From the Department of Molecular Biology and
Biochemistry, University of California, Irvine, California 92697 and
the § Department of Molecular Biology, The Scripps Research
Institute, La Jolla, California 92037
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ABSTRACT |
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In Azotobacter vinelandii, deletion
of the fdxA gene, which encodes ferredoxin I (FdI), leads
to activation of the expression of the fpr gene, which
encodes NADPH-ferredoxin reductase (FPR). In order to investigate the
relationship of these two proteins further, the interactions of the two
purified proteins have been examined. AvFdI forms a
specific 1:1 cross-linked complex with AvFPR through ionic
interactions formed between the Lys residues of FPR and Asp/Glu
residues of FdI. The Lys in FPR has been identified as
Lys258, a residue that forms a salt bridge with one of the
phosphate oxygens of FAD in the absence of FdI. UV-Vis and circular
dichroism data show that on binding FdI, the spectrum of the FPR flavin is hyperchromatic and red-shifted, confirming the interaction region
close to the FAD. Cytochrome c reductase assays and
electron paramagnetic resonance data show that electron transfer
between the two proteins is pH-dependent and that the
[3Fe-4S]+ cluster of FdI is specifically reduced by NADPH
via FPR, suggesting that the [3Fe-4S] cluster is near FAD in the
complex. To further investigate the FPR:FdI interaction, the
electrostatic potentials for each protein were calculated. Strongly
negative regions around the [3Fe-4S] cluster of FdI are
electrostatically complementary with a strongly positive region
overlaying the FAD of FPR, centered on Lys258. These
proposed interactions of FdI with FPR are consistent with cross-linking, peptide mapping, spectroscopic, and electron transfer data and strongly support the suggestion that the two proteins are
physiological redox partners.
Azotobacter vinelandii ferredoxin I
(AvFdI)1 is a
small iron-sulfur ([Fe-S]) protein that has been extensively
characterized by x-ray crystallography and direct electrochemical and
spectroscopic methods (1-6). This seven-iron ferredoxin contains two
different types of [Fe-S] clusters: one [3Fe-4S]+/0
cluster and one [4Fe-4S]2+/+ cluster. The first half of
the polypeptide chain folds around these two clusters in a manner
similar to most eight-iron ferredoxins that contain two
[4Fe-4S]2+/+ clusters. This NH2-terminal
region therefore represents a core structural frame shared among
different classes of ferredoxins (7). However, AvFdI
contains an additional ~60-amino acid extension at the COOH terminus
that is unique to this class of seven-iron ferredoxins. Proteins
homologous to AvFdI have been reported from a number of
organisms including Azotobacter chroococcum,
Pseudomonas stutzeri, Pseudomonas ovalis, Pseudomonas aeruginosa,
Caulobacter crescentus, Rhodobacter capsulatus, Streptomyces griseus,
Thermus thermophilus, and Bacillus schlegelii with the
pseudomonas protein sequences showing >90% identity to
AvFdI (8-13). Although it has been known for some time that
FdI has a metabolic function unrelated to nitrogen fixation that is
important for cell growth (14), the specific cellular function of FdI
and related seven-iron ferredoxins in other organisms has yet to be determined.
In 1988, it was reported that disruption of the fdxA gene
that encodes FdI in A. vinelandii led to a dramatic increase
in the levels of another small acidic protein (13). In order to investigate the mechanism of regulation by FdI, the small acidic protein that is overexpressed in response to fdxA deletion
was purified and characterized (15), and the gene encoding the protein was cloned and sequenced (16). The protein was shown to be a Mr ~29,000 NADPH-ferredoxin reductase that was
designated FPR because its physical properties and amino acid sequence
showed striking similarity to the FPR from Escherichia coli
(15, 17, 18). In E. coli, the protein appears to be part of
a system that activates at least three different enzymes involved in
anaerobic metabolism: anaerobic ribonucleotide reductase (19), pyruvate formate-lyase (20), and cobalamin-dependent methionine
synthase (21). These activation reactions not only require NADPH and FPR but also ferredoxin or flavodoxin and
S-adenosylmethionine. In those systems, FPR functions by
mediating electron transfer between NADPH and the ferredoxin or
flavodoxin (22). In E. coli, the fpr gene is
activated in response to oxidative stress by the SoxRS regulon (23). In
A. vinelandii, the fpr gene is similarly activated in response to oxidative stress; however, in that case the
system is very specific for AvFdI (24, 25), a ferredoxin type that is not found in E. coli.
Here, we have examined the possibility that in A. vinelandii
both the metabolic and regulatory functions of FdI involve direct and
highly specific interactions between FdI and FPR. As confirmed by both
chemical cross-linking and spectroscopic characterization, AvFdI forms a complex with AvFPR at the
stoichiometric ratio of 1:1 and the [3Fe-4S]+ cluster of
AvFdI is specifically reduced by NADPH via FPR. Recently, the x-ray structures of both E. coli and A. vinelandii FPR have been determined (26, 27). Taken together,
these data are used to model the interaction sites between the
two proteins in A. vinelandii.
Materials--
FdI (28) and FPR (15) were purified to
homogeneity, and FdI strain LM100 was constructed (14) as described
elsewhere. FdI concentrations were estimated from the absorbance at 405 nm, using an extinction coefficient of 29.8 mM Cross-linking Experiments--
For these experiments, 60 µM FdI was treated in the presence of 20 µM
FPR with 5 mM EDC in 25 mM MOPS, pH 7.2. The
reaction mixture was incubated for 2 h at room temperature
followed by quenching the reaction with the addition of the
solubilization buffer used for SDS-PAGE. SDS-PAGE (12% acrylamide) was
performed in 80 mM Tris/glycine, pH 8.8. The gel was then
stained with 0.1% Coomassie Blue in a fixative solution (40% methanol
and 10% acetic acid), or the proteins were electrotransferred to
nitrocellulose membranes. Antigen-antibody interaction was visualized
using horseradish peroxidase-conjugated secondary antibody.
Activity Assays--
FdI dependent NADPH-cytochrome c
reductase activity of FPR was performed at room temperature under
strictly anaerobic conditions using both an anaerobic glove box and a
glucose (25 mM)/glucose oxidase (0.5 unit) system. The
reaction mixture consisted of 50 µM cytochrome
c, 2.5 µM FdI, 0.2 mM NADPH, and 3 µM FPR. The reaction was initiated by the addition of 0.2 mM NADPH, and the increase in absorbance at 550 nm was
measured. Acetylene reduction assays for nitrogenase were performed as
described previously (30) under argon in 100 mM TES, pH
7.4, or 100 mM MES, pH 6.5, with 20 mM sodium
dithionite or NADPH-FPR as a reductant.
Chemical Modification of FdI and FPR--
Pyridoxal 5'-phosphate
followed by NaBH4 reduction was used to modify lysine
residues in both proteins. 5 µM FPR or 5 µM
FdI was incubated in the dark in a reaction mixture containing 25 mM HEPES, pH 8.0, 5% glycerol, 0.1 mM EDTA,
and 20 mM pyridoxal 5'-phosphate for 30 min at room
temperature. The pyridoxal 5'-phosphate was then neutralized by
addition of 40 mM fresh NaBH4. Unreacted chemicals were then removed using small Sephadex G-25 columns (0.9 × 15 cm). Phenylglyoxal was used for modification of arginine residues. 5 µM FPR or 5 µM FdI was
incubated in the dark with 20 mM phenylglyoxal for 2 h
at room temperature and made free of unreacted chemicals using Sephadex
G-25 columns (0.9 × 15 cm). 10 mM EDC for
modification of glutamate or aspartate residues was used in 50 mM HEPES, pH 7.5, containing 100 mM NaCl at a 5 µM protein concentration. After a 20-min incubation, the
reaction was terminated with 30 mM ethanolamine.
Spectroscopy--
EPR spectra were obtained using a Bruker
ESP300E spectrometer, interfaced with an Oxford liquid helium cryostat.
Absorptional spectra were recorded in 0.5-ml quartz cuvettes on a
Hewlett Packard 8452A diode array spectrophotometer. CD spectra were
obtained using a Jasco J-500C spectropolarimeter. CD measurements were carried out using small volume cylindrical cells with fused quartz windows. For anaerobic EPR measurements, the samples were loaded into
the EPR tubes in an O2-free (O2 < 1ppm) glove
box (Vacuum Atmosphere, Hawthorne, CA). The tubes were sealed with a
cap, wrapped with parafilm, and then transferred to the liquid nitrogen Dewar.
Proteolytic Cleavage, Peptide Mapping, and Amino Acid
Sequences--
Apoprotein obtained by 10% trichloroacetic acid was
dissolved in 7.6 M urea containing 0.4 M
ammonium bicarbonate phosphate, pH 8.0, and 2 mM EDTA and
reduced by addition of a 5-fold molar excess of dithiothreitol per mol
of SH, under argon at 50 °C for 15 min. The alkylation of the thiol
groups was performed by incubation for 30 min with 10 mM
iodoacetamide at room temperature. After adjusting the urea
concentration of sample solution to <2 M with distilled
water, tryptic digestion of carboxylmethylated apoprotein was carried
out by addition of trypsin (10% w/w) at 37 °C for 20 h.
Peptides were separated by reverse phase high performance liquid
chromatography. An aliquot containing 1-30 nmol of digested protein
was resolved with a HP1090 system with a filter photometer equipped
with a Vydac C-18 column (internal diameter, 0.45 mm). Peptides
separations utilized a solvent system consisting of aqueous 0.1%
trifluoroacetic acid (solvent A) and 90% acetonitrile/0.09% trifluoroacetic acid (solvent B), with the linear gradient changing 1%
per min from 5 to 100% solvent B (31, 32). The
NH2-terminal protein sequencing was performed by the
Biotechnology Resource Facility of the University of California,
Irvine, on a Perkin-Elmer Applied Biosystems Division model 477A
automated protein sequencer with the on-line model 120A
phenylthiohydantoin-amino acid analyzer equipped with strip chart
recorder and 610A data analysis system.
Electrostatic Calculations--
The electrostatic potentials for
FdI and FPR were calculated with the program UHBD (33), which uses
finite difference methods to solve the Poisson-Boltzmann equations for
the electrostatic potential on a three-dimensional grid. Coordinates
for FdI (Protein Data Bank entry 6FD1 (5)) include all hydrogen atoms
except for His and Cys residues. All nonpolar hydrogen atoms were
removed. Polar hydrogen atoms not present in the crystallographic
coordinates were added using the molecular graphics program Insight
(MSI Inc., San Diego, CA). Hydrogen atoms were attached to the sulfur
atoms of the nonligating Cys residues 11 and 24 and to the N
Polar hydrogens were also included in the crystallographic coordinates
of FPR (Protein Data Bank entry 1A8P (27)). Except for
His109, His side chains were modeled with only N
Electrostatic potentials were calculated on a grid with 1.0-Å spacing
in a medium equivalent to 150 mM ionic strength. The electrostatic potential mapped onto the molecular surfaces (see Fig.
12) is the value of potential evaluated at a series of points 1.4 Å above and normal to the surfaces. Molecular surfaces were calculated
with the program MSMS (35) using a 1.4-Å probe sphere, which is the
van der Waals radius of a water molecule.
Interaction of FdI and FPR with the Chemical Cross-linking Reagent
EDC--
While working out the purification scheme for FPR, we
prepared an affinity column that had purified FdI attached to a resin via lysine residues (15). In 0.1 M NaCl, FPR appeared to
bind specifically to that column, giving us the first indication that these two proteins might be redox partners in vivo. To
characterize this interaction further, we undertook a study using the
chemical cross-linking reagent EDC, which links lysine residues in one protein with glutamate or aspartate residues in the other (36). To
begin this study, we examined the behavior of each of these proteins by
itself with EDC.
As shown in Fig. 1A, FdI,
which has an isoelectric point of 4.03, migrates abnormally on SDS-PAGE
as if its molecular weight were 26,000 rather than 12,050 (37), a
phenomenon that also occurs for other highly acidic proteins (38, 39).
Addition of EDC to FdI causes modification of the carboxylate groups,
resulting in the protein migrating as expected based on its molecular
weight. As also shown in Fig. 1A, the FPR monomer does not
show any abnormality on SDS-PAGE either before or after treatment of
the protein with EDC with respect to its molecular weight position.
However, Coomassie-stained gels always also revealed a small amount of
higher molecular weight complexes. The molecular weights of these
complexes suggest that they corresponded to mostly FPR homodimer,
possibly with different orientations, yielding complexes that run is
somewhat different positions on the gel. Fig. 1B shows a
corresponding Western blot, which both confirms that the high molecular
weight complexes react with antibodies raised against FPR and shows
that very small amounts of FdI dimer (not observed on the
Coomassie-stained gel) are also formed by reaction of FdI alone with
EDC. By varying conditions, we found that the amount of FdI dimer could
be increased to the point where it could be easily observed on
Coomassie-stained gels by lowering the pH (Fig.
2). The electrostatic calculations that are discussed below offer a clear explanation for this observation because the extreme negative charge of FdI would have to be neutralized by protonation of surface residues prior to dimer formation. It should
also be noted that we never observed complexes greater than FdI
dimers.
The formation of FdI dimers could be an artifact of nonspecific
cross-linking or it could be physiologically relevant. Although in the
crystalline state AvFdI is clearly a monomer (2-5),
previous studies have suggested that the homologous seven-iron
ferredoxins from S. griseus (40), P. ovalis (41),
and Sulfolobus acidocaldarius (42) might form dimers under
certain conditions in solution. In general, the more physiological
conditions of low salt (40) and, as shown in Fig. 2, lower pH tend to
favor the formation of dimers. In addition, FdI does have a regulatory
function, and it has been proposed that this function might involve
dimer formation (43). None of these data are definitive, but they do
leave open the possibility that either the metabolic or regulatory
functions of FdI may involve dimer formation.
Formation of a 1:1 FdI:FPR Complex Using EDC--
As shown in Fig.
3A, when FdI and FPR are
incubated together with EDC, a new major band having an apparent
Mr ~43,000 appeared, concurrent with a
reduction in the bands corresponding to FdI alone or FPR alone. The
molecular weight of the band is consistent with a 1:1 complex of the
two proteins, and Western analysis (Fig. 3B) using
antibodies raised against the individual FPR and FdI proteins confirmed
that both are present in the complex. The efficiency of cross-linking
was strongly dependent upon the ionic strength, pH, and the molar ratio
of FdI to FPR. The amount of cross-linked product decreased as either
[NaCl] or pH increased. Ionic strength less than 0.2 M,
pH lower than 7.0, and a molar ratio of FdI to FPR greater than 3 are
optimal conditions for complex formation (data not shown). The ionic
strength and pH dependences of the interaction between the two proteins
points to a strong contribution of electrostatic forces in the early
stages of complex formation. The same efficiency in the cross-linking
reaction was observed when the reaction was carried out anaerobically
in the presence of 2 mM
Na2S2O4. Under those conditions,
the [3Fe-4S]+/0 cluster of FdI is reduced, and the FAD of
FPR is in the reduced hydroquinone state. This result shows that the
formation of a 1:1 complex does not depend upon the oxidation state of
the chromophores. The absence of any additional reaction products
indicates that the cross-linking of FdI with FPR is highly specific. In
addition, no cross-linked products were obtained when either FdI
or FPR was heat-denatured prior to addition of EDC, showing that the reaction required both partners to be in their native
conformations.
Identification of FPR as Lysine and FdI as Glutamate/Aspartate
Donor--
EDC is a water-soluble, zero-length carbodiimide
cross-linker that covalently links salt bridges formed between
carboxylate groups from glutamate or aspartate residues in one protein
to amino groups from lysine residues in the other (36). The
pKa values of arginine residues are too high to form
a stable amino bond, so arginines are generally not present in the
cross-linked complexes. The absence of a spacer in the zero-length
cross-linker also increases the probability that the residues that are
involved in the cross-linked product represent the actual sites of
protein-protein interaction. In this case, FPR has 11 lysine and 33 aspartate and glutamate residues, whereas FdI has 6 lysine and 23 aspartate and glutamate residues, so our first experiments were
directed toward determining which protein was the lysine donor and
which was the glutamate or aspartate donor. To obtain this information, we did a series of experiments in which we chemically modified either
the lysine or the glutamate/aspartate residues in FdI and/or FPR prior
to attempting to form the cross-linked complex with EDC.
As shown in Fig. 4, when lysine residues
from FPR and aspartate/glutamate residues from FdI were modified, the
complex formation was greatly reduced. On the other hand, when
aspartate/glutamate residues from FPR and lysine residues from FdI were
modified, the complex formation is indistinguishable from the control
(Fig. 4). Complex formation was also greatly impaired with only lysine modified FPR but was not affected with arginine modified FPR (data not
shown). These results show that the lysine from FPR and
glutamate/aspartate from FdI form a salt bridge at the interface
between the two proteins. This result is also consistent with our
previous observation that when FdI was cross-linked via lysines to a
resin, a reaction that would leave its glutamate/aspartate residues
available, FPR bound tightly to the affinity column (15). We have now
tried the reciprocal experiment and find that if FPR is cross-linked to
a resin via its lysine residues, FdI does not bind to that affinity
column, which also supports our conclusion that in the cross-linked
complex FPR is the lysine donor and FdI is the glutamate/aspartate
donor.
Identification of Lys258 as the Lysine Donated by
FPR--
In order to identify the cross-linking site, the FPR:FdI
cross-linked complex was purified away from the FPR homodimer using FPLC. Then, EDC-treated FPR alone, EDC-treated FdI alone, and the
cross-linked complex were individually S-cysteine-alkylated and digested with trypsin, and the resulting peptides were separated by
high performance liquid chromatography. By comparing the high performance liquid chromatography profiles for the three samples, we
isolated a peak that is observed in the cross-linked product and FdI
alone, but not in FPR alone. The NH2-terminal sequence of
that peak showed that two sets of residues were found for each cycle,
one from FPR and the other from FdI. FVEX matched those of the known
FPR peptide 254AFVEK258. The Lys258
was not observed in the sequencing reaction, which would be the expected result if that lysine were involved in the cross-linked complex. No peptide ran close to the same position in the profile of
trypsin digested FPR alone and the FPR peptide with the same sequence
eluted much earlier in the chromatogram when the tryptic peptides of
FPR were run under the same conditions. This result is also consistent
with Lys258 being the cross-linked residue in FPR. The
other set of residues matched those from a known very large tryptic
peptide of FdI, 13YTDCVEVCPV ... K85.
NH2-terminal sequencing of that fragment was successful
through residue Glu48 when all aspartates or glutamates
were observed, consistent with the FdI site being contained within the
peptide Cys49-Lys85. This site was not further defined.
FPR Cross-linked Residue Lys258 Is Salt-bridged to FAD
in the Absence of FdI--
As shown in Fig.
5, in the crystal structure of
AvFPR (refined at a 2.0-Å resolution) the COOH-terminal
residue, Lys258 interacts via a hydrogen bond (3.2 Å) with
a phosphate oxygen of the AMP portion of the FAD (27). This interaction
is unique among homologous FAD containing enzymes of known structure.
In the most closely related protein, E. coli flavodoxin
reductase (the E. coli fpr gene product), the COOH-terminal
residue, Trp248, stacks on the adenine ring of FAD (26). In
AvFPR, the side chain of Phe255 stacks on the
adenine; consequently, the conformation of the bound FAD is similar in
both structures. However, AvFPR differs in that there are
three additional residues following Phe255:
Val256, Glu257, and Lys258. In
addition to the charged hydrogen bond between Lys258 and
the phosphate group, the aliphatic side chain of this residue, its
amide, and the terminal carboxyl group, all of which lie on the protein
surface, have six van der Waals contacts (3.4-4.7 Å) with the ribose
O2', O3', and C3' atoms of the AMP moiety of FAD. Therefore, it would
be expected that interaction of FdI with FPR via Lys258
would perturb the optical properties of the FAD, as observed below.
FdI Binding Perturbs the Spectrum of the Flavin--
A model that
is consistent with the data presented above is that FdI binding to FPR
disrupts the interaction of cross-linking residue Lys258
with the FAD. Independent evidence for FdI binding perturbing the
environment of the flavin is shown in Fig.
6. In this experiment, UV-visible
spectrophotometry was used to investigate possible changes in the FAD
chromophore upon FdI binding. The absorption spectrum of FdI is
featureless in the 350-600 nm region (Fig. 6A), whereas FPR
exhibits the strong characteristic flavin spectrum in this region (Fig.
6B). The spectrum in Fig. 6C is that of a physical 1:1 mixture of the two proteins, not of a cross-linked complex. The optical difference spectrum shown in Fig. 6D
was obtained by computer subtraction of the sum of the spectra
exhibited by the individual proteins (Fig. 6, A and
B) from the physical mixture shown in Fig. 6C.
This difference spectrum is very similar in shape to that exhibited by
FPR, but it is red-shifted, which indicates that when FdI binds to FPR
in solution, it appears to perturb the spectrum of the flavin. No
difference spectrum was observed when FPR was incubated with bovine
serum albumin for a control experiment. As shown in Fig.
7 this reaction was easily quantitated by
varying the ratio of FdI to FPR and monitoring the absorbance change at
498 nm, the wavelength at which the total change was largest. The
absorbance increased with increasing ratio of [FdI] to [FPR] up to
a maximum when the two proteins were in equimolar concentration. This
result again indicates that FdI forms a specific 1:1 complex with FPR
in solution, strengthening the conclusion from the cross-linking
experiments and extending the result to show that the flavin
environment is altered when FdI binds. Assuming
As shown in Fig. 8, in attempts to see
whether the [Fe-S] clusters of FdI were also perturbed upon complex
formation, we employed CD spectroscopy because the visible region CD
spectrum of oxidized FdI is very unique to this class of ferredoxins.
Fig. 8, A and B, shows the CD spectra for
oxidized FdI and FPR respectively. The oxidized FdI has strong positive
CD bands above 500 nm, but oxidized FPR does not. Furthermore, even at
wavelengths shorter than 500 nm, the two CD spectra were easily
distinguishable because FdI has two strong positive CD bands in this
region, whereas FPR has a broad negative band. The same strategy was
used to obtain the difference spectrum shown in Fig. 8D as
was used for the UV-visible experiment. Again, the difference spectrum
is similar to the FPR CD spectrum, indicating that the FAD environment
is perturbed upon FdI binding but giving no indication of whether the
FdI chromophores are perturbed. Finally, as shown in Fig.
9, the EPR spectrum of FdI that arises
from the [3Fe-4S]+ cluster is also not perturbed by
binding to FPR. This negative result may indicate that the FdI binding
site is distant from the FdI chromophore or simply that the EPR
spectral properties are dominated by the structure of this buried
cluster and are not influenced by the formation of salt bridges between
residues on the surface of the two proteins.
Electron Transfer from NADPH to FPR to FdI--
In the last step
in photosynthesis, proteins that are somewhat homologous to FPR receive
reducing equivalents from low reduction potential [2Fe-2S] containing
ferredoxins and transfer them to NADP+ to form NADPH (44).
On the other hand, for E. coli FPR, which is much more
similar to AvFPR, the reaction appears to go in the reverse
direction, with electrons transferred from NADPH to FPR and onto
ferredoxins or flavodoxins, although in that case, the specific
ferredoxin or flavodoxin has not been identified (23). The reduction
potentials of NADPH and of the FAD in AvFPR are reported to
be similar, at approximately -320 mV versus the standard hydrogen electrode (8, 15, 45), making the reduction of FPR very
sensitive to the ratio of NADPH/NADP+.
In this study, we used two methods to measure electron transfer from
NADPH to FPR to FdI. The reduction potential of the
[4Fe-4S]2+/+ cluster of FdI is far too negative at -650
mV versus standard hydrogen electrode to be reduced by this
system (8). The reduction potential of the [3Fe-4S]+/0
cluster is pH-dependent (pKa = 7.8;
slope = -55 mV per pH unit) due to direct protonation of the
reduced cluster. This proton transfer reaction is known to be
physiologically relevant (8, 45). At high pH, the alkaline limit is
~
As shown in Fig. 10, electron transfer
from NADPH to FPR to FdI can be demonstrated using the nonphysiological
NADPH-cytochrome c reductase assay. The background
activity obtained by mixing NADPH and FPR with oxidized cytochrome
c is specifically enhanced by addition of FdI. The
specific activity of 0.8 µmole cytochrome c
reduced/mg of FPR/min at pH 6.5 is much higher than that of the spinach
ferredoxin-NADP+ reductase (FNR): ferredoxin system
(published value = 1.2 × 10 The Interaction of A. vinelandii FPR:FdI--
To further
investigate the interactions of these two proteins, the electrostatic
potentials of FdI and FPR were calculated (Fig.
12). The calculations were done
assuming a physiological ionic strength of 150 mM salt
concentration; the protonation and oxidation states were taken into
account, with partial charges assigned to all residues and cofactors
(see under "Experimental Procedures"). The electrostatic potential
around each protein at a distance of 1.4 Å (the radius of a water
molecule) from the molecular surface is plotted onto the molecular
surface. In Fig. 12A, FdI and FPR are oriented to suggest a
specific mode of interaction for the two proteins. The electrostatic
potential of FdI is strongly negative on the face of the protein where
the [3Fe-4S] cluster is nearest the surface. The residues on the
surface in this region include the cluster ligand Cys49,
Pro50, and Ala51, and the adjacent loop
containing Glu57, Asp58, Glu59,
Glu62, and Asp63 and are in the peptide
Cys49-Lys85, identified by cross-linking. The
acidic loop participates in defining the negative electrostatic
potential around the cluster, and it is interesting to note that these
residues are all contained within the 60-amino acid COOH-terminal
extension that is unique to this class of ferredoxins. In contrast, the
opposite face of FdI is largely neutral or only weakly negative, except
for a region adjacent to the [4Fe-4S] cluster (Fig. 12B).
The surface directly over the [4Fe-4S] cluster, a large, almost flat
protuberance, is essentially neutral as well, although it is surrounded
by a ring of negative potential (Fig. 12C).
The surface of FPR displays a striking concentration of positive
potential within and around a cavity bounded by the ribityl atoms of
FAD, N10 of the flavin, N6 and N7 of adenine, the side chains of
Arg51 and Lys258, and the amides of
Ala52 and Val256 (Fig. 12A). Also
contributing to the positive potential is the dipole of the only helix
in the NH2-terminal FAD binding domain (Fig.
12A, left of Arg51). A unique
interaction of Arg51 with the phosphate of the FMN portion
of FAD contributes significantly to this electropositive cavity.
Arg51 is a conserved residue in the motif RxYS/T that is
involved in FAD binding in all homologous oxidoreductase structures;
however, AvFPR is unique in that a second basic residue that
was identified as the cross-linking site, Lys258, also
interacts with the FAD phosphates (27). The presence of
Lys258 causes Arg51 to interact with only the
FMN phosphate deeper within the cavity. The methyl groups of the
dimethylbenzene portion of the flavin isoalloxazine ring and the side
chain of Val256 are adjacent to the cavity on the protein
surface. Surrounding this region, the surface is mildly positive over
much of the NH2-terminal domain. The surface of the larger
COOH-terminal domain is mildly negative, whereas the circumference of
the entire protein is weakly negative (Fig. 12A). In
contrast, the opposite face of FPR is virtually neutral, again with the
circumference being weakly negative (Fig. 12B). Therefore,
the only pronounced positive electrostatic potential on the surface of
FPR is directly over the active site.
One way in which the electrostatic surfaces of FdI and FPR may
interact, and in fact the only way with obvious electrostatic complementarity, is for the strongly negative region surrounding the
[3Fe-4S] cluster to dock with the positive region overlaying of FAD.
In Fig. 12A, this can visualized by imagining that the page
is creased between the two surfaces, allowing them to be folded toward
each other. This interaction would provide charge complementarity for
much of the FdI surface, especially that to the right of the [3Fe-4S]
cluster in the view in Fig. 12A, and it brings the cluster
at Cys49 close to the FAD. Simultaneously, such an
interaction also brings the acidic patch,
Glu57-Asp58-Glu59/Glu62-Asp63
into proximity with Lys258. Hydrophobic contacts of
Cys49, Pro50, and Ala51 on FdI with
the flavin methyl groups and Val256 on FPR could stabilize
the interaction and promote electron transfer. The weak negative
potential over the rest of the larger FPR molecule could further act to
repel the very negatively charged, smaller FdI molecule, thereby
focusing FdI toward the positive region of FPR. Therefore, both
attractive and repulsive electrostatic forces appear to play a role in
orienting FdI and FPR for interaction. This orientation is consistent
with our cross-linking data and electron transfer between FAD and the
[3Fe-4S] cluster.
The proposed interaction of FdI with FPR is consistent with several
independent observations and results. The strong electrostatic complementarity of the two protein surfaces would account for the
binding of FPR to FdI on an affinity column where 300 mM
salt concentration is required to displace FPR (15). It is also
consistent with the observation that the formation of a specific 1:1
complex is favored at low ionic strength. As discussed above, the
reduction potential of the [3Fe-4S]+/0 cluster is more
compatible with that of the NADPH/FAD couple than is the [4Fe-4S]
cluster, and electron transfer from flavin to the
[3Fe-4S]+ cluster has been demonstrated directly.
Furthermore, the essentially neutral electrostatic potential on the
surface of FdI at the [4Fe-4S] cluster (Fig. 12C) argues
that this cluster is not involved in the electron transfer reaction.
Electron transfer via exposed methyl groups of the flavin ring has been
proposed in other systems in which protein-protein interactions were
known (46, 47, 26). Mutagenesis studies have implicated the
complementary interaction of basic residues on Anabaena
ferredoxin-NADP+ reductase with acidic residues on the
Anabaena [2Fe-2S] Fd (48, 49).
Fig. 13 compares the sequences of
AvFPR to that of the proteins in the data base that are most
similar to the protein. The sequence from P. aeroginosa is
new and was obtained from the Pseudomonas Genome Project.
7Fe ferredoxins >90% identical to AvFdI have been identified in a number of Pseudomonas species, including
P. aeruginosa, and as shown in Fig. 13, the P. aeruginosa FPR sequence is also highly homologous; it is 85%
identical and 91% similar to AvFPR. The COOH-terminal
Lys258 residue that we have identified as the FPR
cross-linking residue is also conserved in the P. aeruginosa
sequence, strengthening our view that the functions and interactions of
the proteins in the two organisms should be the same. The next most
similar protein in the data base to AvFPR is the protein
from E. coli (Fig. 13). Although this organism does not
appear to synthesize a homologous ferredoxin, there are close parallels
between the regulation of the fpr genes in the two organisms
(25). The specific redox partner of FPR in E. coli has not
been identified but is believed to be flavodoxin or ferredoxin. The
x-ray crystal structure of E. coli FPR has recently been
determined, and three localized arginine residues
(Arg236-Arg237-Arg238) near the
COOH-terminal end were proposed as anchoring points for the redox
partner flavodoxin or ferredoxin (26). As shown in Fig. 13, these
localized basic residues are not conserved in the AvFPR or
PaFPR. The ferredoxin-NADP+ reductases involved
in photosynthesis (designated FNR) are less homologous to the proteins
from A. vinelandii, P. aeruginosa, and E. coli
(Fig. 13). In Anabaena PCC 7119 FNR, the residues
Lys53, Arg77, and Lys294 are
suggested to be involved in ferredoxin binding, and Arg77
directly interacts with FAD (50-52). These three residues are located
in the FAD and NADP+ binding domains, implying that
ferredoxin binds the two domains. For spinach FNR, the ferredoxin
binding site has been suggested to be a loop containing
Lys85 and Lys88, residues that are exclusively
in the FAD domain (53-56). A homologous loop is present in the FNR
from Anabaena but is missing in the AvFPR and
EcFPR structures (Fig. 13).
Physiological Considerations--
Taken together, the above data
show that FPR and FdI bind specifically to one another and clearly
demonstrate that electron transfer can occur from NADPH to FPR to the
[3Fe-4S]+ cluster of FdI, strongly suggesting that this
is the physiological direction of electron transfer. The next step is
to try to identify the protein that accepts electrons from the
protonated reduced [3Fe-4S]0-H+ cluster of
FdI. One possibility is that reduced FdI could serve, at least under
some conditions, as an electron donor to nitrogenase. Indeed, many
years ago, electron transfer from NADPH to crude preparations of
nitrogenase was reported in a reaction that required addition of FdI
and spinach ferredoxin reductase (57, 58). Another report suggested
that FdI, which is not encoded by a nif gene, might
substitute for the nifF gene product flavodoxin under certain conditions because nifF deletion strains (like FdI
deletion strains) still fix dinitrogen at wild-type rates (59). This idea was tested by making a double mutant that did not synthesize either flavodoxin or FdI. That strain still fixed dinitrogen; however,
FPR levels in that strain were greatly overexpressed, leading to the
suggestion that FPR might serve as another substitute electron donor to
nitrogenase (60). In this study, we have directly tested these ideas
using a system composed of all purified components. In these
experiments, we were unable to demonstrate any electron transfer from
NADPH to FPR to nitrogenase in the presence or absence of FdI, leading
to the conclusion that this is not the physiological function of either protein.
A second possibility for a metabolic function for the FPR/FdI system
would be in the activation of enzymes involved in anaerobic metabolism,
because this is known to be the function of the homologous FPR from
E. coli (19-21). Although A. vinelandii is an
obligate aerobe, it is likely to have internal anaerobic metabolism to protect the extremely oxygen sensitive enzyme nitrogenase from oxygen,
and future experiments will be directed toward examining this
possibility. We note here that repeated attempts to delete the
fpr gene from the A. vinelandii chromosome have
been unsuccessful, making it likely that this protein is necessary for
the survival of the cell.
In addition to its metabolic function, which is important for cell
growth, FdI has a regulatory function in controlling the expression of
its redox partner FPR, a system that is considered in detail elsewhere
(24, 25). As in E. coli, the A. vinelandii fpr
gene is activated when the cells are exposed to oxidative stress via a
transcriptional activator that binds to a SoxS-like consensus sequence
just upstream of the fpr gene (24). The transcriptional activator in A. vinelandii has not yet been purified, but it
is known that the role of FdI in the system is to shut down the
activation of fpr transcription. The current working
hypothesis, which is consistent with all of the data presented
here and discussed in detail elsewhere (23-25), is that when oxidative
stress is over, NADPH levels build up, FPR becomes reduced, and the
reduced FPR specifically reduces the [3Fe-4S]+ cluster of
FdI, which then goes on to reduce the next protein in the regulatory
cascade, leading to inactivation of the system. Future experiments are
being directed toward identifying the member of the cascade that is
specifically reduced by FdI.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
1 cm
1. FPR concentrations
were determined by the method of Bradford (29), using bovine serum
albumin as a standard. All chemicals used in the protein modifications
were obtained from Sigma. The chemiluminescence kit used in Western
blot analysis was purchased from DuPont or Amersham Pharmacia Biotech.
Sequencing grade porcine trypsin, modified by reductive methylation to
reduce autolysis of the enzyme and thereby increase its stability, was
purchased from Promega.
atoms of His residues 35 and 103. N
atoms for His35 and
His103 were not protonated, giving both His side chains an
overall neutral charge. The [Fe-S] clusters of FdI were fully
oxidized ([4Fe-4S]2+ and [3Fe-4S]+). The
partial charges of the FdI [Fe-S] clusters and their attached Cys
ligands were based on quantum mechanics calculations (34).
being
protonated, giving them an overall neutral charge. For
His109, both imidazole side chain N atoms were protonated;
His109 is solvent exposed and forms a salt bridge to an Asp
side chain through N
. Polar protons were added to model a fully
reduced FAD hydroquinone. Partial charges for FAD atoms were assigned to be consistent with the AMBER charges for DNA and RNA. All other partial charges for FdI and FPR were assigned using the library of
AMBER partial charges provided with UHBD.
RESULTS AND DISCUSSION
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Fig. 1.
SDS-PAGE of the reaction product before and
after treatment of FdI and FPR with EDC. The proteins were
activated with 5 mM EDC for 2 h; then, the reaction
was terminated by addition of sample buffer for SDS-PAGE. Samples were
analyzed on 12% SDS-PAGE. A compares FdI without EDC
(a), FdI treated with EDC (b), FPR without EDC
(c), and FPR treated with EDC (d) with molecular
weight standard markers, after staining with Coomassie. B
compares FdI treated with EDC (a) and FPR treated with EDC
(b), after blotting and reacting with FdI and FPR
antibodies, respectively.
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Fig. 2.
Cross-linking of FdI dimer as a function of
pH. 20 µM FdI was incubated with 5 mM
EDC for 2 h at room temperature at the indicated pH values, and
the reaction was then terminated by addition of 100 mM
ammonium acetate, pH 7.0. After a 10-min incubation, excess EDC was
removed, and the samples were concentrated with a YM-3 Centricon
(Amicon). 10 µg of each protein sample was analyzed by 12% SDS-PAGE,
and the gel was stained with Coomassie.
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Fig. 3.
Cross-linking between FdI and FPR. The
proteins were activated with 5 mM EDC for 2 h; then,
the reaction was terminated by addition of sample buffer for SDS-PAGE.
Samples were analyzed on 12% SDS-PAGE. A compares FPR
treated with EDC (FPR), FdI treated with EDC
(FdI), and FPR and FdI treated with EDC (FPR + FdI), after staining with Coomassie. B, FPR and FdI
treated with EDC, after blotting and reacting with FdI antibody
(a) and FPR antibody (b).
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Fig. 4.
Cross-linking between chemically modified FdI
and chemically modified FPR. Pyridoxal 5'-phosphate was used for
modification of lysine residues, phenylglyoxal for arginine residues,
and EDC for glutamate or aspartate residues as described under
"Experimental Procedures." Unreacted excess chemicals were then
removed using small Sephadex G-25 columns (0.7 × 15 cm). The
cross-linking was carried out using the conditions described in Fig. 3,
and the samples were analyzed by 12% SDS-PAGE with Coomassie staining.
Lane a, unmodified FPR and unmodified FdI were incubated
with 5 mM EDC; lane b, lysine-modified FPR and
aspartate- or glutamate-modified FdI were incubated with 5 mM EDC; lane c, aspartate- or glutamate-modified
FPR and lysine-modified FdI were incubated with 5 mM
EDC.
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Fig. 5.
The local environment of FAD in
AvFPR showing residues 51-54 of the conserved RxYS/T
motif, Phe37 and residues 254-258, which represent a
unique COOH-terminal extension (27). Arg51 and
Lys258 form charged hydrogen bonds with FAD phosphate
oxygens. Lys258 also makes van der Waals contacts to the
ribose of FAD. Other hydrogen bonds and contacts are indicated in Å.
Tyr53 and Phe255 are involved in stacking
interactions with the isoalloxazine and adenine rings of FAD,
respectively.
A498 comes from the FAD signal of the complex and that its Mr is ~43,000, the value of
498 was calculated to be 1.1 × 103
M
1 cm
1.
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Fig. 6.
UV-visible absorption spectra of oxidized FdI
(A), oxidized FPR (B), a physical mixture of
FdI and FPR (C), and the difference spectrum
(D). FdI was 50 µM and FPR was 55 µM in 25 mM MES buffer, pH 6.0. The ratio of
[FdI] to [FPR] was 0.91. The difference spectrum (D) was
obtained by subtracting the spectrum each protein (A and
B) from the mixture spectrum (C).
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Fig. 7.
The change of differential absorbance at 498 nm as a function of changes in the [FdI]/[FPR] ratio. The FPR
concentration was 50 µM, and the FdI concentration was
changed to produce the desired [FdI]/[FPR] ratio in 25 mM MES buffer, pH 6.0. Inset, the difference
spectrum showing the absorbance difference peak that is being
monitored.
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Fig. 8.
Circular dichroism spectra of oxidized FdI
(A), oxidized FPR (B), a physical mixture of
FdI and FPR (C), and the difference spectrum
(D). FdI was 50 µM and FPR was 55 µM in 25 mM MES buffer, pH 6.0. The ratio of
[FdI] to [FPR] was 0.91. The difference spectrum (D) was
obtained by subtracting the spectrum of each protein (A and
B) from the mixture spectrum (C).
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Fig. 9.
EPR spectra of oxidized FdI without FPR
(solid line) and with FPR (broken line) at 10 K. FdI was 50 µM and FPR was 100 µM in 25 mM HEPES buffer, pH 7.0. Microwave
power, microwave frequency, modulation amplitude, and gain were 1 mW,
9.47 GHz, 5 G, and 5 × 103, respectively.
430 mV versus standard hydrogen electrode, but at pH 6 it is ~
320 mV, or very similar to the potential of the NADPH/FPR
couple. These potentials were measured for free FdI in direct
electrochemical experiments and could be modified somewhat upon FdI
binding to FPR, as has been demonstrated for the photosynthetic
ferredoxin/ferredoxin-NADP+ reductase complex from
Anabaena (39).
4 µmol
min
1 mg
1) but slightly lower than that of
cytochrome P450BM-3 (published value = 19 µmol
min
1 mg
1). As shown in Fig. 10, the
electron transfer is pH-dependent, as is the reduction
potential of the [3Fe-4S]+/0 cluster with the lower, more
physiological values, bringing the FdI reduction potential closest to
that of FPR and giving the highest activities. To confirm that electron
transfer from NADPH to FPR to the [3Fe-4S]+ cluster
occurs in the absence of cytochrome c, we directly monitored the reaction using EPR. As shown in Fig.
11, even in the absence of pulling the
reaction with cytochrome c and with a 8-fold excess of FdI
over FPR present, 60% of the FdI [3Fe-4S]+ clusters
became reduced to the 0 oxidation level. The incomplete reduction of
the [3Fe-4S]+ cluster by NADPH/FPR indicates that the
system is in equilibrium. Within the A. vinelandii cell, the
system may be driven toward complete reduction of the
[3Fe-4S]+ cluster by the as yet unidentified FdI electron
acceptor. No reduction of the cluster was observed upon addition of
either FPR alone or NADPH alone, and the absence of any new signals
demonstrates, as we expected based on reduction potential
considerations, that the [4Fe-4S]2+ cluster was not
reduced under those conditions.
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Fig. 10.
FdI-dependent NADPH-cytochrome
c reductase activity of FPR in the function of pH. The
reaction mixture consists of 50 µM cytochrome
c, 2.5 µM FdI, 0.2 mM NADPH, and 3 µM FPR. The reaction was started by the addition of 0.2 mM NADPH, and the increase in absorbance at 550 nm was
measured. For a control, the same reaction was carried out in the
mixture without FdI. The values shown in the y axis were
corrected by subtracting the value without FdI from the value with
FdI.
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Fig. 11.
EPR spectra of oxidized FdI in the absence
(A) and the presence (B) of FPR and NADPH at 10 K. FdI was 50 µM and FPR was 6 µM in
25 mM MES buffer, pH 6.2. The reaction mixture was
incubated for 15 min before loading the sample into an EPR tube.
Strictly anaerobic conditions were provided by both an anaerobic glove
box and a glucose (25 mM)/glucose oxidase (0.5 unit) enzyme
system. A NADPH regenerating system was provided by glucose 6-phosphate
(10 mM)/glucose-6-phosphate dehydrogenase (2 units).
Microwave power, microwave frequency, modulation amplitude, and gain
were 1 mW, 9.47 GHz, 5 G, and 5 × 103,
respectively.
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Fig. 12.
Molecular surfaces of FdI and FPR colored
according to electrostatic potential. The electrostatic potential
is plotted on a color scale ranging from +5.0 (blue) to
5.0 (red) kcal/mol (scale bar). A,
complementary electrostatic surfaces of FdI (left) and FPR
(right). The coordinates of the crystallographic structures
were translated forward (bottom) to show the molecular
features underlying the surfaces. FdI (left, pink C
trace) displays a region of negative potential on the surface over the
[3Fe-4S] cluster (center of molecule; iron atoms,
green; sulfur atoms, yellow). The cross-linked
peptide fragment of FdI, residues 49-64 (blue C
trace),
includes the solvent-exposed, ligating side chain of Cys49
(yellow, center) and the acidic side chains of residues
57-59, 62, and 63 (white with red carboxylate
oxygen atoms). The [4Fe-4S] cluster (right side of
molecule) is also shown. FPR (right, blue C
trace)
displays a region of positive potential over FAD (yellow
bonds), with the most intense potential created by the side chains of
Arg51 and Lys258 (light blue with
dark blue side chain nitrogen atoms). B, a 180°
rotation from A shows the electrostatic potential on the
opposite faces of the FdI (left) and FPR (right).
This side of FdI has neutral to negative potential, with the greatest
negative potential being adjacent to the [4Fe-4S] cluster, which is
on the left side of the molecule in this view. The back side of FPR
shows a neutral to weakly negative surface, which would be expected to
repel the highly negatively charged FdI. C, the view of the
FdI surface rotated by approximately 90° from the view in
A to show the environment around the [4Fe-4S] cluster
(center of surface; iron atoms, green; sulfur
atoms, yellow). The large protrusion over the cluster is
close to neutral, although it is surrounded by a ring of negative
potential made up, in part, by acidic residues Glu57,
Asp58, and Glu59. The neutral surface of FdI
over the [4Fe-4S] cluster would be expected to have a less favorable
interaction with FPR at the flavin site compared with the surface over
and around the [3Fe-4S]. Figures were rendered with the AVS graphics
program (61) (Advanced Visual systems, Inc., Waltham, MA) using modules
developed by Drs. Thomas Macke and Michel Sanner at The Scripps
Research Institute.
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Fig. 13.
Structure based sequence comparison of FPRs
from A. vinelandii (Av) (16), P. aeruginosa (Pa), and E. coli
(Ec) (17-18) and FNRs from Spinacea oleracea
(So) (62) and Anabaena sp. PCC 7119 (An) (63). The alignment, based upon superpositions of
the structures, was determined as described in Ref. 27. The x-ray
crystal structure of PaFPR is not known, but it was assumed
to be the same as AvFPR for the structure-based sequence
alignment.
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ACKNOWLEDGEMENT |
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We thank Louis Noodleman (The Scripps Research Institute) for assisting with partial charge assignments for the [Fe,S] clusters.
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FOOTNOTES |
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* The work was supported by National Institute of Health Grants GM-45209 (to B. K. B.) and GM-36325 (to C. D. S.) and Department of Energy Grant DE-FG03-96ER62262 (to V. A. R.).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. Tel.: 949-824-4297; Fax: 949-824-8551; E-mail: bburgess{at}uci.edu.
The abbreviations used are: AvFdI, Azotobacter vinelandii ferredoxin I; MOPS, 3-(N-morpholino)-2-hydroxypropanesulfonic acid; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-amino} ethanesulfonic acid; EPR, electron paramagnetic resonance; MES, 2-(N-morpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; EDC, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; CD, circular dichroism; FPR, NADPH-ferredoxin reductase; FNR, ferredoxin-NADP+ reductase.
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REFERENCES |
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