From the CEA, Département de Biologie
Moléculaire et Structurale, Laboratoire
Métalloprotéines, 17 rue des Martyrs,
38054 Grenoble Cedex 9, France and § Institut für
Biochemie I, Molekulare Bioenergetik, Universitätklinikum
Frankfurt, ZBC, D-60590 Frankfurt/Main, Germany
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ABSTRACT |
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The 2[4Fe-4S] ferredoxin from Chromatium
vinosum arises as one prominent member of a recently defined
family of proteins found in very diverse bacteria. The potentiometric
circular dichroism titrations of the protein and of several molecular
variants generated by site-directed mutagenesis have established that
the reduction potentials of the two clusters differ widely by almost
200 mV. This large difference has been confirmed by electrochemical
methods, and each redox transition has been assigned to one of the
clusters. The unusually low potential center is surprisingly the one
that displays a conventional
CX1X2CX3X4C
(Xn, variable amino acid) binding motif and a
structural environment similar to that of clusters having less negative
potentials. A comparison with other ferredoxins has highlighted factors
contributing to the reduction potential of [4Fe-4S] clusters in
proteins. (i) The loop between the coordinating cysteines 40 and 49 and
the C terminus -helix of C. vinosum ferredoxin cause a
negative, but relatively moderate, shift of ~60 mV for the nearby
cluster. (ii) Very negative potentials, below
600 mV, correlate with
the presence of a bulky side chain in position
X4 of the coordinating triad of cysteines.
These findings set the framework in which previous observations on
ferredoxins can be better understood. They also shed light onto
the possible occurrence and properties of very low potential
[4Fe-4S] clusters in less well characterized proteins.
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INTRODUCTION |
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[4Fe-4S] clusters are present in a wide variety of proteins;
they are often coordinated to amino acids organized in characteristic motifs (1) corresponding to protein domains, sometimes identified by
x-ray crystallography (see Ref. 2 and references therein). A pair of
[4Fe-4S] clusters is found in many bacterial ferredoxins (3) and
other electron transfer proteins and enzymes (see Fig. 1 in Ref. 4). In
most cases these clusters are bound to the proteins through a pair of
CXXCXXC ... CP (X, variable amino
acid) motifs containing all necessary cysteine ligands. Extensive work on the simplest ferredoxins over the last 35 years has established that
the two clusters are relatively close, at a distance of approximately 10 Å thus enabling them to interact magnetically, and that they display similar reduction potentials, generally around 400 ± 100 mV (normal hydrogen electrode). Until now, these generic properties have not been conclusively challenged in any fully characterized molecule containing such 2[4Fe-4S] domains. Proteins with homologous coordinating motifs are known in which one of the [4Fe-4S] clusters is substituted by a [3Fe-4S] center, often as a result of the loss of
one of the cysteine ligands (5). Consequently, these proteins
exhibit properties that are clearly different from those of molecules
containing two [4Fe-4S] centers (6).
Recently, the 2[4Fe-4S] ferredoxin from the purple sulfur photosynthetic bacterium Chromatium vinosum has been further investigated (7), following its initial characterization (8), in an effort to assess the influence of specific sequence elements (including a 6-amino acid insertion between two cysteine ligands and a long C-terminal extension) on the properties of the [4Fe-4S] clusters. It was found that, in contrast to other 2[4Fe-4S] ferredoxins without these elements, the electronic communication between the two clusters was apparently impaired (7). The gene encoding C. vinosum ferredoxin (CvFd)1 was later cloned and expressed in Escherichia coli (9), and the structure of the protein was determined (10). These studies provide the necessary background for exploring the structure-function relationships in CvFd by protein engineering, and this report presents our initial efforts toward this aim.
The properties of Fe-S clusters can be probed by a number of spectroscopic methods. The absorption spectra are generally broad as they result from overlapping charge transfer bands in the visible-near UV range. The resolution of these spectra can be significantly improved by the implementation of circular dichroism (CD) which provides both a characteristic pattern for each cluster type and a sensitive monitor of changes affecting either the cluster or the protein (11). The use of the method as an accurate analytical tool is broadened when the CD spectra of electron transfer proteins can be monitored as a function of the applied potential (12). Indeed, both a measure of the reduction potential and an estimate of the structural changes induced by the addition or removal of electrons are then provided.
The results of such studies, together with direct electrochemical measurements, are reported herein for CvFd and a series of molecular variants. The comparison with the well characterized 2[4Fe-4S] ferredoxin from Clostridium pasteurianum (CpFd) reveals that the [4Fe-4S] clusters in CvFd display unusual redox properties which help explain previously observed differences between these proteins (7). These data also provide unprecedented compelling evidence that the protein domains containing two [4Fe-4S] clusters found in widely different biological systems do not always display similar physicochemical and functional properties.
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EXPERIMENTAL PROCEDURES |
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Preparation of Proteins-- Site-directed mutagenesis was carried out on plasmid pCVFD11 encoding CvFd (9) using two rounds of polymerase chain reaction as described previously (13). The oligonucleotides bearing the mutations are listed in Table I. The plasmids were sequenced with the dideoxynucleotide termination method (14). The genes bearing the correct mutations were expressed in E. coli K38/pGP1-2 (15), as described previously (4), and the synthesis of ferredoxin was checked by [35S]cysteine labeling (16). For large scale (20 liters) production, cells were grown at 30 °C until they reached an optical density measured at 600 nm of about 1.5. They were then induced for 1 h at 42 °C, and the synthesis of ferredoxin proceeded for about 5 h at 30 °C before harvest. Purification of the CvFd derivatives was as previously outlined for other 2[4Fe-4S] ferredoxins (17). It is of note that none of the CvFd derivatives studied herein had a significantly different chromatographic behavior compared with the native protein. However, the changes introduced into the amino acid sequence were often easily verified by a range of spectroscopic methods (see below).2 The 2[4Fe-4S] ferredoxin from E. coli (9) was overproduced and similarly purified.
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Electrochemistry--
Cyclic voltammetry and square wave
voltammetry were performed using a previously described device (18).
The potential was controlled through an Autolab PGSTAT-10 potentiostat
(Deutsche Metrohm, Filderstadt, Germany). The working electrode was a
gold disc, the counter electrode a platinum wire, and the reference electrode a standard saturated Ag/AgCl electrode (Radiometer, Copenhagen). All potentials in this work are referenced to the normal
hydrogen electrode, taking E(Ag/AgCl) = 197.6 mV. The
concentrations of the protein samples were 1-2 mM, in 10 mM potassium phosphate buffer, pH 7.5. An attempt to
perform electrochemical studies on a pyridine-3-carboxaldehyde
thiosemicarbazone-modified gold electrode was not successful. However,
a very good response was obtained in the presence of 0.2 mM
viologens; considering the range of potentials investigated, a mixture
of methyl viologen (E10 455 mV)
and 4',4"-dimethyl-1',1"-trimethylene-2',2"-dipyridinium dibromide
(E10
680 mV) was used. The working
electrode was poised at
800 mV before each voltammogram was
taken.
Circular Dichroism and Absorption Spectroscopy--
The setup
for optical measurements and redox titrations was as in previous
studies (12). The concentrations of the protein samples for CD
spectroscopy in the visible range were 1-2 mM, in 10 mM potassium phosphate buffer, pH 7.5, and contained 0.6 mM each of benzyl viologen
(E10 360 mV), methyl viologen
(E10
445 mV),
1',1"-trimethylene-2',2"-dipyridinium dibromide
(E10
550 mV), and
4',4"-dimethyl-1',1"-trimethylene-2',2"-dipyridinium dibromide
(E10
680 mV). The last two
compounds were synthesized according to Ref. 19. The potential was
applied between 15 and 30 min before recording the CD spectra during
titration experiments; it was checked that no further spectral changes
occurred after this equilibration time over the potential range
investigated. The path lengths of the cells were 0.1 mm for work in the
visible range and 10 or 20 µm between 180 and 280 nm. Absorption
spectra were similarly recorded, but the solutions contained the
protein and the four viologens at concentrations of 0.8 mM
and 20 µM, respectively.
Dithionite Reduction of CvFd-- Ferredoxin solutions (concentrations of 0.1-0.2 mM) in 20 mM potassium phosphate buffer, pH 8.2, were kept under argon inside an anaerobic chamber (Jacomex, Livry-Gargan, France) ensuring an oxygen concentration of less than 1.5 ppm. The proteins were reduced by the addition of a 30-fold molar excess of sodium dithionite (Eastman Kodak), and absorption spectra were regularly recorded with a Hewlett-Packard model 8453 spectrophotometer and a previously described set-up (4).
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RESULTS |
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Molecular Variants of CvFd
Fig. 1 highlights the targets for
site-directed mutagenesis investigated in CvFd. The first set of
variants involves modifications of the protein environment around
cluster II which is coordinated by Cys-18, Cys-37, Cys-40, and Cys-49.
In the variant referred to as 1 the loop (residues 41-48) between
two of the cysteine ligands of cluster II (10) has been replaced by two
residues (Ala-Gln), therefore converting the sequence to a more
conventional binding motif (CXXCAQC ... CP) for
[4Fe-4S] clusters. In the K74
variant, half (residues 74-82) of
the C-terminal
-helix has been removed. In
1K74
the two
modifications were combined, and in K74E the buried hydrophilic side
chain of lysine (10) has been substituted by glutamic acid. In two more
variants, Y44C and Y44S, residue Tyr-44 of loop
1 has been replaced
by a cysteine or a serine.
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The second set of variants, namely D12G, V13G, and Y30F, contains single site replacements introduced close to cluster I which is coordinated by Cys-8, Cys-11, Cys-14, and Cys-53.
Circular Dichroism Spectroscopy
Since CvFd apparently deviates from other 2[4Fe-4S] ferredoxins in its ability to convey intramolecular electron transfer between its clusters (7), evidence for unusual spectroscopic properties was sought using CD spectroscopy.
CD Spectra and Electrochemical Titration of CpFd--
For
reference, the visible CD spectra recorded during the redox titration
of native CpFd are shown in Fig. 2. These
data qualitatively confirm and extend previous reports (11, 20). The
spectra of the fully oxidized protein with [4Fe-4S]2+
clusters contain several moderately intense features all over the
spectral range shown. Upon reduction, a significant decrease in the
intensity of all these bands occurs; at 600 mV a positive band with a
maximum at 360 nm and a shoulder at 380 nm is the main feature of the
spectrum (Fig. 2). In contrast, the far UV region (Fig.
3) shows little, if any, change in the
relatively weak negative band around 200 nm. These observations are
consistent with the lack of extensive secondary structure elements in
clostridial ferredoxins (21). Moreover, they suggest that only small
global structural changes are induced by adding one electron to each cluster.
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CD Spectra and Electrochemical Titration of CvFd--
The CD
spectra of oxidized CvFd are almost identical to those of CpFd in the
300-800 nm range (Fig. 2). Therefore, the optical properties of the
oxidized clusters of CvFd do not differ from typical
[4Fe-4S]2+ clusters, such as those of CpFd (Fig. 2).
Below 300 nm, however, significant changes between CpFd and CvFd are
noticed (Fig. 3). The relatively intense negative band around 220 nm in
CvFd is indicative of the presence of the -helix evidenced in the
x-ray structure of the protein (10). This secondary structure element is absent in CpFd and has been found to produce different signatures when either the 2-turn loop or the
-helix are removed or modified in
CvFd (not shown). Another difference between the two proteins occurs at
258 nm where a positive band is found in the spectra of CvFd but not in
those of CpFd (Fig. 2).
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Visible Absorption Spectroscopy
In order to sustain the above observations, the visible absorbance
of CvFd was also monitored as a function of the applied potential. The
decrease of absorbance at 425 nm is a measure of the degree of
reduction of [4Fe-4S]2+/+ clusters, as the spectra of the
reduced clusters are less intense than those of the oxidized ones (24).
Fig. 5 shows the visible absorption spectra of oxidized (applied
potential, E = 200 mV), partially (E =
600 mV), and fully reduced (E =
730 mV) CvFd. Dithionite appears as effective as electrochemical reduction at
600
mV (not shown) but does not provide the largest decrease in absorbance
as judged by comparison with the spectrum recorded at
730 mV. Only
the latter data agree with absorbance changes observed in a sample
reduced by chloroplasts under illumination (8).
Thus, the above titrations carried out with CvFd strongly suggest that the protein displays two redox transitions. Qualitatively similar results were also obtained with molecular variants of CvFd, and all reduction potential values are listed in Table II. However, such results may be biased, for instance by the involvement of a kinetic barrier slowing down the reduction of one cluster and apparently shifting the midpoint potential of the redox transition. This possibility has been addressed by additional experiments.
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Reduction Potentials by Cyclic and Square Wave Voltammetries
Typical voltammograms of native CpFd and CvFd are shown in Fig.
6. CpFd shows only a single transition at
approximately 400 mV, whereas CvFd shows two transitions at
approximately
460 and
655 mV. The strong intensity of the single
wave of CpFd agrees with the above observation that both [4Fe-4S]
clusters react at this potential. Control experiments, with mediator
mixtures (methyl viologen and
4',4"-dimethyl-1',1"trimethylene-2',2"-dipyridinium dibromide) in the
absence of protein, were carried out and showed that there is only a
minor contribution of the mediator mixture to the electrochemical
signal in the presence of the protein (Fig. 6). The reduction
potentials determined for all forms studied are listed in Table II.
From this series of measurements, it appears that the more negative
value of CvFd redox potential (
655 mV) is shifted in variants with
substitutions in the vicinity of cluster I, i.e. D12G and
V13G; in these molecules the potential with the less negative value
(
460 mV) agrees with the value measured for the native protein.
Conversely, shifts of the less negative but not of the more negative
redox potential were observed in variants, such as
1, K74
,
and
1K74
, structurally modified around cluster II. These data
indicate that the redox transition at
460 mV (native CvFd) arises
from cluster II and the lowest transition at
655 mV is due to cluster
I. Proteins carrying the C-terminal truncation displayed partially
broadened and split electrochemical signals, so that some potentials
could not be determined reliably. This electrochemical behavior
correlates with a decreased stability of these proteins which was also
observed during the CD-monitored redox titrations and other experiments
(not shown). No significant changes were found for Y30F, as already
observed with similar variants of other ferredoxins (27).
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Kinetics of the Interaction between the Electrode and the Protein
2[4Fe-4S] ferredoxins show quasi-reversible electrode kinetics
at the gold electrode in the presence of viologens up to scan rates of
1 V/s. At high scan rates, the separation between the anodic and the
cathodic peak increased with increasing scan rate; from this, the
heterogenous electron transfer rate constant k0
(28) could be estimated (Table III),
assuming a diffusion coefficient D = 106
cm2/s which is consistent with the observed peak current.
Quite remarkably, different electrode kinetics were observed for
cluster I (Em =
655 mV) and cluster II
(Em =
460 mV); the heterogenous rate constant for
the oxido-reduction of the latter was determined as approximately
4 × 10
3 cm/s, whereas the former showed reversible
electrode kinetics up to scan rates of 0.5 V/s. Therefore, a lower
limit of 2 × 10
2 cm/s was estimated for the
heterogenous rate constant of cluster I in all variants studied. These
values support the statement that no fast electron transfer takes place
between the clusters (7); otherwise, oxidoreduction of both clusters
could proceed rapidly via cluster I.
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Electron transfer to cluster II is faster in the 1 and
1K74
forms in which it is comparable to electron transfer to cluster I. This
result is consistent with dithionite reduction kinetics discussed
below. The other structural changes studied in this work had less
significant effects on the electron transfer kinetics except for Y44C
which displayed an increased heterogenous rate constant by a
factor of approximately 3-fold. It is conceivable that cysteine in this
position facilitates electron transfer to and from cluster II by
interacting with the gold electrode or that the removal of the aromatic
ring makes cluster II more exposed.
Surprisingly, the heterogenous rate constant determined for CpFd, which agrees with previously reported ones (29, 30), is closer to those of CvFd cluster II than to those of CvFd cluster I (Table III). This is another noteworthy difference between the latter and CpFd clusters, despite their structural similarity (10).
Effect of Ionic Strength and pH
The effect of ionic strength on the values of the reduction potential of native CvFd has been measured in solutions with NaCl concentrations up to 1 M. A linear relationship between the redox potential and the square root of the ionic strength was observed over the whole range studied with a slope of +35 mV/M0.5 for both clusters. The variation is similar to that obtained in previous studies on [4Fe-4S]2+/+ clusters in other proteins (23).
The effect of pH has also been studied. At pH values equal to or below
6, the electrochemical response was not satisfactory in the implemented
conditions. Although the ionic strength was not kept constant at the
different pH values, the weak dependence with ionic strength observed
at pH 7.5 dismisses a major contribution of this parameter. Only at pH
values above 10 do the reduction potentials of both transitions
significantly shift; these data may indicate the binding of hydroxide
ions to the clusters and associated changes in their electronic
properties. At these high pH values, structural modifications of the
protein must also be considered. The case of cluster II is interesting
because, in contrast to cluster I, this group displays an increase of
the redox potential with increasing pH opposite to the effect of
electrostatic repulsion between the
[Fe4S4(Cys)4]2/3
cluster and nearby deprotonated residues, such as His-43.
Kinetics of Dithionite Reduction of CvFd
From the comparison of the absorption spectra described above,
dithionite appears to be able to reduce only cluster II in CvFd. The
decrease with time of the absorbance at 425 nm has been analyzed as a
pseudo first-order kinetic process; the rate constants calculated using
the added dithionite concentration are listed in Table III. It is
easily concluded that in the molecular variants missing either loop
1 or half of the C terminus
-helix, reduction of cluster II takes
place at least 10 times faster compared with native CvFd.
The same kinetic effect is also observed in the case of K74E, thus providing evidence of significant structural changes around cluster II in molecules in which Lys-74 is changed. The side chain of Lys-74 is involved in stabilizing interactions around cluster II (10), and its replacement or its removal are expected to perturb the structural environment of cluster II. Despite the similarities observed between the kinetics of dithionite and electrode reduction of the molecules listed in Table III, these reactions do not exactly obey the same mechanism as shown by the qualitative differences exhibited by CpFd and cluster II of CvFd Lys-74 variants (slow electrode kinetics and fast dithionite reduction).
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DISCUSSION |
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The results presented herein reveal a difference of approximately 200 mV between the reduction potentials of the clusters in a 2[4Fe-4S] ferredoxin. Although previous studies were conducted with CvFd (22, 23), including studies under experimental conditions very similar to those implemented here (25), the very negative redox transition exhibited by CvFd escaped detection.
Existence of Two Redox Transitions--
The voltammograms of CvFd
and all studied derivatives consistently showed the occurrence of two
redox transitions in these proteins. The CD redox titrations fully
agree with these conclusions: very similar CD spectra were obtained for
reduced CpFd and CvFd, but although the oxidized spectra of the former
protein readily disappeared at 500 mV, those of the latter required
potentials below
700 mV. Similarly, the decrease in absorbance at 425 nm exhibited by CpFd upon dithionite reduction (~55%, see Refs. 23 and 31) could only be reached by applying a potential of
730 mV in
the case of CvFd. Dithionite reduction of the latter protein resulted
in a decrease of ~25%, i.e. a value roughly half of that corresponding to full reduction. Therefore, two independent methods of
investigation, voltammetry and optical titrations, point toward the
involvement of two distinct redox transitions in the case of CvFd.
Furthermore, the spectroscopic features observed in the course of the
potentiometric titrations showed no evidence for cluster
interconversion, from a [4Fe-4S] to a [3Fe-4S] cluster for
instance, a now largely documented occurrence in [4Fe-4S] proteins
(32). Indeed, the close similarity of the CD spectra recorded with CpFd
and CvFd and the reversibility of the CD-monitored titration do
indicate that the clusters remain in the [4Fe-4S] form during these
experiments and that the electrochemical signals have to be attributed
to the redox properties of genuine [4Fe-4S] clusters. The formation
of [3Fe-4S]+/0 clusters is expected to be accompanied by
the development of an intense CD band around 450 nm as in the case of
mitochondrial aconitase (33), which has never been detected in our
experiments. The electrochemical observation of separate redox waves
has very recently been obtained with a newly isolated 2[4Fe-4S]
ferredoxin from Azotobacter vinelandii apparently bearing
strong structural similarities with the proteins investigated herein,
but the two redox transitions have not been assigned, and no structural
reasons for their occurrence have been put forward (34).
Assignment of the Redox Transitions to the Two Clusters--
The
two redox transitions of CvFd are sensitive to some extent to the
modifications introduced in the sequence of the protein (Table II).
Remarkably enough, the lowest potential transition is affected when
amino acids close to cluster I are changed, whereas the less negative
one more specifically responds to replacements introduced close to
cluster II. These assignments are supported by additional spectroscopic
evidence to be reported elsewhere. This brings forth the surprising
conclusion that the cluster of CvFd (cluster II), the environment of
which was shown by comparison of oxidized x-ray structures to differ
most from that of clusters in clostridial ferredoxins, exhibits a
moderate shift in potential of approximately 60 mV, whereas the other
cluster for which no obvious structural difference was detected at this
level of resolution displays a huge negative shift of about
250
mV.
Factors Contributing to the Reduction Potential of Cluster
II--
From the comparison of the values measured with native CpFd,
and with the 1, K74
, and
1K74
variants (Table II), the loop between Cys-40 and Cys-49 and the C terminus
-helix clearly
contribute to the negative shift of the potential of cluster II
compared with CpFd. When the two structural features are removed in
1K74
, the reduction potential difference is almost completely
abolished, showing that a structural environment very similar to that
in CpFd has thus been engineered around cluster II in CvFd. It is likely that a hydrogen bond equivalent to that between Gly-41 NH (or
Gly-12 NH) and one inorganic sulfur in clostridial type ferredoxins
(21) that is absent in native CvFd (10) has been recovered in
1K74
.
Why Is the Potential of Cluster I So Negative?--
The very low
potential redox transition in CvFd has been assigned to cluster I based
on the absence of significant potential shifts in the case of molecules
bearing changes close to cluster II and on a selective shift in the
case of the D12G and V13G (Table II) modifications around cluster I. The only marginal (approximately 15 mV) exceptions are for molecules
missing the 1 loop which may undergo a general adjustment of the
protein structure. Contrary to the case of cluster II, no obvious
structural features distinguish CvFd cluster I from the clusters of
clostridial ferredoxins (10). The exposed side chain of Asp-12
contributes some 30 mV to the value of the potential of cluster I in
CvFd, in a way reminiscent, but probably not identical (see below), to
the case of the H35D/D41H form of A. vinelandii ferredoxin I
(38).
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Comparisons with Other Proteins--
To date only a few
2[4Fe-4S] domains in proteins have been reported to contain clusters
with different values of reduction potentials. These range from
moderate differences as in subunit PsaC of photosystem I (44) and a
molecular variant of Rhodobacter capsulatus 2[4Fe-4S]
ferredoxin (45), up to larger ones in the cases of E. coli
nitrate reductases A and Z (46) and dimethyl sulfoxide reductase (47).
Since CvFd is well characterized (7, 9, 10), it could now become a
standard model to study the above unusual redox properties, all the
more so as proteins of this family display the largest potential
difference among all these examples. The quest for a rationale to the
very negative value of the potential of CvFd cluster I may also benefit
from previous observations made with other systems containing low
potential (<600 mV) [4Fe-4S]2+/+ clusters, such as
center Fx of photosystem I (44) or the nitrogenase Fe protein (48).
Functional Consequences-- The function or the redox partners of CvFd are not known, and only some hypotheses have been put forward in the past (49). Contrary to other 2[4Fe-4S] ferredoxins, CvFd is unable to couple electron transfer between C. pasteurianum pyruvate-ferredoxin oxidoreductase and hydrogenase, although the reduction potential of its cluster II lies in the range considered suitable to fulfill this function (50). In the light of the findings presented here (very low potential of cluster I), CvFd behaves in this assay like ferredoxins containing a single [4Fe-4S] cluster. This may indicate that the two nearly isopotential and symmetrical clusters of 2[4Fe-4S] ferredoxins are necessary to complete an efficient electron transfer chain between these enzymes.
The demonstration of an extremely low potential [4Fe-4S] cluster belonging to a tandem of such closely spaced centers has further consequences. For instance, the exact stoichiometry of Fe-S centers in a number of NADH-ubiquinone oxidoreductases remains a matter of some dispute (51), mainly because of the discrepancies between the potential coordinating motifs found in sequences and the relatively low number of signals detected with various spectroscopies. It seems possible that if some of the clusters present in these complex systems display properties similar to those of cluster I of CvFd, they may easily escape observation, hence accounting for part of the discrepancies. It will then be important to establish the functional role of such clusters, and the study of relatively simple molecules like the ferredoxins investigated in the present work may prove easier to fulfill this goal than that of complicated enzymes, such as complex I of the mitochondrial respiratory chain. ![]() |
ACKNOWLEDGEMENTS |
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Professor B. K. Burgess is thanked for communicating unpublished data. Discussions with Professor R. Cammack about the early characterization of CvFd and the content of the manuscript have been very useful. Dr J. Meyer is thanked for reading the manuscript.
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FOOTNOTES |
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* This work was supported in part by Grant Li 474/7 from the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: CEA/Grenoble, DBMS-MEP, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France. Tel.: 33 476885623; Fax: 33 476885872; E-mail jean-marc.moulis{at}cea.fr.
1 The abbreviations used are: CvFd, Chromatium vinosum ferredoxin; Cp, Clostridium pasteurianum.
2 P. Kyritsis, O. M. Hatzfeld, T. A. Link, and J.-M. Moulis, unpublished data.
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REFERENCES |
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