Department of Biochemistry and Molecular
Biology, Eberly College of Science, The Pennsylvania State University,
University Park, Pennsylvania 16802-4500
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INTRODUCTION |
Two-thirds of the biologically
produced methane in nature originates from the methyl group of acetate
in a pathway where acetate is cleaved and the methyl group is reduced
to methane with electrons derived from oxidation of the carbonyl group
to carbon dioxide (7). Much is known concerning the cleavage
of acetate and one-carbon transfer reactions; however, less is known
regarding electron transport. Recently, a novel iron-sulfur
flavoprotein (Isf) from the acetate-utilizing methanoarchaeon
Methanosarcina thermophila was characterized (3,
12). The homodimeric Isf contains two flavin mononucleotide (FMN)
molecules and two 4Fe-4S clusters unequivocally identified by electron
paramagnetic resonance (EPR) and Mössbauer spectroscopy. The
midpoint potential values of the 4Fe-4S cluster and FMN are 
394 and
277 mV, respectively. These results are the basis for a postulated
role for the cluster in electron flow from ferredoxin A, the
physiological electron donor for Isf, to the 4Fe-4S cluster and then to
the FMN of Isf. The physiological electron acceptor for Isf is unknown.
The deduced sequence of Isf contains six cysteines, four of which are
in an unusually compact novel motif with high identity to a motif
(CX2CX2CX4-7C) that is conserved
among all homologous Isf sequences identified in the databases. This
observation suggests that the motif ligates the 4Fe-4S clusters in Isf;
however, corroborating biochemical evidence has not been reported.
The cubane 4Fe-4S cluster is ubiquitous in proteins from all domains of
life, where it mainly functions in electron transfer (17).
The sulfur atom of cysteine is the prominent protein ligand coordinated
to iron atoms in these clusters. The coordination of 4Fe-4S clusters by
amino acids other than cysteine is rather uncommon. Examples of
variations from cysteine ligation include aconitase with oxygen
ligation originating from hydroxide, water, or substrate. The 4Fe-4S
cluster in the ferredoxin from Pyrococcus furiosus is
ligated with oxygen from aspartate (21). An iron atom in the
4Fe-4S cluster of hydrogenase from Desulfovibrio gigas is
coordinated by a histidyl nitrogen (20). A single motif
(CX2CX2C plus a distal C in the polypeptide
chain) coordinates all low-potential, redox-active 4Fe-4S clusters for
which cysteine is the exclusive ligand. Possible exceptions to this
ubiquitous 4Fe-4S motif are found in the corrinoid/iron sulfur proteins
of M. thermophila and Clostridium
thermoaceticum, and a putative iron-sulfur protein from
Rhodobacter capsulatus, where the sequence
CX2CX4CX16C is perfectly conserved
(13); however, investigations of involvement of this motif
in ligation of 4Fe-4S clusters have not been reported. Thus, the highly
conserved CX2CX2CX4-7C motif in
Isf is the most compact motif known with the potential to coordinate a
4Fe-4S center. Although the great majority of iron-sulfur proteins function in electron transfer reactions, the clusters in a few proteins
function in nonredox catalysis or serve a structural role. Still other
iron-sulfur clusters bind nucleic acids or play a regulatory role
(4, 10). Two of these, endonuclease III and MutY, contain a
redox-inert 4Fe-4S cluster coordinated by a compact cysteine motif
(CX6CX2CX5C) (15, 16).
Using site-specific replacement of residues ligating the clusters
(14), much has been learned regarding the coordination of
iron-sulfur clusters, particularly the identities of ligating residues.
Changes in spectroscopic properties and other characteristics have
provided information regarding the polypeptide environment of the
cluster and the effects that the coordinating ligands have on the
biochemical properties of the cluster. Thus, a series of site-specific
replacements in Isf from M. thermophila were performed to
obtain corroborating biochemical evidence for the proposed role of the
novel cysteine motif and further characterize the properties of the
4Fe-4S cluster dependent on ligation. The results support involvement
of the motif in coordination of the 4Fe-4S cluster.
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MATERIALS AND METHODS |
Sequence comparisons.
Microbial genomic sequence databases
were searched online (http://www.tigr.org). Sequences were aligned
using the program Clustal X, version 1.64b.
Plasmid construction and site-directed mutagenesis.
Plasmid
pML701, which contains the entire gene for Isf, was used as a template
to construct mutants. Site-directed mutagenesis was performed using
MORPH as described by the manufacturer (5 Prime 
3 Prime, Inc.,
Boulder, Colo.). Each construct was confirmed for the intended mutation
by sequencing using the automated dideoxy method at the Pennsylvania
State University nucleic acid facility.
Protein production and purification.
Escherichia coli
BL21(DE3) cells transformed with derivative expression plasmids
carrying the designated isf mutations were grown on
Luria-Bertani broth supplemented with ampicillin (100 µg/ml). Once
cells reached an A600 of about 0.8, they were
induced to produce high levels of the Isf variants by addition of 1%
(wt/vol [final concentration]) Bacto-Lactose for 2 h. The cells
were harvested by centrifugation at 11,800 × g for 10 min at 4°C. The cell pellets were frozen at 70°C.
The C16S variant and wild type were purified as described elsewhere
(12). All other variants were purified as follows.
Approximately 5 g (wet weight) of cells was suspended in 6 volumes
(wt/vol) of buffer A (50 mM Tris-HCl [pH 7.6], 200 µg of
lysozyme/ml, 2 mM dithiothreitol [DTT]) and incubated for 20 min at
21°C. Cells were lysed by two passages through a French pressure cell
at 20,000 lb/in2. The lysate was centrifuged at
10,000 × g for 30 min at 4°C. The pellet, containing
inclusion bodies, was washed twice in 30 ml of buffer B (50 mM Tris-HCl
[pH 7.6], 2 M urea, 1% Triton X-100, 2 mM DTT). The protein
aggregates were solubilized in 2 ml of buffer C (50 mM Tris-HCl [pH
7.6], 6 M guanidine-HCl) and incubated for 2 h at 21°C.
Insoluble protein was removed by centrifugation at 10,000 × g for 10 min at 4°C. The protein solution at this stage is
termed denatured. The soluble fraction was then diluted 100-fold in
buffer D (50 mM Tris-HCl [pH 7.6], 500 mM L-arginine, 2 mM DTT) and incubated at 4°C for 12 h. In the following step, the sample was concentrated using polyethylene glycol 8000. The protein
was dialyzed in buffer E (50 mM Tris-HCl [pH 7.6], 250 mM
L-arginine, 200 mM NaCl, 2 mM DTT) and then buffer F (50 mM Tris-HCl [pH 7.6], 200 mM NaCl, 2 mM DTT). The protein at this stage
is defined as renatured. There was no apparent change in subunit size
among the wild type and variants as judged by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The overall
procedure resulted in homogeneous proteins as judged by SDS-PAGE.
Reconstitution of iron-sulfur clusters and FMN into renatured
apoprotein.
Reconstitution of iron-sulfur clusters and FMN was
performed by adding 1 ml of 10 mM FMN, 800 µl of 2-mercaptoethanol,
300 µl of 60 mM FeCl3, and 300 µl of 60 mM
Na2S to 100 ml of renatured apoprotein solution (2,
11). All reagents were added dropwise with 10-min intervals
between steps, and the reconstitution reaction mixture was incubated at
4°C for 12 h. This procedure is identical to the one used for
reconstitution of iron-sulfur clusters into the PsaC subunit of
photosystem I (2, 11). The protein was concentrated with an
ultrafiltration unit fitted with a YM 30 membrane (Amicon, Beverly,
Mass.), and the unbound molecules were removed by a PD10 gel
filtration. The protein at this step is called reconstituted. Iron and
FMN were determined as previously described (12). Reduction
with ferredoxin A was as described elsewhere (12).
Spectroscopy.
UV-visible spectra were obtained with a
Hewlett-Packard 8452A diode array spectrophotometer. EPR signals of
iron sulfur clusters were recorded with a Bruker ECS 106 EPR X-band
spectrometer operating with an ER/4102 ST or 4116 DM resonator and an
Oxford liquid helium cryostat (Oxford Instruments, Oxford, United
Kingdom). The temperature was controlled using an ITC4 Oxford
temperature controller. The microwave frequency was determined with a
Hewlett-Packard 5340A frequency counter. The spectrometer conditions
are described in the figure legends and Results. The term oxidized is
used to denote proteins as they were purified and without the addition
of sodium dithionite. To detect the EPR signal of
[4Fe-4S]1+ clusters, the sample was reduced by the
addition of 0.1 ml of 1.0 M pH 10 glycine buffer to 0.2 ml of sample,
thus changing the pH of the sample to 10, followed by the addition of
approximately 1 mg of sodium dithionite. It was necessary to increase
the final pH of the sample to 10.0, thereby lowering the solution
potential to ensure full reduction of low potential iron-sulfur
clusters (2). This standard procedure is identical to that
previously described for analysis of PsaC (2). Binary EPR
data were processed with a macro command program written by I. Vassiliev using Igor Pro, version 3.14 (Wavemetrics), as described
elsewhere (19). EPR spectra were recorded at different times
using several different X-band resonators, which operate on slightly
different microwave frequencies. This resulted in small differences in
the positions of identical signals when plotted against the magnetic
field axis. Therefore, we plotted spectra against the
g-value axes by converting experimental data points recorded
against magnetic field reference values using the formula
g = 714.484·f/H, where f is
microwave frequency in megahertz and H is magnetic field in
Gauss. This procedure allowed the direct comparison of EPR spectra on
the same scale. The top axes of the EPR spectra show the magnetic field
scales, which were calculated back from the g-value scale by
employing the same formula and a frequency of 9.4676 MHz (4102 EPR
cavity) (11). This procedure allowed plotting signals using identical g-value and magnetic field scales, which allowed
for a direct comparison of the signals. Apparent g values
are used for the description of EPR data.
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RESULTS |
Sequence comparisons of Isf from M. thermophila with
Isf homologues.
Figure 1 shows that
metabolically diverse species contain open reading frames with deduced
sequence identity to M. thermophila Isf, suggesting that it
functions in carbon dioxide-reducing (Methanococcus jannaschii and Methanobacterium thermoautotrophicum)
and sulfate-reducing (Archaeoglobus fulgidus) members of the
domain Archaea, and also in metabolically diverse
procaryotes from the domain Bacteria (Chlorobium
vibrioforme, Chlorobium tepidum, and Clostridium
difficile). Comparison of these sequences with Isf from M. thermophila shows an unusually compact N-terminal cysteine motif
with a strictly conserved spacing of
CX2CX2CX4-7C, which is atypical of the cysteine motif coordinating all known redox-active 4Fe-4S centers.
The absence of motifs known to ligate redox-active 4Fe-4S clusters and
conservation of this novel motif among Isf sequences suggest
involvement in ligation of the 4Fe-4S clusters in Isf; thus, we
undertook a biochemical approach to obtain corroborating experimental
evidence for the proposed role of the motif in the Isf from M. thermophila and further investigate properties of the 4Fe-4S
cluster dependent on ligation.
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FIG. 1.
Multiple amino acid sequence alignment of Isf from
M. thermophila (MST) with sequences deduced from open
reading frames identified in the genomic sequences of
Methanococcus jannaschii (MCJ), Methanobacterium
thermoautotrophicum (MBT), Archaeoglobus fulgidus (AF),
Chlorobium vibrioforme (CV), Chlorobium tepidum
(CT), and Clostridium difficile (CD). Numbers after the
abbreviated organism names indicate the different protein isoforms.
Database codes for proteins: MST, GenBank U50189; MCJ-1 and -2, GenBank
C64391 and B64435; MBT-1, -2, and -3, GenBank AE000802, AE000908, and
AE000919; AF-1, -2, and -3, GenBank AE0010041, AE0009971, and
AE0009721; CV, EMBL Z83933.1; CT, C tepidum gct10; CD, CD shotgun.dbs
cd2h6.q1t. Cysteines (at positions 16, 47, 50, 53, 59, and 180) in Isf
of M. thermophila are numbered above the top line. Residues
conserved in at least 7 out of 10 sequences are shaded in gray.
Putative FMN binding regions in Isf are underlined.
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Heterologous production, purification, and reconstitution of
wild-type Isf and variants.
All six cysteines present in Isf (Fig.
1) were individually altered to either alanine or serine. When produced
in E. coli, all variants except C16S were contained in
inclusion bodies. The soluble C16S was purified the same as for the
wild type. The presence in inclusion bodies is indicative of protein
misfolding, a characteristic of variant iron-sulfur proteins in which
the cluster is absent or inserted incorrectly (14). Indeed,
no iron-sulfur clusters were detected by EPR spectroscopy for any of
the isolated inclusion bodies containing variants. These results are
consistent with the proposal that the conserved cysteine motif
comprised of Cys 47, Cys 50, Cys 53, and Cys 59 ligates the 4Fe-4S
cluster in Isf from M. thermophila. However, it is also
possible that any of the variants misfolded as a consequence of factors
unrelated to the inability to incorporate the 4Fe-4S cluster; thus,
reconstitution of an iron-sulfur cluster and FMN was attempted. The
isolated inclusion bodies were extracted in guanidine hydrochloride,
which solubilized the proteins. At this juncture, no discrete bands were detected by native PAGE, suggesting that the proteins were denatured. The solubilized variants were diluted in buffer containing arginine, which was necessary to prevent protein aggregation during renaturation (1, 6, 18). After removal of the arginine by
dialysis, native PAGE indicated no discrete bands, suggesting that the
proteins had not achieved the native state. Iron-sulfur clusters and
FMN were not detected by UV-visible spectroscopy; thus, the apoproteins
were incubated in the presence of ferric iron, sulfide,
2-mercaptoethanol, and FMN in an attempt to reconstitute the redox
centers. Native PAGE (Fig. 2) indicated a
discrete band for each variant migrating to approximately the same
position as the purified wild type, a result which suggested that all
were in the native conformation and dimeric, in accord with the wild type (12). UV-visible spectroscopy (Fig.
3 and 4)
indicated incorporation of FMN and iron-sulfur clusters. Neither FMN
nor iron-sulfur clusters could be reconstituted separately, suggesting that both are required for proper folding of the protein. There was no
apparent change in subunit size among wild-type Isf and variants as
judged by SDS-PAGE (data not shown). There was intermittent success and
low yields in the reconstitution of C59A/S and C180A/S, indicating that
these variants were unstable. The identical
denaturation/renaturation/reconstitution process was performed for the
wild type. As for the variants, only the reconstituted wild type
exhibited a discrete band after native PAGE (Fig. 2). These results
suggested overall structural integrity of the C16A/S, C47A/S, C50A/S,
and C53A/S variants; thus, the presence in inclusion bodies is likely
the result of the inability to incorporate the 4Fe-4S cluster or FMN,
or both.
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FIG. 2.
Native PAGE of wild-type Isf and variants. (A)
As-purified wild type (Isf), reconstituted wild type (RC-Isf), and
alanine variants. Each lane was loaded with 25 µg of protein except
for C180A, which was loaded with 18 µg. (B) As-purified wild type
(Isf) and serine variants. The Isf, C47S, C53S, and C180S lanes were
loaded with 18 µg of protein; all other lanes were loaded with 25 µg of protein.
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FIG. 3.
UV-visible absorption spectra of wild-type Isf and
alanine variants. The samples (500 µl) were in 50 mM Tris-Cl (pH 7.6)
containing 200 mM NaCl. Spectra were recorded at 21°C. The amount of
as-purified wild-type Isf was 80 µg; the amount of each variant was
120 µg.
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FIG. 4.
UV-visible absorption spectra of wild-type Isf and
serine variants. The samples (500 µl) were in 50 mM Tris-Cl (pH 7.6)
containing 200 mM NaCl. Spectra were recorded at 21°C. The amount of
as-purified wild-type Isf was 80 µg; the amount of each variant was
120 µg.
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Characterization of reconstituted wild-type Isf and variants.
The UV-visible spectra of denatured and renatured wild-type Isf showed
no absorbance characteristic of either iron-sulfur clusters or FMN
(Fig. 5), suggesting the complete loss of
both redox components. The UV-visible spectrum of the reconstituted wild-type Isf was nearly identical to that of the as-purified wild
type, suggesting that the properties of both proteins were similar.
However, the intensity of absorbance between 350 and 500 nm was
threefold less for reconstituted than for as-purified Isf, suggesting
incomplete incorporation of FMN and iron-sulfur centers. This trend
applied to all of the reconstituted variant proteins. Both iron and FMN
were present in reconstituted wild type, C16A/S, C47A/S, C50A/S, and
C53A/S. The contents ranged from 3.7 to 7.2 iron atoms/dimer and from
0.5 to 1.7 molecules of FMN/dimer; however, the content of iron and FMN
was variable between preparations, precluding accurate comparisons
among the reconstituted variants and wild type. The iron and FMN
contents were not determined for the remaining variants due to low
yield and instability. The UV-visible absorption spectra for the
reconstituted C16A/S variants were also similar to the wild-type
spectra (Fig. 3 and 4); however, the absorbance maxima and relative
intensities in the spectra for the C47A/S, C50A/S, C53A/S, C59A, and
C180A variants were a departure from spectra for reconstituted
wild-type Isf and the C16A/S variants. These results suggest
alterations in the properties of either or both redox centers in all
except the C16A/S variants. The UV-visible spectrum of C59S contained no features characteristic of iron-sulfur cluster incorporation, a
result that was confirmed by EPR spectroscopy (data not shown).
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FIG. 5.
UV-visible absorption spectra of as-purified, denatured,
renatured, and reconstituted wild-type Isf. The denatured protein was
in 50 mM Tris-Cl (pH 7.6) containing 6 M guanidine HCl. The
as-purified, renatured, and reconstituted proteins were in 50 mM
Tris-Cl (pH 7.6) containing 200 mM NaCl. The spectra were recorded at
21°C. The amounts used were 80 µg (as-purified wild-type Isf) and
150 µg (all others).
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Ferredoxin A reduced the as-purified and reconstituted wild type at
similar rates when normalized to the FMN content (as purified, 
A476 = 0.52 ± 0.03/min/µmol of
FMN; reconstituted, A476 = 0.59 ± 0.03/min/µmol of FMN). The C16A/S, C47A/S, C50A/S, and C53A/S variants were all reduced with ferredoxin A; however, the rates were
highly variable between preparations, precluding accurate comparisons.
Measurements for reduction of C180A/S or C59A/S were not possible due
to instability of these variants.
EPR spectroscopy of reconstituted wild-type and variants.
EPR
spectroscopy was performed for the wild type and variants to further
characterize the reconstituted iron-sulfur centers. A summary of the
results is presented in Table 1. The full
power and temperature dependence profiles of the signals were recorded for all reduced resonances discussed. All resonances had temperature optima of around 15 K, and none were saturated under the following conditions: temperature, 15 K; microwave power, 20 mW; modulation amplitude, 10 G. Thus, none of the signals shown in Fig.
6 and 7 are perturbed due to saturation.
Resonances of wild-type Isf and all variants are shown under identical
experimental conditions, allowing direct comparisons (Fig. 6 and 7).
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FIG. 6.
EPR spectra of wild-type Isf and alanine variants. (A)
Reduced as-purified wild-type Isf. (B to F) Reduced reconstituted
wild-type Isf, C16A, C47A, C53A, and C180A variants. (G and H) Oxidized
reconstituted C50A and C59A variants. EPR conditions: temperature, 15 K; microwave power, 20 mW; modulation amplitude, 10 G. The intensity of
the EPR signal presented on trace H was multiplied by 10 for
presentation purposes.
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FIG. 7.
EPR spectra of reduced reconstituted C16S (A) and C180S
(B) variants. EPR conditions: temperature, 15 K; microwave power, 20 mW; modulation amplitude, 10 G.
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The spectra recorded for reduced samples of wild-type Isf, C16A/S,
C47A, C53A, and C180A/S were broadened beyond observation above 40 K, a
result typical for reduced [4Fe-4S]1+ clusters. We were
unable to saturate these signals even when high microwave powers were
applied to the samples, a quality also typical for
[4Fe-4S]1+ centers. These two lines of evidence rule out
the possibility that the observed signals derived from 2Fe-2S centers
in the wild type or any of the variants and provide strong evidence
supporting the presence of 4Fe-4S centers in these proteins.
The reduced as-purified wild-type Isf exhibited a spectrum with
apparent g values of 2.06, 2.03, 1.92, 1.86, and 1.81 (Fig. 6A), results that are nearly identical to those in a previous report
(3) in which the authors attributed the complexity of the
spectrum to heterogeneity of the sample. We were able to distinguish two distinct species based on power and temperature dependencies (data
not shown), one with apparent g values of 2.06, 1.92, and 1.81 and another with apparent g values of 2.03, 1.92, and
1.86. The ratio of these species varied in different Isf preparations, suggesting that the as-purified wild-type protein exists in two distinct conformational states. Reconstitution of wild-type Isf that
had been denatured and renatured also exhibited a
[4Fe-4S]1+ EPR spectrum with apparent g
values and linewidths identical to those for the wild-type spectrum
(Fig. 6B), with evidence for both species originally present in the
wild-type as-purified Isf spectrum.
The reduced C16A/S variants exhibited EPR spectra with apparent
g values of 2.06, 2.04, 1.92, 1.86, and 1.82 (Fig. 6C and 7A), identical to results for wild-type Isf with two distinct 4Fe-4S
species. These results indicate that Cys 16 does not participate in
ligation of the 4Fe-4S cluster of Isf, consistent with sequence comparisons showing that Cys 16 is not conserved with Isf homologues (Fig. 1). The reduced C180A/S variants showed EPR spectra (Fig. 6F and
7B) with linewidths and apparent g values (2.04, 1.93, and
1.86) nearly identical to those for one of the 4Fe-4S species present
in the as-purified wild-type Isf spectrum. A minor contribution of the
other species was also observed as a shoulder at g = 2.06.
Except for C59S, UV-visible spectroscopy of the reconstituted variants
suggested that an iron-sulfur cluster was present; thus, these variants
were further examined by EPR spectroscopy. Spectroscopy of the reduced
C50A or C59A variants detected no [4Fe-4S]1+ cluster;
however, spectra of the oxidized variants showed a low-intensity signal
with linewidths and g values typical for
[3Fe-4S]1+ clusters (Fig. 6G and H). EPR spectra of
oxidized as-purified or oxidized reconstituted wild-type Isf showed no
features indicating a [3Fe-4S]1+ cluster. These results
show that removal of Cys 50 and Cys 59 causes formation of the 3Fe-4S
cluster in place of the 4Fe-4S cluster, a result which clearly
establishes that these cysteines are involved in ligation of the 4Fe-4S
cluster. A [4Fe-4S]1+ cluster was detected in both of the
reduced C47A and C53A variants by EPR spectroscopy (Fig. 6D and E);
however, there were significant differences in the linewidths and
g values between the spectra of the two variants. The
linewidths and g values for both variants were also
significantly different from the spectra of either of the two species
present in the reduced form of as-purified or reconstituted wild-type
Isf (compare Fig. 6A and B and Fig. 6D and E). A low-intensity
[3Fe-4S]1+ EPR signal was detected in the oxidized C47A
and C53A variants (data not shown).
The EPR spectra (not shown) of the oxidized and reduced C47S, C50S, and
C53S variants indicated only the presence of 4Fe-4S clusters; however,
the spectral features did not contribute toward understanding the
identity of the ligands that replaced cysteine.
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DISCUSSION |
Several lines of evidence support the view that the cysteine motif
comprised of Cys 47, Cys 50, Cys 53, and Cys 59 ligates the 4Fe-4S
cluster in Isf from M. thermophila. EPR spectroscopy strongly indicates involvement of Cys 50 and Cys 59. First,
low-potential 4Fe-4S clusters are observed by EPR spectroscopy in only
the reduced +1 state; thus, the inability to detect
[4Fe-4S]1+ clusters in reconstituted C50A and C59A serves
as strong evidence that 4Fe-4S centers are not formed in these
variants. Second, there are several examples of 4Fe-4S3Fe-4S
conversions resulting from the substitution of a ligating cysteine
(14); thus, EPR evidence for [3Fe-4S]1+
clusters in oxidized C50A and C59A adds convincingly to the evidence that Cys 50 and Cys 59 are involved in ligation of the 4Fe-4S cluster
in Isf. The results also show that other ligands cannot substitute for
Cys 50 and Cys 59 to preserve the 4Fe-4S cluster in these variants.
EPR results strongly support a role for Cys 50 and Cys 59 in ligation
of the 4Fe-4S cluster, and together with Cys 47 and Cys 53, they
comprise an unusually compact motif with high identity to a motif
(CX2CX2CX4-7C) present in
homologous Isf sequences. The conservation of this motif in Isf
homologues from metabolically diverse species in the domains
Bacteria and Archaea, representing the extremes
of evolution, is consistent with all four cysteines of the motif
serving a common function. Nonetheless, EPR spectra for the
reconstituted C47A and C53A variants indicated the presence of
[4Fe-4S]1+ clusters which could be used as an argument
against involvement of these residues in ligation of the iron-sulfur
center. The linewidths and apparent g values for the spectra
of C47A and C53A (Fig. 6D and E; Table 1) differ significantly from
those of wild-type Isf (Fig. 6A and B). This difference implies that
either the ligands to the 4Fe-4S clusters in C47A and C53A changed or
there was an overall change in the protein conformation. The occurrence
of 4Fe-4S clusters in the C47A and C53A variants could be explained by
other residues replacing Cys 47 and Cys 53 in a process called ligand
swapping, for which there is precedent in other iron-sulfur proteins
(14). Although ligand swapping is one possibility, it is
also possible that 2-mercaptoethanol is an external thiolate ligand
replacing Cys 47 and Cys 53 in these variants for the following reasons. First, reconstitution of the variants required
2-mercaptoethanol, a compound which has been shown to serve as an
external ligand to the 4Fe-4S cluster in the C51D and C14G variants of
the PsaC subunit of photosystem I in Synechococcus sp.
strain PCC 7002 (2, 11). Second, the reconstitution
conditions used in this work were nearly identical to the conditions
used to reconstitute iron-sulfur clusters in PsaC. Clearly, a more
rigorous investigation, most likely involving other techniques, is
needed to determine the role of Cys 47 and Cys 53 in ligation of the
4Fe-4S cluster and the possibility of ligand swapping occurring in the
C47A and C53A variants.
The compact nature of the cysteine motif proposed to coordinate the
4Fe-4S cluster in Isf is unusual compared to motifs known to coordinate
low-potential redox-active 4Fe-4S clusters where one of the cysteines
are located remote in the sequence from the other three (typically
CX2CX2C and a distant C). Thus, it is possible that each of the two 4Fe-4S centers in Isf is coordinated by three cysteines in the motif from one subunit and the fourth cysteine from
the adjacent subunit. Examples of 4Fe-4S clusters bridging protein
subunits are the nitrogenase Fe-protein and the FX cluster in photosystem I (8, 9). If the iron-sulfur clusters bridge the subunits in Isf, this would be the first example of a protein with
two bridging iron-sulfur clusters. Unfortunately, the failure of
approaches to separate the subunits with iron-sulfur clusters intact
precluded resolution of this question.
In addition to the proposed motif ligating the 4Fe-4S cluster, two
other cysteines (Cys 16 and Cys 180) are present in the Isf sequence
(Fig. 1). Similar properties (including linewidths and g
values of the EPR signals) between the C16A/S variants and wild type
serve as clear evidence that Cys 16 is not involved in coordination of
the 4Fe-4S cluster. It should be noted, however, that variant C16A was
produced in inclusion bodies, suggesting that this residue is important
for proper folding of Isf. The C16S variant was produced as a soluble
protein, and sequence comparisons (Fig. 1) indicate that a threonine
residue in Isf homologues replaces Cys 16 of the M. thermophila Isf. These results suggest that a hydroxyl or
sulfhydryl group is important in this position for maintaining a native
conformation. Although the evidence supports noninvolvement of Cys 16 in ligation of the 4Fe-4S cluster, the evidence for noninvolvement of
Cys 180 is weaker. The presence of C180A/S in inclusion bodies and
instability of the reconstituted variants indicates improper folding
which could be due to the loss of Cys 180 as a ligand to the 4Fe-4S
cluster or an overall conformational change in the protein due to
substitution of Cys 180. There is a possibility that Cys 180 is a
ligand for about 50% of the clusters, accounting for the two distinct
EPR species in the wild type. This possibility, however, is weakened by
the fact that, in addition to the major species, there is a minor species in the spectra of the C180A/S variants with at least one of the
three apparent g values (g = 2.06) that is
clearly equivalent to one of the two species present in the wild type.
Another possible explanation would be that substitution of Cys 180 changes the overall protein conformation, making one of the two protein
conformations present in the wild type more preferable than the other.
Hence, we observe one of the species present in the wild-type Isf, with only a minor contribution from the other. Unfortunately, our results do
not allow us to unequivocally distinguish between these two possibilities.
This investigation has also provided insight into 4Fe-4S cluster and
FMN self-assembly for Isf in vitro. The results show that insoluble
wild-type apo-Isf can be solubilized and refolded to a conformational
state that can be reconstituted with the 4Fe-4S cluster and FMN which
is reduced with ferredoxin A at a rate comparable to as-isolated Isf.
The results further show that both a 4Fe-4S cluster and FMN must be
incorporated to adopt the native conformation. The presence of arginine
was essential to achieve the conformational state necessary for
reconstitution. It is proposed that arginine helps to reshuffle
molecules trapped in nonproductive reactions, which results in
increased refolding efficiency (5). The EPR properties of
reconstituted wild-type Isf indicated that the procedure for refolding
and reconstituting apo-Isf yielded a 4Fe-4S cluster with an environment
identical to that for the wild type, suggesting that reconstituted Isf
has a conformation similar, if not identical, to that of as-purified
Isf. The ability to reconstitute apo-Isf in vitro provides a tool for
investigating properties of the iron-sulfur cluster and FMN, for
example, reconstitution with flavin analogs to probe the function of
FMN. The ability to refold and reconstitute apo-Isf suggests that
accessory factors are not essential for in vivo synthesis of Isf;
however, the results showed that in vitro reconstitution was mostly
incomplete, suggesting that a significant fraction of apo-Isf was
improperly refolded. This result, and the requirement for arginine,
suggests the possibility that a chaperonin may be important for
efficient folding in vivo. It also cannot be ruled out that accessory
proteins are necessary for efficient incorporation of the 4Fe-4S
cluster and FMN.
We are indebted to John Golbeck, in whose laboratory the EPR
spectra were recorded and analyzed. Also we thank him for advice regarding experiments and preparation of the manuscript. We also thank
R. C. Thauer for suggestions concerning the arginine refolding method and extend a special thank you to Rebecca D. Miles and Squire
Booker for critical reading of the manuscript.
Work described here was partially supported by the Department of Energy
Basic Energy Sciences grant DE-FG02-95ER20198 (J.G.F.) and National
Science Foundation grant MCB-9723661 to John H. Golbeck. U.L. was
supported by a Thailand MOSTE grant.
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Abstract
Introduction
