(Received for publication, January 10, 1997, and in revised form, March 3, 1997)
From the Department of Molecular Biology and
Biochemistry, University of California, Irvine, California 92697-3900, § Department of Molecular Biology, The Scripps Research
Institute, La Jolla, California 92037, and ¶ Department of
Chemistry, Oxford University, Oxford, United Kingdom
Ferredoxins that contain [4Fe-4S]2+/+ clusters often obtain three of their four cysteine ligands from a highly conserved CysXXCysXXCys sequence motif. Little is known about the in vivo assembly of these clusters and the role that this sequence motif plays in that process. In this study, we have used structure as a guide in attempts to direct the formation of a [4Fe-4S]2+/+ in the [3Fe-4S]+/0 location of native (7Fe) Azotobacter vinelandii ferredoxin I (AvFdI) by providing the correct three-dimensional orientation of cysteine ligands without introducing a CysXXCysXXCys motif. Tyr13 of AvFdI occupies the position of the fourth ligating cysteine in the homologous and structurally characterized 8Fe ferredoxin from Peptococcus aerogenes and a Y13C variant of AvFdI could be easily modeled as an 8Fe protein. However, characterization of purified Y13C FdI by UV-visible spectra, circular dichroism, electron paramagnetic resonance spectroscopies, and by x-ray crystallography revealed that the protein failed to use the introduced cysteine as a ligand and retained its [3Fe-4S]+/0 cluster. Further, electrochemical characterization showed that the redox potential and pH behavior of the cluster were unaffected by the substitution of Tyr by Cys. Although Y13C FdI is functional in vivo it does differ significantly from native FdI in that it is extremely unstable in the reduced state possibly due to increased solvent exposure of the [3Fe-4S]0 cluster. Surprisingly, the x-ray structure showed that the introduced cysteine was modified to become a persulfide. This modification may have occurred in vivo via the action of NifS, which is known to be expressed under the growth conditions used. It is interesting to note that neither of the two free cysteines present in FdI was modified. Thus, if NifS is involved in modifying the introduced cysteine there must be specificity to the reaction.
Azotobacter vinelandii ferredoxin I (AvFdI)1 is a Mr 12,000 monomer that contains one [3Fe-4S]+/0 cluster and one [4Fe-4S]2+/+ cluster. The sequence for the NH2-terminal half of AvFdI is homologous to the sequences of the much smaller clostridial-type ferredoxins that contain two [4Fe-4S]2+/+ clusters (1). Comparisons of these and many other [Fe-S] protein sequences have shown that the presence of a highly conserved CysXXCysXXCys motif can be used to predict that a [4Fe-4S]2+/+ cluster will be present (for reviews, see Refs. 2-6). During the evolution of the 7Fe ferredoxins from the 8Fe ferredoxins, two residues were inserted into this motif between the second and third cysteines to form a CysXXCysXXXXCys motif (1). This insertion moved the second cysteine out of reach of the cluster resulting in the inability to form a [4Fe-4S]2+/+ cluster and the appearance of a [3Fe-4S]+/0 cluster in that position (7-13).
The structural consequences of inserting the two additional residues
between the second and third cysteines are shown in Fig. 1, which compares the structure of native
AvFdI (7-9) to that of Peptococcus aerogenes
ferredoxin (PaFd) (10-13) in the [3Fe-4S]+/0
cluster region of AvFdI, which has the sequence
Cys8XXCys11XXXXCys16.
The comparison in Fig. 1 reveals that the chain trace of
PaFd is puckered out in AvFdI, leaving the
AvFdI Cys11 removed away from the position of
the ligating cysteine in PaFd. Cys11 could
therefore not be used as a cluster ligand without substantial structural rearrangement. A further structural difference in this region is that the COOH-terminal half of AvFdI, which is
absent in the much smaller PaFd, wraps around the
AvFdI residue 8-16 loop shielding it from solvent (9). In
this study, we have used structure as a guide in attempts to direct the
formation of a [4Fe-4S]2+/+ cluster in the
[3Fe-4S]+/0 location of native AvFdI by
providing the correct three dimensional orientation of cysteine
ligands. As shown in Fig. 1, Tyr13 of AvFdI
occupies the position of the fourth ligating cysteine in
PaFd so here we report the purification and characterization of Y13C FdI.
Native AvFdI was purified as described previously (14). Ammonium sulfate was from Fisher and all other materials were obtained from the vendors listed previously (15).
Mutagenesis of fdxA and Expression and Purification of Y13C FdIThe oligonucleotide used for the mutagenesis had the sequence GCAAGTACTGCCATTGTGTTGGTGCAAGTGCACCGATTG. This sequence differs from the wild-type sequence (16) by the change of codon 14 from TAC to TGC, resulting in the change of FdI residue 13 from a tyrosine to a cysteine. The success of the mutagenesis was confirmed at the DNA level by dideoxy-DNA sequencing and at the protein level by NH2-terminal protein sequencing (which was carried out in the protein sequencing facility in the department of Molecular Biology and Biochemistry at the University of California, Irvine) and by x-ray crystallography. Unless otherwise indicated, oligonucleotide-directed in vitro mutagenesis (15), FdI overexpression (17), and cell growth (15) were performed as described previously. The purification of Y13C was carried out anaerobically using a modification of purification method two described by Stephens et al. (14). A. vinelandii containing the overexpression vector for Y13C FdI was grown and cell-free extracts were prepared from 1.1 kg of cell paste. No sodium dithionite was used in the purification of Y13C FdI because it became obvious after repeated attempts to purify the protein according to the native FdI purification scheme that the presence of 2 mM sodium dithionite in the buffers denatured this variant of FdI. All steps, however, were carried out anaerobically using thoroughly degassed buffers. The cell-free extracts were not heat treated as described in the purification of nitrogenase (18) due to the possibility that Y13C FdI would be heat-labile. Cell-free extracts were loaded onto a 5 cm × 20-cm DE52 cellulose column equilibrated with extensively degassed 0.025 M Tris-HCl, pH 7.4. Following loading and washing, the column was chromatographed with a 0.1 to 0.4 M NaCl gradient (1.6 liters of each, 570 ml/h) in the same buffer. After this initial gradient Y13C FdI was eluted from the column batchwise with 0.5 M NaCl. The ferredoxin fraction was immediately diluted 1:2 in the same buffer without salt and loaded onto a 2.5 cm × 10-cm DE52 cellulose column. After loading, the column was washed with 4 liters of 0.025 M Tris-KCl, pH 7.4, 0.12 M KCl, at a rate of 300 ml/h. A gradient from 0.12 to 0.3 M KCl (800 ml of each) followed, and Y13C FdI eluted close to 0.25 M KCl. The protein was concentrated on a DE52 cellulose column, and 10-ml samples of concentrated protein were then loaded onto a 1 m × 2.5-cm Sephadex G75 superfine gel filtration column equilibrated in 0.025 M Tris-HCl, pH 7.4, 0.1 M in NaCl. Following elution from that column, 40 ml of 70-80% saturated ammonium sulfate in 0.1 M Tris-HCl, pH 7.4, was added slowly with stirring to 13 ml of Y13C FdI. The protein precipitated over the course of 2 h and was centrifuged in airtight centrifuge tubes in a SS34 rotor at 6,000 rpm for 35 min. The A280/A400 ratio of Y13C FdI following ammonium sulfate precipitation was 1.7 and the A280/A260 ratio was 1.2, values very similar to those reported for the native protein (14). The final yield of Y13C FdI was 19 mg.
SpectroscopyFor spectroscopic studies, all samples were prepared anaerobically under argon in a vacuum atmospheres glove box (O2 < 1 ppm) using degassed buffers. Where indicated samples were first concentrated using a Centricon-10 microconcentrator and were then buffer exchanged into 0.1 M potassium phosphate buffer, pH 7.4, or into a mixture of 50 mM PIPES and 50 mM TAPS buffers at pH 6.0 or 8.3. Electron paramagnetic resonance spectra were obtained using a Bruker 300 Ez spectrophotometer, equipped with an Oxford Instrument ESR-9002 liquid helium continuous flow cryostat. Circular dichroism spectra were obtained using a JASCO J720 spectropolarimeter, and UV-visible spectra were obtained with a HP 8452A diode array UV-visible spectrophotometer.
ElectrochemistryPurified water of resistivity ~18 ohm/m (Millipore) was used in all experiments, and each of the reagents was at least analytical grade. DC (staircase) cyclic voltammetry was carried out using an Autolab electrochemical analyzer (Eco-Chemie, The Netherlands). The all-glass cell, electrodes, and application of protein film voltammetry have been described previously (15, 19). The saturated calomel reference electrode was held at 22 °C, and potential values were converted to the standard hydrogen electrode scale by using E (saturated calomel reference electrode) = +243 mV. The pyrolytic graphite edge working electrode (area approximately 0.18 cm2) was prepared for protein film formation by polishing with an aqueous alumina slurry (Buehler Micropolish 1.0 µm) and sonicating extensively in water to remove traces of Al2O3. All voltammetric experiments were carried out in an anaerobic glove box (Belle technology) with the O2 < 2 ppm. To prepare films, ~1 µl of protein solution (typically 100 µM in 20 mM Hepes, pH 7.0, containing 0.1 M NaC1 with 200 µg/ml polymyxin as co-adsorbate) was applied to the surface of the chilled pyrolytic graphite edge electrode using a glass capillary tip. The buffer-electrolyte solution in the cell contained 0.1 M NaCl, 20 mM mixed buffer (5 mM of acetate, Mes, Hepes, and TAPS) and 200 µg/ml polymyxin. Reduction potentials were determined from the average of peak positions measured in the directions of increasing (Epa) and decreasing (Epc) potentials.
CrystallizationAll crystallization experiments were done in an anaerobic glove box at <1 ppm O2 using degassed solutions. Y13C FdI is less soluble than native FdI so we were unable to induce nucleation using seed crystals of native FdI or other mutants as done previously (20, 21). Refinement of the native crystallization conditions (8, 9) did not yield crystals. Subsequently, crystallization screening was done using the vapor diffusion method and conditions developed by E. A. Stura (22). Clusters of small needle-like crystals were observed using sodium citrate as a precipitant. However, refinement of these conditions did not produce larger crystals, although the crystals could be grown reproducibly. On an attempt to induce nucleation in droplets that had not formed the small needle-like crystals after 18 days, several of the crystallization plates were transferred in sealed containers from the glove box at 27 °C to a room at 18 °C for 7 days, then to an incubator at 8 °C for 10 days, and then returned to the glove box. Temperature shifts are known to induce nucleation of the native protein (8). After five months (nine months total elapsed time) large, black rod shaped single crystals were observed in several of the drops.
Two crystals were mounted in sealed capillaries for data collection
(Table I). Both crystals were grown from a 13 mg/ml
protein solution in 25 mM Tris-HCl, pH 7.4, and in both
cases the droplets consisted of 2 µl of protein solution mixed with 2 µl of a 1.0-ml reservoir solution. For the first crystal mounted, the
reservoir solution was 1.26 M sodium citrate, pH 7.0, containing 0.5 mM -mercaptoethanol (
ME). For the
second crystal mounted, the reservoir solution was 1.32 M
sodium citrate, pH 7.0, without
ME added.
|
Diffraction data were collected
using CuK radiation from a Ru200 x-ray generator operated at 40 kV,
80 mA, and equipped with a graphite monochromator and Siemens area
detector mounted on a four-circle goniostat. Data were collected by
both
and
scans in increments of 0.25° with exposure times of
400 s/frame. To minimize the effect of decay, the first crystal was
translated in the beam twice. The total time for data collection was 9 days for the first crystal (with
ME) and 4 days for the second
crystal (without
ME). The data from both crystals were indexed,
integrated, merged, and scaled using the Xengen suite of programs
(Table I) (23). The space group of both crystals is P21
with a = 39.3, b = 73.7, c = 45.1A, and
= 98.4 ° and two
molecules (A and B) of Y13C FdI (2.67 Å3/Da) per asymmetric unit.
The structure was solved by molecular replacement using the data set
from the first crystal and the Xplor version 3.1 suite of programs
(24). The native FdI structure from tetragonal crystals (8, 9) with
residue 13 modeled as a glycine was used as a search model. Rigid body,
positional, and isotropic B factor refinement of the
structure representing the two best solutions to the rotation and
translation searches resulted in an R factor of 0.23 for all
data in the resolution range 8.0-2.5 Å. An unbiased 2|Fo|-|Fc|
Fourier map computed with all data in the range 20.0-2.5 Å revealed
5 electron density at residue 13 in place of tyrosine of native FdI
in both molecules A and B, indicative of cysteine persulfide. The electron density was characteristic of both a cysteine
side chain and a disulfide (Fig. 2). A model for a
cysteine persulfide residue (CSS) was constructed and fit to the
density using the XtalView suite of programs (25). The model consists of a cysteine amino acid side chain (C
, C
, S
) with an
additional sulfur atom (S
) bonded at 2.02 Å to S
). The
C
-S
-S
bond angle is tetrahedral. The CSS residues were refined
in Xplor using charge and energy restraints of a disulfide for the
S
-S
bond and a protonated cysteine sulfur for the terminal S
atom. The side chains of a total of 34 Glu, Asp, Lys, Arg, and Gln
residues on molecules A and B were adjusted to
fit the electron density in 2|Fo|-|Fc|
maps due to changes in their conformation in the monoclinic crystal
form relative to the tetragonal form. The final model was refined
against all observed data to 2.35 Å (Table I). There are no outliers
in the Ramachandran plot except for four glycines in both molecules
A and B. Co-ordinates have been deposited with the Protein Data Bank.
Because the first crystal was grown from a droplet containing ME,
the possibility existed that the additional density observed on the
cysteine introduced at position 13 was due to
ME, if slow oxidation
of the cysteine had occurred over the extended crystallization time.
Although no additional density was observed for carbon and oxygen atoms
of
ME on either molecule A or B, it was
possible that these atoms could have been disordered while sulfur
bonded to Cys13 was not. Therefore, a second data set was
collected from a crystal grown in the absence of
ME (Table I). The
data from this crystal was scaled to the first, and a
|Fo|-|Fc|
map was calculated using data in the range 20.0-3.0 Å and phases
based on the refined model of Y13C FdI, including CSS residues on both
molecules A and B. The difference Fourier map was
featureless at residue 13, indicating no difference in the crystals
grown without
ME versus
ME. Subsequently, a
2|Fo|-|Fc|
map calculated with all data in the range 20.0-2.67 Å revealed a very
similar density for the CSS residue on molecules A and
B as observed in the first crystal (Fig. 2). Therefore, we
conclude that
ME is not a factor in the formation of monoclinic Y13C
FdI crystals, and it is not present as a modification of Cys13. The Y13C FdI structure was also refined against all
observed data to 2.67 Å (Table I).
In
addition to the shape and height of the electron density for both CSS13
and CSS213 in both the structure with and without ME added (Fig. 2),
the presence of a cysteine persulfide was demonstrated by a series of
refinement calculations in which each sulfur or oxygen of each
non-ligand cysteine and serine was modeled as oxygen and sulfur,
respectively (Table II). For example, Cys11
was modeled as "Ser11," and Ser56 was
modeled as "Cys56." Similarly, the terminal atom on the
Cys13 side chain was modeled as sulfur (the CSS residue) or
oxygen, a possible Cys13-SOH residue. The only other free
Cys or Ser in FdI is Cys24, which was also modeled as
"Ser24." The corresponding residues in molecule
B were modified in the same way. A series of refinement
calculations were carried out using the 2.3 Å data from the crystal
grown with
ME (Table I), Xplor3.1, and the same charge and energy
restraints used to refine the Y13C FdI structure (Table I). For the
test Cys13-SOH side chain, the residue was defined to have
a S
-O
bond of 1.8 Å, tetrahedral angle for C
-S
-O
and
OH group as in serine. The eight models, in which only one sulfur or
oxygen atom at a time was changed, were independently refined. Three of
these models with Ser11, Ser24, and
Cys56 were also refined against the native FdI data (9) at
the same resolution by the same protocol as a further control, since
the native FdI crystallizes in a different space group. Finally, the correct native structure with Cys11, Cys24, and
Ser56 was refined at 2.3 Å for comparison.
|
Table II summarizes the results of the test refinement calculations.
The height of the electron density of the atom in question in a
2|Fo|-|Fc|
map, calculated in each case with all data in the range 20.0-2.3 Å,
is given in intervals of 1 of the electron density, and the
isotropic temperature factor (B factor) for each atom in
question is given. In each and every case where a sulfur atom was
modeled as an oxygen, the B factor refined to 2.0 Å2, the minimum allowed by the program, indicative that
the model had insufficient electron density at that site. In
particular, a model with oxygen bonded to S
of Cys13
refines to 2.0 Å2 on both molecules A and
B, while the S
atom retains a B factor similar
to that observed with a persulfide (CSS residue) as a model.
Conversely, when an oxygen is modeled as a sulfur (Ser
"Cys")
the B factor always becomes much larger, indicative of excess electron density at the site in the model. Thus, the fact that a
S
atom on CSS13 and CSS213 refines to a reasonable B
factor indicates that it is not oxygen. Finally, the electron density in each and every case is 4-5
for sulfur, whether modeled as sulfur
or oxygen, and 1.5
for oxygen in serine, whether modeled as sulfur
or oxygen. Together, these data demonstrate that Cys and Ser can be
discriminated at 2.3 Å resolution, and that the atom bonded to S
of
Cys13 is more massive than oxygen and has electron density
comparable to that of sulfur in other cysteine residues with comparable
B factors. In principle, this atom could be aluminum,
silcon, phosphorus, chlorine, or argon in terms of x-ray scattering;
however, none of these atoms are likely or able to bond singly to
sulfur. Therefore, we conclude that the CSS residue is a cysteine
persulfide.
Ferredoxins are small acidic electron transfer proteins that contain [Fe-S] clusters attached to the polypeptide via cysteine residues. When a ferredoxin contains a [4Fe-4S]2+/+ cluster three of the four cysteine ligands are usually supplied by a highly conserved CysXXCysXXCys sequence motif (1, 6). Little is known about the in vivo assembly of these clusters and the role that the sequence motif plays in that process. Where x-ray structures are available it is clear that the clusters are buried (7-13), which suggests that they may be assembled during the protein folding process rather than after the protein is fully folded. For the simpler ferredoxins the protein can fold in vitro from apoprotein with the correct [4Fe-4S]2+/+ in place (26, 27), but in vivo other proteins are likely to be involved in the delivery of iron and sulfide (28). Whether the normal assembly process requires a specific sequence motif or just a specific three dimensional orientation of protein cysteine ligands is not known.
AvFdI is a 7Fe protein that contains a [3Fe-4S]+/0 cluster ligated by cysteinyl residues at positions 8, 16, and 49 (7-9). Although there is a free cysteine at position 11, that cysteine is far removed from the [3Fe-4S]+/0 cluster and could not be used as a fourth ligand to the cluster in the absence of substantial structural rearrangement. In this study the x-ray structures of AvFdI and PaFd (7-13) were used as a guide in attempts to obtain the correct three-dimensional orientation of four cysteines without creating a CysXXCysXXCys sequence motif.
Fig. 1 shows the structures of one of the
CysXXCysXXCys loops of PaFd
(11, 12), the corresponding
Cys8XXCys11XTyr13XXCys16
loop from AvFdI (9), and a model for the
Cys8XXCys11XCys13XXCys16
loop in a Y13C variant of AvFdI. The model was obtained by
replacing the Tyr13 side chain with that of cysteine,
without alteration of the main chain atoms, and rotating the
1 torsion angle from g+ to g
to illustrate the potential for proximity of the Cys13 S
atom to the [3Fe-4S] cluster. The three models are shown in the same
orientation to emphasize their overall similarity and to point out that
in AvFdI the two residues between Cys8 and
Cys11 represent a bulge expanding the upper half of the
loop. Therefore, in structural terms Cys11 corresponds to
residue 9 of PaFd, and Tyr13 corresponds to the
Cys11 ligand to a [4Fe-4S] cluster in PaFd. If
the two residue bulge in AvFd is deleted, and the remaining
atoms superposed onto PaFd, the main chain
,
angles
of the two loops are seen to be very similar, as evidenced by a
root-mean-square deviation of 31 pairs of corresponding atoms after
least squares fit of 0.53 Å. Based on this observed structural
similarity, it was anticipated that a Tyr213 to Cys
replacement in AvFdI should result in a protein capable of
forming a [4Fe-4S] cluster as in PaFd.
As described under
"Experimental Procedures" and shown in Table I, the x-ray crystal
structures for two crystals of Y13C, one grown with ME and one grown
without
ME, were solved. Both crystals had two molecules
(A and B) in the asymmetric unit. Fig. 2 shows
the electron density for the refined Cys13 residue of Y13C
in both molecules A and B in the crystallographic asymmetric unit in both a crystal grown with or without
ME present. Two conclusions arise from analysis of these data. First, the engineered Cys13 fails to serve as a cluster ligand and
second, it has been modified to become a cysteine persulfide. The
structure of Y13C FdI refined against the higher resolution, more
complete data set collected from a crystal grown in the presence of
ME (Table I) will be used as a basis for further discussion. The
geometry of the persulfides is normal and their conformations are very
similar in molecule A and B: S
-S
bonds
2.00, 2.01 Å and C
-S
-S
angles 104.4° and 108.8°,
respectively. The B factors for the persulfides are also similar, being 4.3, 7.3, and 11.9 Å2 for C
, S
, and
S
respectively, on molecule A, and 6.9, 9.9, and 13.6 Å2 for these atoms on molecule B. Overall
molecules A and B are very similar with an
root-mean-square deviation for 106 C
atoms after least squares fit
of 0.126 Å and a very similar distribution of average B
factor per residue over the length of the polypeptide chain. The two
copies of FdI in the asymmetric unit are also very similar to native
FdI in tetragonal crystals (9) with rms deviations for 106 C
atoms
after least squares fit of 0.179 and 0.183 Å. However, the
conformations of several surface side chains, in particular
Lys98, are rearranged due to the alternate crystal packing.
In the monoclinic unit cell similar contacts occur between residues at the C terminus and the [4Fe-4S] cluster binding loop as in tetragonal crystals, while a local 2-fold axis relating molecule A and
B is orthogonal to b, inclined 55° from
a, but does not intersect the 21-screw axis.
This interaction creates favorable contacts involving
Lys10, Thr82, Glu83,
Asp93, Gly96, Lys98,
Gln102, and His103.
Fig. 3 shows the environment of the cysteine persulfide
residue relative to Tyr13 in native FdI. The persulfide
retains hydrophobic contacts to Cys11, Pro50,
Pro87, and Ala91 present in native FdI for
Tyr13; a hydrogen bond involving Tyr13 and
Asp95 is lost. The van der Waals surface of the persulfide
occupies a similar volume as the tyrosine, except that, by virtue of
being a shorter side chain, the space filled by the tyrosine hydroxyl and C atoms and some of the space filled by the C
1 and C
2
atoms, is vacant. Consequently, the [3Fe-4S] cluster remains shielded from solvent in Y13C FdI, but is closer to the solvent accessible surface of the protein. In effect, residue 13 in both structures fills
a small cleft formed by the cysteine containing loops of residues 8-16
and 47-54 on either side of the [3Fe-4S] cluster (Fig. 3).
The occurrence of persulfides in protein structures is not without
precedent. In the Q206C mutant of subtilisin, a persulfide is observed
at the introduced cysteine (29). In this case, as well as in human
superoxide dismutase (30), a persulfide apparently arises as an
artifact of the purification procedure. In bovine liver rhodanese, a
persulfide is observed at the catalytically essential
Cys247 in the active site; the electron density of this
residue at 2.5 Å resolution is very similar to that shown in Fig. 2
(31). In Desulfovibrio gigas ferredoxin II (FdII) additional
electron density is observed on Cys11, a residue analogous
to Cys11 in AvFdI (32). In D. gigas
FdII Cys11 is not a ligand and a [3Fe-4S] cluster is
present in the protein; the additional density was modeled and refined
as methane thiol (32). In Y13C FdI there is no additional electron
density beyond the S atoms of the persulfides. At this point it is
not clear how the Y13C FdI persulfide arose but one possibility is that it was formed in vivo via the action of NifS or a
NifS-type enzyme (33). These pyridoxal phosphate-containing
enzymes appear to assist [Fe-S] cluster assembly by donating sulfide
(33), possibly by formation of persulfides on ligand cysteines. It is
interesting to note that neither of the free cysteines present in FdI
is modified, whereas the engineered Cys13 is a persulfide.
This suggests that if NifS is involved in modifying Cys13 there must be specificity to the reaction, either
involving a Cys ligand motif or a so far unrecognized structural
motif.
Fig. 4 shows the immediate environment of the CSS13
residue on molecule A of Y13C FdI. The structure of molecule
B is virtually identical. Considering the three divalent,
inorganic sulfur atoms of the [3Fe-4S] cluster, S of
Cys11 and CSS13, there are six sulfur atoms in close
proximity in this structure, and seven interatomic distances less than
6 Å. The conformation of Cys11 is very similar to that in
native FdI. S
of CSS13 has the most contacts (four), consistent with
the expectation that, if residue 13 were a cysteine, then this sulfur
atom could potentially be a ligand to a fourth iron atom, forming a
[4Fe-4S] cluster. In particular, the contact between this S
and
S12 of the [3Fe-4S] cluster is short, 3.44 Å, or 0.26 Å less than
the combined van der Waals radii of two sulfur atoms. This distance is
comparable to those between S9, S11, and S12 (average, 3.47 Å) and
within the range of S-S distances observed in other [3Fe-4S] and
[4Fe-4S] clusters (32). For comparison, the contact between
Cys24 S
, a non-ligand cysteine, and inorganic sulfur of
the [4Fe-4S] cluster in native FdI is 3.42 Å. Therefore, under
oxidizing conditions it could be expected that a S-S bond could form
between CSS13 S
and S12, as indeed occurs between Cys24
and the [4Fe-4S] cluster in the presence of ferricyanide (34). The
structure does not, however, explain the observation described below
that Y13C FdI is unstable under reducing conditions.
Absorbance and CD Spectra
The UV-visible absorption spectra
of Y13C FdI are shown in Fig. 5 compared with those of
native FdI. In the O2 oxidized state these spectra are
indistinguishable. These spectra were also unchanged by the anaerobic
addition of 2 mM dithiotheritol, which was added in an
attempt to reduce the persulfide. Native FdI is an air-stable protein.
Y13C is also fairly air stable when compared with some other FdI
mutants (20), but it is not as stable as the native protein. For
example, Y13C FdI could be stored at room temperature in air for about
24 h without cluster destruction, but longer storage eventually
led to complete bleaching of the protein. Therefore the protein was
purified anaerobically. As shown in Fig. 5 the addition of dithionite
to either native or Y13C FdI resulted in partial bleaching of the
spectrum. For the native protein this is known to arise from the
reduction of the [3Fe-4S]+ cluster to the 0 oxidation
state, because the reduction potential of the [4Fe-4S]2+
cluster is too low to be reduced by dithionite at this pH (35). The
data in Fig. 5 show that the Y13C FdI dithionite reduction behavior is
parallel to that of native FdI; however, we were surprised to observe
that the Y13C FdI variant was extremely unstable in the
dithionite-reduced state with complete destruction of both clusters
occurring within 90 min of the addition of dithionite. Thus, although
the protein was purified anaerobically, dithionite was not added to any
of the buffers. It should be noted that the instability on reduction is
not dependent upon the presence of dithionite as it is also observed in
the direct eletrochemical experiments described below.
CD spectra of oxidized and reduced native FdI and Y13C FdI obtained at
pH 7.4 are compared in Fig. 6. The wavelength dependence and form of the oxidized spectra are again extremely similar, strongly
suggesting that the two proteins should have the same cluster
composition. For the dithionite-reduced protein, the wavelength dependence is also similar but not identical. However, the amount of
time required to prepare the samples and collect the data was sufficiently long that some cluster destruction may have occurred in
the reduced state so that the minor differences may not be significant.
To obtain more information about the reduced [3Fe-4S]0
cluster, we carried out the CD measurements at both high and low pH.
The wavelength dependence and form of the CD spectra of native
AvFd are quite different depending upon the pH due to the direct protonation of the reduced cluster at low pH (15, 36). The same
changes in CD are observed for the Y13C FdI on going from high to low
pH, indicating that its reduced [3Fe-4S]0 can also be
directly protonated (data not shown). However, again the experiments
must be carried out quickly, because the Y13C protein is extremely
unstable in the reduced state.
Electrochemistry
Further confirmation of the similarities
between the native and Y13C variant of FdI was obtained by
electrochemical characterization. Despite the instability of the
protein, it was possible to determine reduction potentials over a
limited pH range using protein-film voltammetry in which a monolayer or
less of sample molecules contacting different solutions can be examined
in a short period of time (19). Fig. 7 shows base
line-corrected voltammograms, measured at 20 mV s1 for
the direction of increasing potential, which reveal the general manner
in which the different cluster redox transitions in the Y13C FdI vary
with pH. The most prominent feature, by reference to previous studies
(37), is assigned to the novel pH-dependent two-electron
couple [3Fe-4S]0/2
and is designated C
.
Likewise, the signal with the most positive potential is designated
A
and is assigned to the well documented couple
[3Fe-4S]1+/0, while the remaining signal (B
)
overlaid completely by C
at pH < 7.0 corresponds
closely to the potential values of the couple [4Fe-4S]2+/1+ in the native protein.
Fig. 7 shows the pH dependence of reduction potentials determined from
the average of the oxidative and reductive peak potentials. No
differences were observed between values obtained at scan rates 20, 50, and 100 mV s1. Notwithstanding the narrow range over
which data were obtained, the curve for couple A
could be
fit to a scheme consistent with protonation of the "0" oxidation
level at pK = 7.2 ± 0.3, and E0
(alkaline form) =
417 ± 10 mV. The
line for couple C
is very steep, and, overlaying couple
B
, it was not realistic to attempt analysis beyond linear
regression, which gave a slope of
74.5 mV/pH unit. At pH 7.0, couples
A
, B
, and C
have reduction
potentials
402 ± 5,
640 ± 10, and
740 ± 10 mV,
respectively, values previously obtained for the native protein. These
data therefore indicate that the replacement of tyrosine by a cysteine
persulfide does not affect either the pH dependence or reduction
potential of the [3Fe-4S] cluster. Attempts were made to detect iron
uptake into the [3Fe-4S]0 cluster using the procedure
previously described (38) for observing cluster transformations in
FdIII from Desulfovibrio africanus. No changes in signals
were observed in experiments carried out at pH 7.0, with Fe(II)
concentrations of 370 and 690 µM in the buffer
electrolyte and holding the potential at
460 mV for various times
before cycling.
In general, the presence of a [3Fe-4S]+
cluster in any protein is easily identified by the appearance of a
characteristic g = 2.01 EPR signal (14). As shown in Fig.
8 the EPR spectrum of oxidized Y13C FdI was found to be
both qualitatively and quantitatively indistinguishable from that of
native FdI. As is the case for native FdI, Y13C FdI is EPR silent in
the dithionite-reduced state, consistent with the formation of
[3Fe-4S]0. No evidence for the conversion of that cluster
to a [4Fe-4S]2+/+ cluster was obtained when the reduction
was carried out in the presence of excess iron.
Taken together these data demonstrate that, despite the introduction of an additional Cys residue in the correct structural location, a [4Fe-4S]2+/+ cluster fails to form in that location.
Why does Y13C FdI not form a [4Fe-4S]2+/+ cluster in the [3Fe-4S]+/0 location? One possibility is that a CysXXCysXXCys motif is actually required for the in vivo assembly of a [4Fe-4S] cluster in that location. Another is that the presence of the persulfide may prevent Cys13 from serving as a ligand. Alternatively it may be that the presence of the persulfide indicates that the assembly process was initiated by addition of a sulfide to Cys13, but that the subsequent use of that residue as a cluster ligand was somehow prevented. For example, the [3Fe-4S] cluster may already have had its full complement of inorganic sulfur and therefore have been unable to use CSS13 to insert an inorganic sulfur.
Analysis of the structure also provides clues as to why
Cys13 might not be able to serve as a cluster ligand even
if the persulfide could be cleaved in vivo. Thus, residue 13 and its neighbors are involved in a number of contacts with the
surrounding protein that may restrain Cys13 from undergoing
small but necessary conformational changes. That some adjustment is
needed is indicated by the S to [3Fe-4S] cluster divalent sulfur
distances which cannot be made symmetrical by simple rotation of the
Cys13
1 torsion angle. In the Y13C FdI
structure these distances at best range from 2.5 to 4.2 Å, whereas in
the PaFd structure where an iron atom of the [4Fe-4S]
cluster is ligated, the corresponding distances are 3.5 to 4.1 Å.
Therefore, even if the additional sulfur of the persulfide were removed
Cys13 would have to shift ~1 Å to ligate Fe as
Cys11 does in PaFd. In the structure of
AvFdI (9) at least 11 hydrogen bonds may restrain such a
shift; the carbonyls and amides of residues 11-15 are involved in
hydrogen bonds with the main chain atoms of Cys8,
Val17, Glu18, and Leu88, and with
the side chains of Thr14 and Lys84; and the
side chains of Lys12, Thr14, and
Asp15 are involved in hydrogen bonds with the carbonyl of
Gly28, amide of Lys85, and the side chains
Glu27 and Lys84, forming two salt bridges. In
previous studies we have shown that the [4Fe-4S]2+/+
cluster of AvFdI is able to switch one remote Cys ligand for another Cys residue (23, 38, 42). It should be noted, however, that in
the structures of those C20A and C20S FdI variants considerable rearrangement takes place to accommodate a new ligand
(Cys24) to a [4Fe-4S] cluster (20, 39). In those cases
the loop undergoing the conformational change,
Cys20XXXCys24, is on the surface of
the protein and is involved in only two hydrogen bonds with other
residues.
The data just described demonstrate that Y13C FdI is extremely similar to native FdI in its cluster organization and redox properties, but that it differs from native FdI in being unstable in the reduced state and in the presence of oxygen. The protein also accumulates to only about 5% of wild-type levels in vivo, which may reflect either an initial folding problem or increased oxygen sensitivity leading to protein destruction. In native FdI, Tyr13 is partially exposed on the surface of the protein but also is in contact with the [3Fe-4S] cluster (9). As indicated above, the replacement of this large, aromatic residue with a cysteine persulfide creates a cavity adjacent to the cluster. Although the cluster is not directly exposed to solvent, the smaller CSS13 chain is unlikely to be as effective in shielding the cluster, perhaps explaining the increased oxygen sensitivity of this mutant and its instability in the reduced state. The Tyr13 side chain also has hydrophobic contacts with Cys11, Pro50, Pro87, and Ala91, and is involved in a hydrogen bond with Asp95. The loss of these contacts may expose additional hydrophobic surface area in Y13C FdI as evidenced by the reduced solubility of the mutant protein at high ionic strength, as observed in our early unsuccessful crystallization trials. The exposure of this additional hydrophobic surface might also contribute to the lowered stability of the Y13C FdI variant.
In previous studies we have shown that AvFdI has an electron-transfer function that is important for cell growth (35, 40) and a regulatory function in controlling the expression of the fpr gene product (40-42). Y13C FdI appears to be able to carry out both of these functions as evidenced by the normal growth of the Y13C overproduction strain and by the observation that FPR levels are the same in strains overproducing the native and Y13C variants of FdI. If the in vitro instability of the reduced protein discussed above is due to increased solvent exposure, then something appears to prevent this exposure from becoming a problem in vivo.
Atomic coordinates and structure factors (Code 1FTC) were deposited in the Protein Data Bank, Brookhaven National Laboratory.
We thank E. A. Stura for invaluable discussions and Hayley Angove for assistance with EPR spectroscopy.