(Received for publication, September 1, 1995; and in revised form, November 20, 1995)
From the
The transcription factor FNR from Escherichia coli regulates transcription of genes in response to oxygen
deprivation. To determine how the activity of FNR is regulated by
oxygen, a form of FNR had to be isolated that had properties similar to
those observed in vivo. This was accomplished by purification
of an FNR fraction which exhibited enhanced DNA binding in the absence
of oxygen. Iron and sulfide analyses of this FNR fraction indicated the
presence of an Fe-S cluster. To determine the type of Fe-S
cluster present, an oxygen-stable mutant protein LH28-DA154 was also
analyzed since FNR LH28-DA154 purified anoxically contained almost
3-fold more iron and sulfide than the wild-type protein. Based on the
sulfide analysis, the stoichiometry (3.3 mol of
S/FNR monomer) was consistent with either one
[4Fe-4S] or two [2Fe-2S] clusters per
mutant FNR monomer. However, since FNR has only four Cys residues as
potential cluster ligands and an EPR signal typical of a 3Fe-4S
cluster was detected on oxidation, we conclude that there is one
[4Fe-4S] cluster present per monomer of FNR LH28-DA154.
We assume that the wild type also contains one [4Fe-4S]
cluster per monomer and that the lower amounts of iron and sulfide
observed per monomer were due to partial occupancy. Consistent with
this, the Fe-S cluster in the wild-type protein was found to be
extremely oxygen-labile. In addition, molecular-sieve chromatographic
analysis showed that the majority of the anoxically purified protein
was a dimer as compared to aerobically purified FNR which is a monomer.
The loss of the Fe-S cluster by exposure to oxygen was associated
with a conversion to the monomeric form and decreased DNA binding.
Taken together, these observations suggest that oxygen regulates the
activity of wild-type FNR through the lability of the Fe-S
cluster to oxygen.
The ability to sense and adapt to changes in oxygen concentration is critical to the survival of many organisms. In Escherichia coli, the transcription factor FNR plays a central role in allowing the bacterium to adapt to changes in oxygen availability in its environment(1) . When oxygen is limiting, FNR activates synthesis of many enzymes required to generate energy by anaerobic respiration and also represses synthesis of some enzymes involved in aerobic respiration. Since the levels of FNR do not significantly differ between aerobically and anaerobically grown cells, oxygen deprivation must modulate FNR-dependent transcription by regulating the activity of FNR(2) . Determining how oxygen regulates FNR activity should provide fundamental information regarding the strategies available to cells to sense changes in oxygen levels.
The isolation and analysis of FNR mutant proteins that are active in the presence of oxygen (FNR*) has provided important clues as to how wild-type FNR activity might be regulated by oxygen(3, 4, 5) . For example, the in vitro analysis of one FNR* mutant protein, FNR-DA154 which has an Asp to Ala substitution at position 154, led to the initial proposal that oxygen regulates the oligomeric state of FNR. When purified in the presence of oxygen, FNR-DA154 protein showed increased dimerization (3) and DNA binding (3, 5) relative to the monomeric wild-type protein(3, 6) . This suggested that wild-type FNR becomes active under anaerobic conditions because of an increase in the formation of FNR dimers that presumably have maximal DNA binding affinity. In addition, the analysis of another FNR* mutant protein, FNR-LH28-DA154 which contains a Leu to His substitution at position 28 in addition to the DA154 substitution, identified a cofactor, an Fe-S cluster. This Fe-S cluster was associated with aerobically purified FNR-LH28-DA154 protein, and the association of this cluster enhanced DNA binding by the mutant protein(4) .
Taken together, the results from the analysis of the FNR* mutant
proteins led us to consider that, under anaerobic conditions, wild-type
FNR contains an Fe-S cluster that increases dimerization and DNA
binding. Consistent with this proposal, wild-type FNR requires
Fe for activity in
vivo(7, 8) , and FNR possesses four cysteines
that are essential for FNR function and that could serve as potential
ligands for an Fe cofactor(9, 10, 11) .
However, previously purified wild-type FNR did not contain an
Fe-S cluster but contained Fe
at a
stoichiometry of up to 1 mol of Fe
per monomer of
FNR(6) . This Fe
appeared to be loosely
associated since its presence depended on providing Fe
in the purification buffer. Although Fe
bound
to FNR in this way had little effect on DNA binding and FNR was still
monomeric(6) , some functional significance was attributed to
the presence of this loosely bound Fe
, because this
form of FNR was reported to increase open complex formation at an
FNR-dependent promoter(12, 13) . To reconcile the
apparent differences in the type of Fe cofactor associated with
wild-type FNR and the FNR* mutant protein, we have considered that
wild-type FNR may contain an oxygen-labile Fe-S cluster which is
required for increased DNA binding and dimerization in the absence of
oxygen, and that the Fe
found associated with
purified wild-type FNR may be a remnant of a labile cluster.
A potential reason for the different properties of purified FNR* proteins and wild-type protein is that, until recently, a reliable functional assay was lacking to monitor purification of FNR. Thus, previously isolated FNR may have lost a component(s) during purification, such as an Fe-S cluster, necessary for maximum FNR activity. Therefore, we developed an assay to monitor DNA binding by FNR in cell extracts in the presence and absence of oxygen, because analysis of the FNR* mutants suggested that DNA binding by wild-type FNR should be regulated by oxygen. Using this assay, we purified and characterized a form of FNR which exhibited increased DNA binding under anoxic conditions. This preparation of FNR was also found to have an increased ability to dimerize and contained an Fe-S cluster.
FNR protein was partially purified from cell extracts by fractionation over a Bio-Rex 70 column essentially as described previously (3) but employing the following modifications. All steps in the purification were done under anoxic conditions at room temperature. The buffers contained 1.7 mM sodium dithionite and lacked EDTA and dithiothreitol. One-ml fractions were assayed for DNA binding, and the fractions containing the peak of DNA binding by FNR were pooled.
The purity of the FNR protein preparations was estimated from Coomassie-stained SDS-polyacrylamide gels. The percentage of the cell extract protein which was FNR protein was determined by analyzing Western blots (17) of SDS-polyacrylamide gels run with multiple concentrations of cell extract protein and purified FNR protein. The gels or blots were scanned using a Color One Scanner (Apple Computer Inc.) and processed using Scan Analysis software (BioSoft) (data not shown).
Figure 1:
DNA binding by FNR present in
anoxically prepared cell extracts. A, gel-retardation assays
were performed as described under ``Materials and Methods''
under anoxic conditions (lanes 1-6) and under aerobic
conditions (lanes 7-12). The concentrations of cell
extract protein assayed in lanes 1-6 and 7-12 are 0, 14,
28, 56, 110, and 220 ng/µl respectively. The migration of the
2.7-kilobase pair plasmid DNA (Vector DNA), the 48-base pair
DNA fragment containing the consensus FNR site (FNR-Target
DNA), and the FNR-DNA complex (Complex) are indicated. B, quantitation of the percentage of the 48-base pair DNA
fragment bound by FNR assayed under anoxic conditions () or
aerobic conditions (
) as a function of the concentration of
protein in anaerobic cell extracts.
The amount of DNA binding by FNR that was present in the cell extracts, was also regulated by oxygen (Fig. 1). Under anoxic conditions, FNR bound approximately 90% of the DNA at the highest concentrations of protein assayed (Fig. 1A, lanes 2-6, and Fig. 1B). However, assaying these same extracts in the presence of oxygen decreased the amount of DNA bound to 20% even at the highest concentrations of protein assayed (Fig. 1A, lanes 8-12, and Fig. 1B), which is similar to the amount of DNA bound by FNR purified from aerobic cells(5) .
Figure 2:
Fractionation of anoxically prepared cell
extracts by cation-exchange chromatography (Bio-Rex 70). FNR fractions
that contained oxygen-regulated DNA binding were identified by assaying
1 µl of every third fraction and plotting the percentage of the FNR
target DNA bound by these fractions either under anoxic conditions plus
dithionite () or aerobic conditions (
). The molar
concentration of KCl used to elute FNR(- - - - -) is also plotted.
Shown above the graph is the section of a SDS-polyacrylamide
gel which contains 10 µl of every third fraction to show the amount
of FNR protein in each fraction.
Figure 3: SDS-polyacrylamide gel electrophoresis analysis of FNR that was anoxically purified by cation-exchange chromatography. Lane 1 contains the cell extract (10 µg) loaded on to the Bio-Rex 70 column and lane 2 shows 10 µg of the pool of Bio-Rex 70 fractions containing the peak of oxygen-regulated DNA binding by FNR.
Figure 4:
DNA binding by anoxically purified FNR.
Quantitation of the percentage of the FNR DNA-target site bound by FNR
under anoxic conditions in the presence of dithionite () or
aerobic conditions (
) as a function of protein
concentration.
Despite the recovery of a form of FNR which behaved similar to that found in the cell extracts, only a fraction of the oxygen-regulated DNA binding activity that was originally present in cell extracts was recovered in this purification step (Table 1). This was determined by two criteria. First, the 5-fold increase in the purity of the FNR protein following cation-exchange chromatography (Fig. 3) does not correspond to the 1.5-fold decrease in the amount of FNR protein required for half-maximal DNA binding. Second, only approximately 30% of the oxygen regulated DNA binding activity present in cell extracts was recovered in the purified fraction. This loss of FNR which possessed oxygen-regulated DNA binding activity suggests that this activity is labile. In addition, it was difficult to further purify this form of FNR, since such attempts resulted in a substantial loss of oxygen-regulated DNA binding.
Figure 5: Gel-filtration analysis of anoxically purified FNR. The absorption at 280 nm of the eluent from the Sepharose 12 HR column has been plotted versus the elution volume in milliliters. Shown are the elution profiles of the pooled Bio-Rex 70 fractions run on the column in the absence (A) and presence (B) of oxygen as described under ``Materials and Methods.'' The presence of FNR in the two fractions indicated by arrows was verified by SDS-PAGE. C, the molecular weight of the protein in these two fractions was determined by comparison to the following standards: BSA, carbonic anhydrase, and cytochrome c. The peaks corresponding to an apparent dimer and monomer of FNR are indicated by the arrows. The large slowly eluting peak at 18 ml is dithionite.
Figure 6: Ultraviolet/visible absorption spectrum of anoxically purified FNR protein analyzed at 2.0 mg/ml.
Figure 7: Effect of oxygen on ultraviolet/visible spectrum of FNR. Shown are the spectra of FNR (1.2 mg/ml) in the absence of oxygen (solid line) and 1 min (stippled line) and 20 min (dashed line) after exposure to oxygen.
Low temperature EPR spectroscopy
was performed on the anoxically purified FNR-LH28-DA154 protein (data
not shown). Under anoxic conditions, the protein was EPR silent as
would be expected for a [4Fe-4S] cluster. When the protein was oxidized by the addition of
potassium ferricyanide, a signal typical of a
[3Fe-4S]
cluster was detected. This
strongly indicates that the type of cluster which was present under
anoxic conditions and was EPR silent must have been a
[4Fe-4S]
cluster (see above). Thus,
it appears that the amino acid substitution in the FNR* mutant protein
stabilizes the association of the same [4Fe-4S] cluster
that is found with wild-type FNR. To determine the stoichiometry of the
[4Fe-4S] cluster relative to the FNR-LH28-DA154
protein, iron and sulfide analyses were performed. Iron and sulfide
were found associated at maximally 4.0 mol of iron and 3.3 mol of
inorganic sulfide/mol of FNR-LH28-DA154 monomer. Based on the
concentration of inorganic sulfide, the stoichiometry was calculated to
be 1.65 mol of [4Fe-4S] cluster/mol of FNR dimer which
indicates that there are two [4Fe-4S] clusters per
dimer of FNR-LH28-DA154 protein.
The aim of this study has been to gain additional insight into the mechanism of the regulation of FNR activity by oxygen. The development of an in vitro functional assay, which mimics how regulation of FNR occurs in vivo, provides an important tool to dissect what factors are involved in mediating oxygen regulation. The purification of a form of FNR which has oxygen-regulated activity has shown that an Fe-S cluster is associated with wild-type FNR and that this Fe-S cluster appears to be required for the increased DNA binding and dimerization of this protein in the absence of oxygen. This study also extends the previous analysis of an FNR* mutant protein, FNR-LH28-DA154, which was more suitable for determining the stoichiometry of the Fe-S cluster relative to the FNR protein.
Figure 8:
Model for the association of the
Fe-S cluster with FNR and regulation of FNR activity by oxygen. A, a hypothetical model of an FNR monomer. This was
constructed by attaching the N-terminal extension of FNR (depicted by a thin line) to the existing ribbon diagram of the CAP
structure(22) ; the letters and numbers are the CAP
nomenclature for -helices and
-sheets, respectively.
Indicated in gray is the dimerization, C-helix. The N terminus
of FNR in the presence of the Fe-S cluster (solid line)
and in the absence of the Fe-S cluster (dashed line) is
indicated, as well as the structure of a [4Fe-4S]
cluster. B, a model for regulation of FNR activity by the
stability of a [4Fe-4S] cluster to oxygen. Inactive
refers to the form of FNR that is predominant under aerobic conditions
and shows decreased DNA binding activity. Active refers to the form
that shows enhanced DNA binding activity and is present predominantly
under anaerobic conditions.
If the structure of FNR is similar to that of CAP, then dimerization should occur along the C-helix (Fig. 8). Therefore, it seems most likely that one monomer would provide all the ligands to the cluster since the location of the cysteines would be too far apart to allow the Fe-S clusters to bridge the subunits. To confirm the location of the Fe-S cluster in the FNR protein requires a three-dimensional structure of FNR.
Although both the
FNR-LH28-DA154 and wild-type FNR proteins appear to contain a
[4Fe-4S] cluster, the percentage of protein containing
a cluster was considerably lower for the wild-type protein (32% based
on the S) as compared to the FNR-LH28-DA154 protein
(82% based on the S
). One possible explanation for
this result is that the amino acid substitutions in the FNR-LH28-DA154
protein stabilize the cluster against degradation in the presence of
oxygen. This is supported by the observation that an Fe-S cluster
is associated with the aerobically purified FNR-LH28-DA154 mutant
protein but not with wild-type FNR(3, 6) . However,
the lability of the Fe-S cluster to oxygen does not seem to fully
explain why in wild-type FNR not all the protein contains an Fe-S
cluster. Even in anaerobic cell extracts, where enzymes and substrates
should be present to assemble an Fe-S cluster, a large percentage
of the FNR protein appears to be inactive for DNA binding, suggesting
that not all the FNR protein originally present in the cell extracts
contains an Fe-S cluster. It may be that naturally only a
fraction of the FNR protein contains an Fe-S cluster. Such a
situation is seen with the iron-regulatory protein(26) , which
occurs in tissues in both the iron-regulatory protein (apo) or
aconitase (containing Fe-S) forms. It is also possible that,
because FNR, under the control of the T7 promoter, is
expressed to high levels in these cells, a factor may be limiting which
is required for association of the Fe-S cluster. It may be that
the amino acid substitutions in FNR-LH28-DA154 bypass the requirement
for this factor. Further experiments will be necessary to determine why
the wild-type protein is not fully occupied by the Fe-S cluster.
The fact that the Fe-S-containing form of FNR was not the
major FNR fraction observed by cation-exchange chromatography may
explain why this form of FNR has not been previously isolated and
underscores the importance of having a functional assay for FNR
activity. Previously, FNR was purified based on immunoblotting of
SDS-polyacrylamide gels for FNR
protein(2, 6, 27) , and therefore, it is not
surprising that FNR purified based on this criterion did not contain
detectable amounts of Fe-S cluster. In addition, it is likely
that the lability of the Fe-S cluster to oxygen was also partly
responsible for the previous failure to isolate a form of FNR
containing the cluster(6) . The one mol of
Fe/mol of FNR found in previous studies (6) may be a remnant of an oxygen-labile Fe-S cluster.
The inclusion of a strong reductant, dithionite, in the purification
buffers may have improved our ability to stabilize the Fe-S
cluster.
The Fe-S clusters of hydrolyase proteins and glutamine phosphoribosylpyrophosphate amidotransferase have been shown to be degraded by oxygen both in vitro and in vivo(29, 30, 31, 32) . A mechanism for this degradation of the Fe-S clusters in the hydrolyase proteins involves the attack by superoxide(29) . Consistent with this, the clusters of some of these proteins have been shown to be protected from degradation by the presence of superoxide dismutase both in vitro and in vivo(29, 32, 33) . It must be pointed out, however, that the Fe-S clusters in hydrolyases have only three Cys ligands and, therefore, one accessible cluster iron to which substrate can be bound. Although it is not certain, it is nevertheless likely that the Fe-S cluster in FNR has four Cys ligands so that a different mechanism of degradation may have to be invoked. Whether superoxide is involved in the degradation of the Fe-S cluster of FNR is not known, and it will be interesting to determine if superoxide dismutase protects the Fe-S cluster of FNR from degradation by oxygen. If FNR is inactivated by superoxide, then the Fe-S cluster of FNR must be exquisitely sensitive to superoxide because FNR activity is lost in vivo under normal atmospheric conditions, whereas the hydrolyase proteins are inactivated in vivo only when the concentration of oxygen is increased.
When the observed in vitro properties of the wild-type and FNR* mutant proteins are considered, the association of an Fe-S cluster appears to stabilize a conformation that increases the dimerization constant. This increase in the number of FNR dimers, which presumably have maximal DNA binding affinity, would result in an increase in transcriptional regulation under anaerobic conditions in vivo. Analysis of FNR* mutants has indicated that amino acid 154 is a critical determinant in dimer formation(3, 25) . This amino acid is located at the putative dimerization interface (as shown in Fig. 8), and in wild-type FNR, this amino acid is a negatively charged aspartic acid. Changing this position to a neutral amino acid, alanine, also results in an increase in dimerization which suggests that under aerobic conditions, the aspartic acid is inhibitory to dimer formation. Therefore, a possible mechanism for the Fe-S cluster increasing the ability of FNR to dimerize is that a long range conformational change is induced by the presence of the Fe-S clusters that either moves the aspartic acid side chain away from the dimer interface or brings in close contact a positively charged group to neutralize Asp-154.
In summary, we have provided evidence that anoxically purified FNR contains a [4Fe-4S] cluster, with a stoichiometry of apparently two clusters per FNR dimer. This Fe-S-containing FNR is capable of oxygen-regulated DNA binding and dimerization. On this basis, we propose that the Fe-S cluster is required for FNR activity and the regulation of this activity by oxygen. The data presented in this study provide important information about how FNR may be functioning as an oxygen sensor. Recently, Fe centers have been found in other regulatory proteins. As far as involvement of Fe-S clusters in regulation is concerned, the iron-regulatory protein is an excellent example as to how Fe-S cluster formation or degradation can determine its function (26) . In addition, the SoxR transcription factor has been shown to contain a [2Fe-2S] cluster(35, 36) . In this case, the protein is activated by oxidative stress in vivo, but the role of the Fe-S cluster in mediating regulation has yet to be elucidated. A different Fe center, a heme moiety, is utilized by the sensor component of the FixJK two-component system of Rhizobium meliloti to regulate the kinase activity of FixL on FixJ in response to oxygen deprivation(28, 37) . Thus, it appears that FNR, as do other proteins, utilizes the versatile coordination and redox chemistry of iron for regulatory functions.