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INTRODUCTION |
Ferredoxins are small, generally acidic, electron transfer
proteins that are found in all types of living systems (1). They
contain iron-sulfur ([Fe-S]) clusters ligated to the polypeptide backbone primarily via cysteine residues. When the first ferredoxin was
discovered in Clostridium pasteurianum, it was thought to be
a general cellular electron carrier that could participate in several
different metabolic processes (2). Today it is clear that a single
organism may have numerous ferredoxins, each distinguished by sequence,
[Fe-S] cluster type, and [Fe-S] cluster reduction potential (1, 3,
4).
The most common cluster type is the [4Fe-4S]2+/+ cluster,
and the first ferredoxins isolated were found to contain two such
clusters in an ~6,000 molecular weight polypeptide (1, 5). These "clostridial-type" ferredoxins, represented by the structurally characterized Peptococcus aerogenes ferredoxin, contain two
Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Cys-Pro motifs for ligation of
the two [4Fe-4S]2+/+ clusters (6, 7) which are believed
to have evolved by gene duplication (8). The two clusters have
reduction potentials that are effectively identical and in the region
of
0.4 V versus SHE.1 These proteins evolved
further to produce other types of ferredoxins. Replacement of the
central Cys in one of the two Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys motifs by
another residue or insertion of two residues between the second and
third Cys resulted in formation of a [3Fe-4S]+/0 cluster
and a new class of ferredoxins, the so-called 7Fe ferredoxins, as
represented by the structurally characterized FdI from
Azotobacter vinelandii (9). The two different cluster types
in these proteins have two very different reduction potentials (10).
Another evolutionary modification of clostridial-type ferredoxins
involved retention of the two [4Fe-4S]2+/+ clusters but
insertion of additional residues into one of the two Cys motifs to give
a Cys-Xaa-Xaa-Cys-Xa7-9-Cys-Xaa-Xaa-Xaa-Cys-Pro motif (1).
This class is represented by the structurally characterized 8Fe
ferredoxin from Chromatium vinosum (11). Surprisingly,
despite the considerable difference in binding motifs for the two
clusters, it has been reported that they have the same reduction
potentials (12-14). We have now discovered an 8Fe ferredoxin (FdIII)
from A. vinelandii which contains two different binding
motifs analogous to the Chromatium protein. As described in
this paper, FdIII is the first example of any 8Fe ferredoxin having two
[4Fe-4S]2+/+ clusters with very different reduction
potentials.
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EXPERIMENTAL PROCEDURES |
Cell Growth and Protein Purification--
The A. vinelandii strain used in this study is designated DJ138/pBS122.
The parent strain DJ138 was described previously (15). The plasmid
pBS122, which was constructed as described elsewhere (16, 17), is a
derivative of pKT230 with insertion of a site-directed mutant
fdxA gene encoding a C16S variant of FdI. For protein
purification, cells were grown under N2-fixing conditions
in a 200-liter New Brunswick fermentor (17). For the experiment to
determine the effect of ammonia levels on the intracellular FdIII
levels, a separate batch of cells was grown in the presence of excess
ammonium acetate.
Cells were harvested, cell-free extracts were prepared, and the first
DEAE-cellulose column was run as for the purification of nitrogenase
(18) and FdI (16, 19) except that the heat step was omitted. FdIII
eluted at 70% of the linear 0.1-0.5 M NaCl gradient as a
well resolved brown peak exactly where the FdI peak was expected (16).
This fraction was then diluted with 2 volumes of 0.1 M
potassium phosphate buffer (pH 7.4) and loaded onto a 2.5 × 12-cm
DEAE-cellulose column that was then washed slowly with 4 liters of 0.12 M KCl in the same buffer. The greenish brown fraction
containing FdIII was then eluted with 0.3 M KCl, and
ammonium sulfate was added to 75% saturation. After centrifugation at
8,000 × g for 20 min, the pellet was resuspended in
0.025 M Tris-HCl (pH 7.4), and the resulting solution was
loaded onto a 2.5 × 100-cm Sephadex G-75 superfine gel filtration
column. FdIII eluted as a well resolved greenish brown band.
Protein Characterization--
NH2-terminal protein
sequencing was carried out after the protein was reduced and alkylated
by
-mercaptoethanol and 4-vinylpyridine at the Biotechnology
Resource Facility at the University of California, Irvine. For
spectroscopic studies all samples were prepared anaerobically under
argon in a Vacuum Atmospheres glove box using fully degassed buffers.
The protein was initially purified in the presence of dithionite. To
prepare oxidized samples the dithionite was first removed by gel
filtration, and then the samples were exposed to oxygen for at least
2 h. Circular dichroism (CD) and electron paramagnetic resonance
(EPR) samples of oxidized FdIII were then prepared by concentrating the
protein and exchanging it into 0.1 M potassium phosphate
(pH 7.4) using a Centricon-3 microconcentrator. The reduction of FdIII
was carried out by mixing well degassed FdIII, 5'-deazariboflavin and
EDTA (final concentrations 100 µM, 200 µM,
and 20 mM, respectively) in 0.1 M potassium
phosphate (pH 7.4) and then illuminating for 1 min using white light
from a slide projector. UV-visible absorption spectra were obtained with a Hewlett-Packard 8452 diode array UV-visible spectrophotometer, CD spectra were recorded using a JASCO J720 spectropolarimeter, and EPR
spectra were obtained using a Bruker 300 Ez spectrophotometer. To
determine iron content, samples were digested, and the analysis was
carried out as described elsewhere (18) using
FeCl3·6H20 to generate a standard curve with
FdI (9) and FixFd (3) as controls. Matrix-assisted laser desorption
ionization-time of flight mass spectrometry was conducted at the
Protein/Peptide Micro Analytical Facility, California Institute of
Technology.
Electrochemistry--
Purified water (~18 megohms·cm;
Millipore) was used in all electrochemical experiments. The buffers
Mes, Hepes, and Taps, and co-adsorbates neomycin sulfate and polymyxin
B sulfate, were purchased from Sigma. Other reagents were purchased
from Aldrich or British Drug House and were of at least analytical
grade. Neomycin and polymyxin solutions were prepared as concentrated
stocks (0.2 M and 15 mM (i.e. 20 mg/ml), respectively) and adjusted to pH 7.4.
An AutoLab electrochemical analyzer (Eco Chemie, Utrecht, The
Netherlands) was used to measure cyclic voltammograms. Controlled potential reductions were carried out using an Ursar Instruments potentiostat in conjunction with a Kipp and Zonen YT recorder. The
all-glass cell and three-electrode system used for protein film
voltammetry and the bulk solution voltammetry have been described previously (10, 17, 20). All potential values are given with reference
to the SHE. The saturated calomel reference electrode (SCE) was held at
22 °C at which we have adopted E(SCE) = +243 mV
versus SHE. Reduction potentials were calculated as the
average of the anodic and cathodic peak potentials, E°' = 1/2(Epa + Epc). The
sample compartment was maintained at 0 °C. The pyrolytic graphite edge electrode (surface area typically 0.18 cm2) was
polished prior to each experiment with an aqueous alumina slurry
(Buehler Micropolish: 0.3 µm for solution electrochemistry or 1.0 µm for protein film voltammetry) and then sonicated extensively to
remove traces of Al2O3.
Controlled potential electrolysis was carried out using a cell that
featured a graphite pot constructed so that the internal walls project
edge surface to the solution. The cell, which has been described
previously (21), was set up in an anaerobic glove box (Belle
Technology, Poole, U. K.) with an inert atmosphere of N2
(O2 < 2.0 ppm). For cyclic voltammetry, only the base was connected. For controlled potential electrolysis, all parts of the
electrode were used, the solution being stirred by magnetic microflea.
As a precaution to ensure homogeneity in electrochemical studies,
samples of FdIII were prepurified using FPLC under an anaerobic
atmosphere. Solutions were dialyzed into the required buffer solutions
using an Amicon 8MC unit equipped with a microvolume assembly and a YM3
membrane. For protein film experiments, the ferredoxin solution
(80-100 µM) used to coat the electrode contained 0.1 M NaCl, 25 mM Tris-HCl, and polymyxin (200 µg/ml). The pH of this solution was adjusted to 7.0 at 0 °C. The
buffer-electrolyte solution in the electrochemical cell consisted of
0.1 M NaCl, a 60 mM mixed buffer system (15 mM in each of acetate, Mes, Hepes and Taps), 0.1 mM EGTA, and 0.2 mg/ml polymyxin, adjusted to the desired
pH using HCl or NaOH at 0 °C. The freshly polished electrode surface
was painted with about 1 µl of chilled protein solution from a fine
capillary and then placed promptly into the cell solution. To stabilize
protein films, the cell solution also contained 200 µg/ml polymyxin
or 2 mM neomycin. The pH of this solution was checked after
each set of experiments, with the pH electrode calibrated at 0 °C.
For bulk solution voltammetry and controlled potential electrolysis,
ferredoxin solutions contained 0.1 M NaCl with 60 mM mixed buffer. Small aliquots of neomycin stock solution
were added (final concentration 2.0 mM) to promote a strong
and persistent electrochemical response.
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RESULTS AND DISCUSSION |
The Discovery of FdIII and Its Relationship to FdI--
When crude
extracts from nitrogen-fixing cells of wild-type A. vinelandii are separated on DEAE-cellulose with a 0.1-0.5
M NaCl gradient, three major brown peaks are observed (Fig.
1). The first two peaks correspond to the
MoFe and Fe proteins of nitrogenase, respectively, and the third
corresponds to FdI (16). The size of the third peak is proportional to
and therefore an indicator of the FdI level present. In recent years we
have constructed and purified many site-directed mutant variants of FdI
(e.g. 9, 10, 17) some of which accumulate to much lower
levels than the wild-type protein. We have observed a strong
correlation between the size of the FdI peak on the first column (Fig.
1) and the amount of material present in the cell-free extracts which
cross-reacts with polyclonal antibodies raised against denatured
gel-purified native FdI (Fig. 2). We were
therefore very surprised to find that cells expressing one particular
variant, C16S FdI, which had only very low levels of FdI that
cross-reacted to the antibody (Fig. 2), had a normal, wild-type FdI
size peak on the first DEAE-cellulose column (Fig. 1).

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Fig. 1.
Elution profiles of DEAE-cellulose columns
from three A. vinelandii strains. wt is
wild-type. FdI contains the fdxA gene, which
is interrupted with a kanamycin resistance gene. C16S, DJ138/pBS122,
harbors a plasmid expressing a FdI mutant variant C16S.
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Fig. 2.
Western blot analysis to compare the levels
of FdI and FdIII in different A. vinelandii strains.
wt is wild-type. FdI contains the interrupted
fdxA gene. C16S contains a plasmid expressing a FdI mutant
variant C16S. Equal volumes of the FdI fractions from the
DEAE-cellulose column were loaded in each lane. Upper panel, detected by the anti-FdIII antibodies; lower
panel, detected by the anti-FdI antibodies.
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Further purification of the brown "FdI" fraction from the C16S
preparation (as described under "Experimental Procedures") resulted
in a greenish brown protein solution that exhibited a single band on
Coomassie-stained SDS-polyacrylamide gels (Fig. 3). This protein did not cross-react with
antibodies raised against FdI, and for reasons described below the new
protein was designated FdIII. Once purified FdIII was available,
polyclonal antibodies were raised, and we reexamined cell-free extracts
from wild-type, FdI
, and C16S strains of A. vinelandii using the FdIII antibody. As illustrated in Fig. 2, the
results show that the levels of FdIII are much greater in cells
expressing the C16S variant of FdI than they are in either wild-type
cells or in cells that make no FdI. The levels of FdIII in the C16S
variant are also observed to be much higher than in all other FdI
mutants tested to date based on Western analysis (data not shown). Thus
FdIII specifically accumulates in response to expression of the C16S
FdI variant. The reason for this is not currently understood. However,
it should be noted that the [3Fe-4S]+/0 cluster is
implicated in a regulatory function carried out by FdI in A. vinelandii and that C16 is a ligand to this cluster (9, 15, 17,
22-24). The purification yields of FdIII from cells expressing C16S
FdI are typically 15 mg/1 kg, wet weight, of cells, compared with the
approximately 8 mg of FdI usually obtained from 1 kg, wet weight, of
wild-type cells (19).

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Fig. 3.
The native FdI, FdIII, and FixFd separated on
an 18% SDS-polyacrylamide gel stained with Coomassie Blue R-250.
FdIII, 5 µg. M.W., molecular mass standards (Novex).
FixFd, 4 µg; FdI, 10 µg. The numbers on the
left correspond to the molecular masses of the protein
standards. The numbers on the right indicate the molecular masses of FdI and FixFd apoproteins.
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FdIII Is Closely Related to C. vinosum Ferredoxin--
To identify
FdIII, the first 56 NH2-terminal amino acid sequence of the
purified protein was obtained after modification of cysteine residues
by 4-vinylpyridine (Fig. 4). Before this
study 11 small [Fe-S] proteins had been identified in A. vinelandii, many by gene sequencing (3). Surprisingly, the
sequence shown in Fig. 4 did not correspond to any of the known
ferredoxin-like proteins from this organism. Data base searches,
however, revealed that the first 56 amino acids of FdIII exhibit 77%
identity and 88% similarity with the low potential 8Fe ferredoxin from
C. vinosum. Fig. 4 compares the sequence obtained here with
sequences from other ferredoxins in the C. vinosum class. In
general, ferredoxins that contain 2[4Fe-4S]2+/+ clusters
can be divided into two classes. The clostridial-type ferredoxins have
two Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Cys-Pro motifs, whereas the
ferredoxins in the "chromatium" class shown in Fig. 4 have one
motif of that type and one more unusual
Cys-Xaa-Xaa-Cys-Xa7-9-Cys-Xaa-Xaa-Xaa-Cys-Pro motif (1).
The ligand assignment shown in Fig. 4 is derived from the x-ray
structure of C. vinosum ferredoxin (11). All eight ligand
cysteine residues lie within the available FdIII sequence and coincide
in position with the [Fe-S] cluster ligand cysteines of C. vinosum ferredoxin, thereby eliminating the possibility that FdIII
is a clostridial-type ferredoxin. Most of the ferredoxins shown in Fig.
4 contain nine Cys residues, one of which is not a cluster ligand. This
ninth Cys is also conserved in FdIII and is in a position identical to
that of the ninth Cys of C. vinosum ferredoxin.

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Fig. 4.
Sequence alignment of FdIII and bacterial
2[4Fe-4S]2+/+ ferredoxins. CvFd, protein sequence of
C. vinosum ferredoxin (48); FtFd translated from
the Francisella tularensis ferredoxin gene (49);
HiFd, translated from the Hemophilus influenzae
ferredoxin homolog gene (50); EcORF86, translated from the
Escherichia coli ORF-086 (G. Plunkett III,
GenBankTM locus ECU36841); ClFdII, protein
sequence of Chlorobium limicola FdII (51); ClFdI,
protein sequence of C. limicola FdI (52); RrFdxN,
Rhodospirillum rubrum FdI (53); AvFix, translated
from the A. vinelandii fixFd gene (28); RpFdI,
protein sequence of Rhodopseudomonas palustris FdI (54);
RcFdxN, translated from the R. capsulatus fdxN
gene (55); RlFdxN, translated from the Rhizobium
leguminosarum biovar trifoli fdxN gene (56);
RmFdxN, translated from the R. meliloti fdxN gene
(57); AvFdxN, translated from the A. vinelandii
fdxN (25); AvVnfFd, translated from the A. vinelandii vnfFd gene (27). The cluster is based on the crystal structure of C. vinosum ferredoxin (11). The calculated
molecular mass of polypeptides (the NH2-terminal Met is not
included) is from the amino acid sequence or translated nucleotide
sequence of the gene.
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The FdIII sequencing data presented here appear to bring to four the
number of ferredoxins from one organism, A. vinelandii, which have a chromatium-type sequence (3). The other three were
identified originally by gene sequencing, and all appear to be related
somehow to nitrogen fixation based on their relationships to other
genes. For example, fdxN is cotranscribed with
nifB (25), vnfFd is cotranscribed with
vnfH (26, 27), and fixFd is cotranscribed with
fixABCX (28). Only FixFd has been purified to date (3). Because of the relationship of these other proteins to nitrogen fixation we tested whether or not FdIII was nif-regulated by
growing the cells in the presence and absence of ammonia. There was no difference in the amounts of FdIII present under the two conditions as
measured either by Western analysis or by the purification of the
protein. Thus, like FdI (29), FdIII is not
nif-regulated.
FdIII Is a Monomer with a Molecular Mass of 9,920 Da--
Although
SDS-polyacrylamide gel electrophoresis is often used to determine
subunit molecular masses, we have found that it is not useful when
studying small acidic [Fe-S] proteins. This is illustrated in Fig. 3,
which compares the migration of the denatured FdIII with the migration
of molecular mass standards and two A. vinelandii
ferredoxins of known molecular mass, FdI and FixFd. As shown in Fig. 3,
FdI and FixFd migrate as if their molecular masses were 18,800 and
9,000 Da, respectively, whereas their actual polypeptide molecular
masses are known to be 12,071 (29) and 7,758 (28). Therefore other
methods were employed to determine the molecular mass.
First, matrix-assisted laser desorption ionization-time of flight mass
spectrometry shows that the FdIII apoprotein has a mass of 9,220 ± 2 Da. Based on a composition of 2[4Fe-4S]2+/+ clusters
(see below) this method gives a molecular mass of 9,924 Da for the
holoprotein. To obtain a second estimate and to compare the native
protein with the denatured protein, an FPLC-Superdex 75 gel filtration
method was used, with FdI and FixFd as standards in addition to the
commercially available standards. Again the migration of FdI and FixFd
deviated significantly from the standard curve derived from the other
four non-iron-sulfur proteins. Using only FdI and FixFd as standards,
the native molecular mass of FdIII by this method was calculated to be
10,600 ± 680 Da. Thus, like the other proteins in Fig. 4 and most
ferredoxin-like proteins, FdIII is a monomer.
Fig. 4 compares the molecular mass of FdIII with values reported for
homologous chromatium-type ferredoxins. Clearly this class can be
subdivided further into two groups, proteins with molecular masses of
6,000-7,000 Da and proteins with molecular masses of 9,000-10,000.
The increase in molecular mass results from a COOH-terminal extension.
Even when only the NH2-terminal sequences are considered,
however, the four proteins having greatest sequence similarity to
FdIII, including C. vinosum ferredoxin, also fall into the
same molecular mass class (Fig. 4).
FdIII Contains Two [4Fe-4S]2+/+ Clusters--
The
UV-visible absorption spectra of FdIII are shown in Fig.
5. The spectrum of air-oxidized FdIII
contains a broad peak at 390 nm and a shoulder at 315 nm. The shape of
this spectrum is indistinguishable from that obtained for air-oxidized
C. vinosum ferredoxin (30), FixFd (3), and other proteins
that contain two [4Fe-4S]2+/+ clusters and is quite
different from that obtained for the 7Fe FdI (10). The spectrum shown
in Fig. 5 has an
A390:A280 ratio of 0.74, which is the same as reported for C. vinosum FdI and for
other 8Fe ferredoxins. The iron content was confirmed by direct iron
analysis using A. vinelandii 7Fe FdI and the 8Fe FixFd as controls. The results gave 7.7 ± 0.4 atoms of iron/molecule of FdIII, 7.7 ± 0.1 atoms of iron/molecule of FixFd, and 6.6 ± 0.4 atoms of iron/molecule of FdI.

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Fig. 5.
UV-visible absorption spectra of FdIII in 0.0 25 M Tris-HC1, pH 7.4. Thick line, air-oxidized;
thin line, incubated in the presence of 2 mM
sodium dithionite for 45 min.
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The visible-near UV CD spectrum of oxidized FdIII is shown in Fig.
6. It exhibits two major positive
features in the visible region, one at 420 nm and the other at 580 nm.
The wavelength dependence and form of the CD spectrum are typical of
[4Fe-4S]2+/+ clusters and quite different from the
spectra exhibited by [1Fe-0S], [2Fe-2S], and
[4Fe-4S]3+/2+ clusters (30).

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Fig. 6.
Visible-near UV CD spectra of FdIII.
Protein samples are 50 µM FdIII in 0.1 M
potassium phosphate, pH 7.4. Trace a, air-oxidized;
trace b, in the presence of 50 µM
5'-deazariboflavin and 5 mM EDTA (pH 7.4), illuminated with
white light (58); and trace c, the reduced sample was
exposed to air for 5 min. The spectra were recorded with 1-nm
increments for 10 scans.
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[4Fe-4S]2+ clusters do not exhibit EPR signals at low
temperature (31). As shown in Fig.
7a, oxidized FdIII is
EPR-silent at liquid helium temperatures consistent with the presence
of [4Fe-4S]2+ clusters and showing that FdIII does not
contain a [3Fe-4S]+ cluster, which would exhibit a very
characteristic g = 2.01 EPR signal under the conditions
used (32). This further eliminates the possibility that FdIII is a 7Fe
protein and also shows that unlike some 8Fe ferredoxins that lose iron
to form 3Fe clusters upon exposure to air (5), the 4Fe clusters of
FdIII are extremely stable. Some protein-bound [4Fe-4S] clusters can
also convert to [3Fe-4S]+ clusters upon addition of
ferricyanide (33). However, incubation with a 10-fold excess of
ferricyanide produced no new EPR signals attributable to
[3Fe-4S]+ or indeed to [4Fe-4S]3+ (34), the
latter observation eliminating the albeit remote possibility (based on
sequence comparisons and visible spectra (1, 30)) that FdIII is
actually a HiPIP.

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Fig. 7.
EPR spectra of FdIII. The protein
samples are 100 µM FdIII in 0.1 M potassium
phosphate, pH 7.4. Trace a, air-oxidized, 10 K; trace
b, after reduction with 200 µM 5'-deazariboflavin, 20 mM EDTA, and white light 5.2 K. The microwave power was
2 milliwatts, the modulation amplitude was 5.1 G, and the microwave
frequency was 9.43 GHz. mT, millitesla.
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Taken together the above data lead to the conclusion that the new
protein isolated here is a 2[4Fe-4S]2+/+ ferredoxin of
the chromatium-type. Ferredoxins that are first identified by gene
sequencing are often named based on the location of the gene relative
to other known genes (e.g. FdxN (25), FixFd (28)).
Ferredoxins that are first identified by protein purification, as is
the case here, are generally numbered in the order in which they are
identified. We have chosen to name this ferredoxin FdIII because FdI
and a [2Fe-2S]-containing protein that is designated [Fe-S]II but
is sometimes referred to as FdII have both been characterized extensively from A. vinelandii (1, 35-37).
Unlike All Known 8Fe Ferredoxins, FdIII Has Two Very Different
Reduction Potentials for the Two [4Fe-4S]2+/+
Clusters--
Of the proteins shown in Fig. 4, the following have been
isolated and at least partially characterized with respect to reduction potentials: Rhodobacter capsulatus FdI (the fdxN
gene product) (38); C. vinosum ferredoxin (11-14, 39, 40);
FixFd from A. vinelandii (3) and recombinant Rhizobium
meliloti FdxN (41). As monitored by the appearance of a
g = 1.94 EPR signal due to [4Fe-4S]+
clusters, at least three of these proteins could be at least partially
reduced in solution by dithionite or a combination of dithionite,
methyl viologen, and zinc (14). As originally reported for C. vinosum ferredoxin (12), the addition of dithionite (at different
pH values ranging from 6.0 to 9.0) or electrochemically reduced methyl
viologen to FdIII did not lead to the reduction of its
[4Fe-4S]2+ clusters as evidenced by the lack of change in
either the UV-visible (Fig. 5), CD, or EPR spectra. Attempted reduction
with dithionite/methyl viologen/zinc, or Ti(III) citrate unfortunately
led to irreversible denaturation of the protein. Fig. 6 shows that the
protein could be reversibly reduced with a 5'deazariboflavin/EDTA/light
system. However, the CD data show that unlike the situation with 8Fe
ferredoxins that have two clusters with potentials around
400
versus SHE even this powerful reductant fails to reduce the
protein fully (3).
Using the same 5'-deazariboflavin/EDTA/light system, EPR samples
were prepared of FdIII (Fig. 7b). In general,
[4Fe-4S]+ clusters exhibit EPR signals with the
g values and the intensity depending on the spin state. Most
[4Fe-4S]+ clusters exhibit S = 1/2 spin and
anisotropic EPR with gav ~1.94. If two S = 1/2 clusters are present, separated by short distances, the signals
are broadened and show additional structure (3, 14, 31). The complex
gav = 1.94 (Fig. 7) exhibited by reduced FdIII
is therefore consistent with the presence of two
[4Fe-4S]+ clusters. Again the reduction is not complete,
and the spectrum integrates to only about 1.2 spins/molecule. This
result combined with the complex nature of the spectrum would be
consistent with complete reduction of one cluster and partial reduction
of the other cluster to give a mixture of FdIII molecules, either with only one cluster reduced or with both clusters reduced. This in turn
leads to the conclusion that the two clusters must have substantially different reduction potentials. Direct electrochemical methods were
employed to measure those potentials.
Fig. 8a shows the cyclic
voltammetry measured for a 60 µM solution of FdIII at pH
7.0, 0 °C, at a scan rate of 10 mV s
1. Two well
defined redox couples (we refer to these as A and B) are observed which
have reduction potentials of
486 ± 10 and
644 ± 10 mV,
respectively. The corresponding peak separations (oxidation minus
reduction) are 120 and 60 mV, and peak currents are proportional to the
square root of the scan rate up to 20 mV s
1. These
observations are as expected for a freely diffusing redox couple at a
planar electrode (42). Fig. 8b shows the voltammetry obtained for a film of FdIII measured at 100 mV s
1. Two
symmetrical signals, pairs of oxidation and reduction peaks, are
observed (A' and B', the "prime" denoting film configuration) with
reduction potentials of
466 ± 10 and
681 ± 10 mV
versus SHE, and approximately equal areas, consistent with
both couples being present in a 1:1 ratio. Small differences are
commonly observed when comparing reduction potentials measured for bulk
solution versus the protein film. The former values should
correspond to potentiometric data, whereas the advantages of the film
configuration lie in economy, sensitivity, the capability of probing
kinetics and easy determination of relative stoichiometries of
different electron transfer reactions as shown here. The generally
sharp appearance of the signals (particularly A') with just modest peak separations at 100 mV s
1 shows that the clusters in the
film-bound protein behave uniformly and are able to shuttle rapidly
between redox levels (43). As measured also by film voltammetry, each
couple exhibits a pH dependence of approximately
15 mV/pH unit with
no obvious pKa. A pH dependence of similar magnitude
has been observed for the [4Fe-4S]2+/+ cluster of
A. vinelandii FdI (10). Controlled potential coulometry at
pH 7.0 gave 1.1 ± 0.3 electron equivalents for the first electron and 1.4 ± 0.3 electrons for the second electron. These results are consistent with 1:1 stoichiometry if allowance is made for interference by trace O2.

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Fig. 8.
Cyclic voltammograms of A. vinelandii FdIII at a pyrolytic graphite edge electrode.
Buffer/electrolyte consists of 0.1 M NaC1, 60 mM mixed buffer (15 mM in each of Hepes, Mes,
acetate, and Taps), 0.1 mM EGTA, pH 7.0, 0 °C.
Panel a, solution voltammetry with scan rate 10 mV/s.
[FdIII] = 60 µM. Neomycin (2 mM) was added to promote and stabilize the response. Reduction potentials are 486 ± 10 and 644 ± 10 mV versus SHE for
couples A and B, respectively. Panel b, film voltammetry
with scan rate 100 mV/s. Polymyxin (200 µg/ml) was used as a
coadsorbate. Reduction potentials are 466 ± 5 mV and 681 ± 10 mV versus SHE for couples A' and B',
respectively.
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In general, the reduction potentials of [4Fe-4S]2+/+
clusters are known to be very sensitive to protein structure with
potentials ranging from
280 to
700 mV for different proteins (44).
It is therefore surprising that for all 8Fe proteins that have been characterized to date both [4Fe-4S]2+/+ clusters are
reported to have similar or even identical reduction potentials (44).
This is true not only for the clostridial-type ferredoxins that have
the same Cys motif (although different sequences) for both clusters,
but also for the chromatium-type ferredoxins that have two very
different motifs for the two clusters. For example, FixFd, which is in
the 6,000-7,000-Da molecular mass chromatium-type class, very clearly
shows only a single signal in cyclic voltammetry, i.e. two
couples having indistinguishable reduction potentials. For C. vinosum ferredoxin, which is in the larger molecular mass group,
the two clusters are also reported to have the same reduction
potential; this is surprising since the local structures around the two
clusters are now known to be very different (11).
The data presented here establish for the first time that a ferredoxin
can contain two [4Fe-4S]2+/+ clusters having two very
different reduction potentials. In contrast to typical 8Fe ferredoxins
in which the two clusters have indistinguishable reduction potentials
and are thus suited to both one-electron or two-electron transfers,
FdIII is expected to be more suited to undertake independent
one-electron processes. This is similar to the situation for 7Fe
ferredoxins in which replacement of one [4Fe-4S]2+/+ by
[3Fe-4S]+/0 results in about a 200-mV difference in
potential for the two clusters. Thus, the 8Fe FdIII and the 7Fe FdI are
very similar to each other not only in size and charge but also in the
reduction potentials for their two [Fe-S] clusters. The results also
highlight the increasing number of Fe-S clusters being identified which have extremely low reduction potentials, i.e. below the
thermodynamic limit for water. These include A. vinelandii
FdI (10), Center X of photosystem I (45), "hyper-reduced"
[3Fe-4S]2
clusters (21), and most recently the
[4Fe-4S] cluster of nitrogenase iron protein, which can be generated
in the all-Fe(II) level (46, 47). It is highly probable that these
newly discovered activities underpin important physiological functions
yet to be established.
We thank Professor Gordon Tollin, Department
of Chemistry, University of Arizona, for providing 5'-deazariboflavin
and for helpful discussions. We thank Professor Brian Hales, Department of Chemistry, Louisiana State University, for helpful discussions concerning interpretation of EPR data and Dr. Sarah E. J. Fawcett for
assistance with the electrochemistry.