. coli corresponding to 25-50% occupancy of the SoxR
[2Fe-2S] centers when SoxR is overproduced to 1% of the
cell protein. Thus, the nonactivated state of SoxR could be a
hemi-metalloprotein with a single [2Fe-2S] center per
(SoxR)
dimer. Still another possibility is that Fe-SoxR in
the reduced form is constantly being generated, but is unstable and
rapidly converted to the apoprotein. In this mechanism, oxidative
stress accompanied by superoxide or nitric oxide would oxidize Fe-SoxR
to stabilize the metalloprotein. In fact, oxidized SoxR is sufficiently
stable to withstand considerable handling during purification (Hidalgo
and Demple, 1994; Wu et al., 1995). Other [2Fe-2S]
proteins, such as E. coli biotin synthase (Sanyal et
al., 1994), are also more stable in the oxidized than the reduced
form. In such a fashion, the activity of SoxR in vivo would be
connected to both the redox activity of its [2Fe-2S] centers
and to their assembly.