(Received for publication, March 13, 1995; and in revised form, June 15, 1995)
From the
The dsr gene from Desulfovibrio gigas encoding
the nonheme iron protein desulforedoxin was cloned using the polymerase
chain reaction, expressed in Escherichia coli, and purified to
homogeneity. The physical and spectroscopic properties of the
recombinant protein resemble those observed for the native protein
isolated from D. gigas. These include an tertiary structure, the presence of bound iron, and absorbance
maxima at 370 and 506 nm in the UV/visible spectrum due to
ligand-to-iron charge transfer bands. Low temperature electron
paramagnetic resonance studies confirm the presence of a high-spin
ferric ion with g values of 7.7, 5.7, 4.1, and 1.8.
Interestingly, E. coli produced two forms of desulforedoxin
containing iron. One form was identified as a dimer with the
metal-binding sites of both subunits occupied by iron while the second
form contained equivalent amounts of iron and zinc and represents a
dimer with one subunit occupied by iron and the second with zinc.
Desulforedoxin from Desulfovibrio gigas is a small,
homodimeric, nonheme iron protein containing a single iron
atom/subunit(1, 2, 3, 4, 5) .
The presence of four cysteine residues/subunit in desulforedoxin has
led to a model in which the iron is coordinated by four cysteinyl
sulfur atoms, analogous to the tetrahedrally coordinated iron atom of
rubredoxin(6) . However, the spectral properties of
desulforedoxin (1, 2, 4) are distinctly
different than those observed for rubredoxin(7, 8, 9, 10, 11, 12) indicating
that the metal-ligand geometry of these two proteins differ. In fact,
the crystal structure of desulforedoxin, recently determined to
1.8-Å resolution, has found that the desulforedoxin metal is
coordinated by four cysteine residues in a tetrahedral arrangement
significantly distorted when compared with rubredoxin(13) .
This distortion is likely due to a difference in metal binding motifs
for rubredoxin versus desulforedoxin. In rubredoxin, this
motif consists of two pairs of cysteine residues at the NH-
and COOH terminii with the sequence C-X-X-C. In
desulforedoxin, this motif is conserved at the amino terminus but the
carboxyl-terminal pair consists of adjacent
cysteines(3, 5) , leading to the hypothesis that the
lack of intervening residues between carboxyl-terminal cysteines
imparts geometric strain on the metal(4) .
In this report,
we describe the cloning of the dsr()gene from D. gigas and the expression of the recombinant protein in Escherichia coli. Recombinant desulforedoxin contained iron
and exhibited optical and EPR properties identical to that observed
with the native D. gigas protein. Interestingly, at least two
distinct species were resolved during purification. One of these was
identified as a dimer with the metal site of each subunit occupied by
iron and is presumably identical to the D. gigas protein
previously isolated(1, 2, 3, 4) .
Surprisingly, the second species contained half an equivalent each of
iron and zinc. This species represents a mixed-metal iron/zinc dimer in
which zinc has been incorporated into one of the iron-binding sites
during expression.
A typical purification
of recombinant desulforedoxin involved anion exchange and gel
filtration chromatographies of crude extracts obtained from E. coli cells overexpressing the dsr gene. Eighteen liters of
BL21(DE3) cells containing DSRT77-2 were grown at 37 °C with
shaking in Luria broth containing ampicillin (100 µg/ml) until the
absorbance at 595 nm reached 0.7 at which point IPTG was added to a
final concentration of 1.0 mM. The cell cultures were grown
for an additional 6 h and harvested by centrifugation at 3,400
g, 15 min. The cells were washed once in 0.10 M Tris-Cl, pH 7.5, and suspended in buffer containing 0.10 M Tris-Cl, pH 7.5, at about 0.4 g wet cell weight/ml. The cells were
lysed by three passages through a French pressure cell at 17,000
pounds/square inch. A solution of 2% protamine sulfate in 0.1 M Tris-Cl, pH 7.5, was added dropwise with stirring at 0 °C for
30 min, and the resulting solution was centrifuged at 39,000
g for 30 min to obtain crude extract. Subsequent purification
was carried out at 4 °C.
The crude extract was loaded onto a
column (2.6 35 cm) containing DEAE-Sepharose CL-6B and the
resin washed with 400 ml of 25 mM Tris-Cl, pH 7.5. The protein
was eluted with 25 mM Tris-Cl, pH 7.5, buffer containing 0.2 M NaCl. Fractions were collected and assayed for
desulforedoxin by measuring the absorbance at 370 nm. Pink-colored
fractions were combined and concentrated to 11 ml using an Amicon
stirred cell equipped with a YM3 Diaflo membrane (Amicon, Inc.,
Beverly, MA). The protein was then loaded onto a column (2.6
100 cm) containing Sephadex G-75 gel filtration resin equilibrated with
25 mM Tris-Cl, 0.15 M NaCl, pH 7.5, and eluted with
the same buffer at 20 ml/h. Fractions containing desulforedoxin were
identified by their optical absorbance spectra(1) , combined,
and dialyzed at 4 °C against two changes of 1.0 liter of 25 mM MOPS, pH 7.0. The dialyzed protein was loaded onto a Q-Sepharose
column and the resin washed with 100 ml of 25 mM MOPS, pH 7.0.
The protein was then eluted with 25 mM MOPS, 0.15 M NaCl, pH 7.0, at which point, desulforedoxin split into two
distinct pink-colored bands as it migrated down the column. After the
majority of the faster moving peak had eluted from the column, the
second fraction of desulforedoxin was eluted with 25 mM MOPS,
0.3 M NaCl, pH 7.0. The two fractions resolved in this manner,
corresponding to the first and second eluting peaks from the
Q-Sepharose ionexchange step, are designated desulforedoxin fractions 1
and 2, respectively.
Alternatively, smaller amounts of desulforedoxin could be purified after gel filtration chromatography using an HR5/5 Mono-Q anion-exchange column. With this column, desulforedoxin fractions 1 and 2 were eluted with a 30-ml gradient of NaCl from 0.1 to 0.4 M. A typical purification yielded approximately 0.4 mg for desulforedoxin fraction 1 and 1.2 mg for desulforedoxin fraction 1, per liter of culture.
Samples of
desulforedoxin fractions 1 and 2 were desalted prior to ESI-MS using a
microcolumn containing silica-based C resin as described
previously(22) . Specifically, the microcolumn was conditioned
with acetonitrile/water/acetic acid (2:97:1, v/v/v,
50 µl)
followed by acetonitrile/water/acetic acid (80:19:1, v/v/v,
25
µl). A final conditioning step using
25 µl of the
predominantly aqueous solution was used prior to loading 6 µl of a
20 µM solution of desulforedoxin onto the column. Sample
cleanup was effected using 20 µl of the predominantly aqueous
solution and the sample eluted directly into the ESI source using
acetonitrile/water/acetic acid (49:50:1, v/v/v) at a flow rate of 2
µl/min. A sheath liquid of isopropyl alcohol/water/acetic acid
(60:40:1, v/v/v) flowing at 5 µl/min and a sheath gas of dry
nitrogen (2 bar) were used to ensure stable ESI spray conditions
throughout the sample analysis.
All analyses were performed using a
Finnigan MAT ESI source and model 900 mass spectrometer (Finnigan MAT,
Bremen, Germany) of EB configuration. Analyte detection was by the
PATRIC focal plane detector using an 8% mass window. ESI
spray voltage was 2.8 kV referenced against an accelerating voltage of
4.75 kV. Scan range was 500-2500 Da at a rate of 2 s/decade with an
instrument resolution of 1200 and an expected mass accuracy of ±
2 Da. Calculated average molecular weights (M
) do
not take into account differences in ligand protonation state expected
between apo versus metal-bound forms of desulforedoxin.
Figure 1: Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis of desulforedoxin fractions 1 and 2. Lane 1, molecular mass standards with corresponding molecular masses noted to the left; lane 2, purified desulforedoxin fraction 1 (8 µg); lane 3, purified desulforedoxin fraction 2 (8 µg).
Interestingly, recombinant desulforedoxin produced
in E. coli resolved into at least two fractions which differed
in their elution times during anion-exchange chromatography. Both
fractions had identical amino termini; NH-terminal
sequencing of both fractions gave the expected sequence ANEG,
indicating that the first methionine residue was removed during
expression in E. coli. The amino acid composition of both
fractions was also identical (Table 1). Lastly, ESI-MS
measurements of carboxymethylated apo-desulforedoxin prepared from both
fractions yielded the same molecular ion of 4036.7 (calculated M
= 4036.5), indicating that both had
identical polypeptide chains.
Figure 2:
UV/visible spectrum of desulforedoxin
fractions 1 and 2 in 25 mM MOPS, pH 7.0. Molar extinction
coefficients () calculated based on protein concentration
determined by amino acid analysis.
where D and E are the zero field splitting parameters. With an E/D value of 0.08, this Hamiltonian reproduces the observed g values.
Figure 3: Deconvoluted ESI-MS data of desulforedoxin fractions 1 (A) and 2 (B) in the 3750-3950 and 7500-7900 mass range as described under ``Experimental Procedures.''
Originally isolated from D. gigas,
desulforedoxin was identified as an unusual nonheme iron
protein(1, 3) . Initial characterizations indicated
that the metal-binding site of desulforedoxin resembled the
iron-binding site of rubredoxin since desulforedoxin contained two iron
atoms, eight cysteine residues, no labile sulfide, and exhibited
absorbance in the UV/visible spectrum due to ligand to metal charge
transfer(1, 2, 4) . Further characterization
of desulforedoxin indicated that the protein was a homodimer and
confirmed the presence of two pair of cysteine residues/subunit,
presumably in coordination with a single iron atom, in a motif similar
to that found in rubredoxins. Thus, an NH terminus pair of
cysteine residues at positions 9 and 12 (CELCG)
and adjacent cysteines at positions 28 and 29 in desulforedoxin
resembled the motif observed in the rubredoxins which have two cysteine
pairs at the amino (CXXCG)
and carboxyl
(CXXCG)
termini providing tetrahedral
coordination to an iron atom(6) .
Although there are
similarities between rubredoxin and desulforedoxin as noted above,
optical, EPR, and Mössbauer spectra of
desulforedoxin differ from those observed for rubredoxin. Thus,
although both desulforedoxin (1, 4) and rubredoxin (7, 8, 9) exhibit ligand-to-iron charge
transfer bands, differences in and molar extinction
coefficient (
) values are clearly evident. EPR spectra of
rubredoxin, with g values observed at 4.3 and 9.4, are
essentially rhombic (E/D = 0.28) (8, 11) while EPR spectra of desulforedoxin are more
axial with E/D = 0.08(2, 4) .
Mössbauer data also indicate that, although the
iron atom of desulforedoxin is probably coordinated by four sulfur
ligands, the degree of metal-ligand covalency compared to rubredoxin
differs(4, 10, 12) .
Two hypotheses have
been put forth to explain the unique spectroscopic properties of
desulforedoxin. The first proposed that the iron atom is coordinated by
four cysteine residues in a geometrically strained manner, with the
strain likely imposed by the lack of intervening residues between
juxtaposed cysteine residues 28 and 29(4) . The crystal
structure of desulforedoxin from D. gigas has now been
determined at 1.8-Å resolution. The iron center is located at the
periphery of the molecule and is bound to four cysteinyl sulfur atoms
with no other ligands in close vicinity to the metal. All four Fe-S
bonds are nearly identical in length (2.30 ± 0.02 Å) while
the S-Fe-S
angles range from 104°
to 121.5°. The S
-Fe-S
angle
involving vicinal cysteines, Cys
and Cys
, is
about 121°, substantially widened when compared to an exact
tetrahedral geometry(13) . A distorted tetrahedral sulfur
coordination has also been supported by resonance Raman of the protein
isolated from D. gigas(24) and
Cd-NMR
of the Cd
-substituted recombinant
protein(25) .
Recently, another non-heme iron protein has
been identified in Desulfovibrio species which exhibits
spectroscopic properties nearly identical to the iron site of
desulforedoxin(26, 27) . That protein, designated as
desulfoferrodoxin, is a monomer containing an NH terminus
domain sharing 67% sequence identity with desulforedoxin (including the
spacing between cysteine residues) (5, 28) and a
carboxyl terminus domain which binds a second iron atom with unique
spectroscopic properties. A comparison of the EPR spectra of the
desulforedoxin-like site in desulfoferrodoxin with the model complex
Fe(III)-diethylenetriaminepentaacetic acid led Hagen et al.(29) to propose that a higher coordination number for the
iron site in desulforedoxin could not be excluded and that other
ligands besides cysteinyl sulfur might be present. With the available
x-ray structure of desulforedoxin(13) , it is proven
unambiguously that the previous model of coordination by four cysteinyl
residues based on sequence and spectroscopic data was correct.
With
desulforedoxin subcloned and expressed in E. coli, we can
explore and modulate the properties of the iron center. Production of
mutants introducing a different spacing between Cys and
Cys
will be very interesting and very useful.
Spectroscopic and x-ray studies of these mutants will help clarify
whether the spectroscopic differences observed between the
desulforedoxin and rubredoxin structures are directly due to the
vicinal cysteines or are due to other structural features. Mutagenesis
studies investigating these hypotheses are currently in progress.
Quite interestingly, expression in E. coli yielded two
iron-containing forms of desulforedoxin, designated as fractions 1 and
2. Analytical ultracentrifugation confirmed that both fractions 1 and 2
have an tertiary structure. Interestingly, the
molecular masses measured by centrifugation for fractions 1 and 2 were
12 and 28% larger, respectively, than the predicted molecular mass of
metal-bound dimers. Since both fractions had identical elution volumes
during gel filtration chromatography (data not shown), these
disparities are likely due to differences in water of hydration and/or
partial specific volumes. The fact that fraction 1 contained 1 mol of
iron/mol of desulforedoxin monomer indicates that this fraction
corresponds to a homodimer with iron occupying both metal-binding
sites. With iron and zinc stoichiometries/desulforedoxin monomer of
0.5, fraction 2 represents a dimer with one metal site occupied by
Fe
and the second with Zn
. This
model is confirmed by ESI-MS of desulforedoxin fractions 1 and 2 which
revealed a molecular mass corresponding to a desulforedoxin dimer
containing either two iron atoms for fraction 1 or one atom each of
iron and zinc for fraction 2. Substitution of one of the trivalent
ferric ions with Zn
would also lower the pI of the
protein, consistent with a later elution time during anion-exchange
chromatography for fraction 2 relative to fraction 1.
Both fractions had identical optical spectra from 320 to 700 nm, the region attributed to ligand-to-iron charge transfer bands, although the molar extinction coefficients in this region (based on protein concentrations) for fraction 2 were about half those measured for fraction 1. These results are as expected for replacement of one of the ferric ions with zinc in fraction 2. Both recombinant desulforedoxin fractions 1 and 2 showed EPR spectra characteristic of a high-spin ferric ion and are identical to those obtained for the D. gigas protein(2, 4) . As was observed previously with desulforedoxin isolated from D. gigas, the EPR intensity of recombinant desulforedoxin is not proportional to iron content (data not shown).
The generation of a mixed metal iron/zinc derivative of
desulforedoxin (fraction 2) was somewhat surprising since this species
has not yet been isolated from D. gigas. In addition to this
species and the di-iron form (fraction 1), it is possible that a
desulforedoxin dimer containing two zinc ions was produced during
expression in E. coli. A form containing two Zn ions would not exhibit the characteristic pink color and visible
absorbance at 370 and 506 nm observed for the ferric ion containing
species. Thus, it is likely that a di-zinc form was disregarded during
purification. In fact, we have observed a species containing two zinc
ions during purification of desulforedoxin mutant proteins. (
)
In addition to desulforedoxin, overexpression of other
iron-containing metalloproteins in E. coli does not always
lead to a correct metal-reconstituted protein. To wit, ribonucleotide
reductase expressed in E. coli is produced largely as an
apoprotein and exogenous Fe needs to be added to
convert the bulk of the recombinant protein to a native-like and
catalytically active form (30) . Recently, overexpression of
rubredoxin in E. coli by Eidsness et al.(31) also resulted in the production of both
Fe
and Zn
-containing species. In
that study, approximately 70% of the total rubredoxin produced
contained Zn
rather than Fe
.
Substitution of Fe
by Zn
in
desulforedoxin and rubredoxin is probably a result of the relative
abundance of these metals during growth and expression in E.
coli, and it may be possible to alter growth conditions to favor
the expression of one form relative to others.
Although unexpected,
the production of Zn substituted forms of
desulforedoxin and other metalloproteins could find great utility in
future spectroscopic studies, e.g. multidimensional NMR
methods. The presence of Fe
leads to paramagnetic
effects in NMR, but substitution with a diamagnetic metal such as
Zn
can be useful for three-dimensional structure
determination via these NMR methods. Thus, the production of these
Zn
substituted derivatives in vivo can
provide material which may not be otherwise available by in vitro metal reconstitution.