©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of Desulfovibrio gigas Desulforedoxin in Escherichia coli
PURIFICATION AND CHARACTERIZATION OF MIXED METAL ISOFORMS (*)

(Received for publication, March 13, 1995; and in revised form, June 15, 1995)

Christopher Czaja (1) Robert Litwiller (1) Andy J. Tomlinson (2) Stephen Naylor (2) Pedro Tavares (3) Jean LeGall (4) José J. G. Moura (3) Isabel Moura (3) Frank Rusnak (1)(§)

From the  (1)Section of Hematology Research, the (2)Biomedical Mass Spectrometry Facility, and the Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, the (3)Departmento de Qu&ıacute;mica and Centro de Qu&ıacute;mica Fina e Biotecnologia, Faculdade de Ciéncias e Tecnologia, Universidade Nova de Lisboa, 2825 Monte de Caparica, Portugal, and the (4)Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alpha(2) 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.


INTRODUCTION

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(2)- 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(^1)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.


EXPERIMENTAL PROCEDURES

Materials

Sephadex G-75 resin (10-40 µm), the HiLoad 26/10 Q-Sepharose Fast Flow column, and the mono-Q HR5/5 columns were obtained from Pharmacia Biotech Inc. Acetonitrile and water for HPLC purification were purchased from Baxter (Minneapolis, MN). Acetic acid was obtained from Aldrich. Protein low molecular mass SDS-PAGE standards (2.5-17 kDa) and isopropyl alcohol were purchased from Sigma. The silica-based C(4) resin used for mass spectrometry of desulforedoxin was purchased from Waters Corp. (Milford, MA). The oligonucleotide primers used to amplify the dsr gene were synthesized by the Molecular Biology Core Facility at the Mayo Clinic and Foundation. The vector pT7-7 was a generous gift of Dr. Stan Tabor, Harvard Medical School. E. coli strain BL21(DE3) was obtained from Novagen Inc. (Madison, WI).

Methods

DNA sequencing was carried out using the Sequenase version 2.0 DNA sequencing kit (United States Biochemical Corp., Cleveland, OH) and [S]deoxyadenosine 5`-(alpha-thio)triphosphate (500 Ci/mmol, DuPont NEN) according to the manufacturer's instructions. Optical spectra were recorded using a Cary 1 UV/visible spectrophotometer. SDS-PAGE was carried out in the presence of Tricine as described(14) . Genomic DNA was isolated from D. gigas as described(15) . EPR measurements were made on a Bruker ER200D-SRC spectrometer equipped with an Oxford Instruments Continuous flow cryostat. Iron and zinc analyses were carried out by the Metals Laboratory at the Mayo Clinic using inductively coupled plasma atomic emission spectrometry(16, 17) . Iron and zinc stoichiometries were determined using protein concentrations determined by amino acid analysis.

Cloning of the Gene for D. gigas Desulforedoxin (dsr)

The dsr gene was cloned from D. gigas genomic DNA using polymerase chain reaction and two oligonucleotide primers homologous to its sequence(5) . The two primers, 5`-CCATGGATCCTCTAGAAGGAGATATACATATGGCGAACGAAGGCGAC-3` and 5`-TTAAGCTTCTGCAGTCGACTTACTGCTGCACCATATC-3`, are homologous to opposite ends of the dsr gene sequence at their 3` termini and contain restriction endonuclease sites for subsequent subcloning at their 5` termini. Following polymerase chain reaction amplification, the resultant 156-base pair fragment was purified using a nondenaturing 8% polyacrylamide gel in 89 mM Tris, 89 mM boric acid, 2.0 mM EDTA (TBE buffer) as described (18) and electroeluted using an IBI model UEA electroeluter (International Biotechnologies, Inc., New Haven, CT) according to the manufacturer's instructions. The dsr DNA fragment was then digested with XbaI and PstI and subcloned into the ampicillin-resistant plasmid pUC19 digested similarly. Recombinant clones were screened by restriction digest analysis and the complete gene sequence of positive clones verified by DNA sequencing.

Overexpression and Purification of Desulforedoxin

Following verification by DNA sequencing, the dsr gene was excised from pUC19 using the restriction endonucleases NdeI and HindIII and subcloned into NdeI and HindIII-digested pT7-7 (19) to give the recombinant vector DSRT77-2. Recombinant plasmids from ampicillin-resistant colonies containing the dsr gene were identified by restriction digest analysis and subsequently transformed into competent BL21(DE3) cells(20) .

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 approx0.7 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.

Mass Spectrometry of Desulforedoxin Fractions 1 and 2

Apo-desulforedoxin (approx200 µg) was prepared as described (4, 21) and brought up in 0.5 M Tris-Cl, pH 7.5, after the last trichloroacetic acid precipitation. Apo-desulforedoxin was reduced by incubating for 30 min at 37 °C with a 50-fold molar excess (over the number of cysteine residues) of beta-mercaptoethanol and alkylated by adding a 3-fold molar excess (over beta-mecaptoethanol) of iodoacetic acid. The pH of the solution was adjusted to neutrality with Tris base and incubated at room temperature for 30 min. Carboxymethylated apo-desulforedoxin was purified by reverse-phase HPLC chromatography.

Samples of desulforedoxin fractions 1 and 2 were desalted prior to ESI-MS using a microcolumn containing silica-based C(4) resin as described previously(22) . Specifically, the microcolumn was conditioned with acetonitrile/water/acetic acid (2:97:1, v/v/v, approx50 µl) followed by acetonitrile/water/acetic acid (80:19:1, v/v/v, approx25 µl). A final conditioning step using approx25 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(r)) do not take into account differences in ligand protonation state expected between apo versus metal-bound forms of desulforedoxin.

Analytical Equilibrium Ultracentrifugation

Samples of desulforedoxin fractions 1 and 2 from the last ion exchange step were subject to analytical equilibrium ultracentrifugation in a Beckman rotor and L8-60 M ultracentrifuge. The protein in 25 mM MOPS, 0.15, M NaCl, pH 7.0, was centrifuged for 47 h at 16.4 °C and 28,000 revolutions/min. The absorbance as a function of radius was obtained using a Beckman Prep UV Scanner equipped with a 545-nm bandpass filter.


RESULTS

Expression of Desulforedoxin in E. coli and Purification to Homogeneity

The gene for D. gigas desulforedoxin was cloned using the polymerase chain reaction with primers homologous to opposite ends of the gene and subcloned into the expression vector pT7-7. A culture of BL21(DE3) cells harboring this plasmid produced desulforedoxin containing iron after induction with IPTG, as evident by the appearance of pink-colored chromatographic fractions which had optical absorbance resembling desulforedoxin purified from D. gigas(1, 4) . The protein was subsequently purified to homogeneity using three chromatographic steps including anion-exchange and gel filtration chromatographies and a final anion-exchange step using either Q-Sepharose Fast Flow or Mono-Q columns. The protein appeared homogeneous as judged by SDS-PAGE (Fig. 1).


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(2)-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(r) = 4036.5), indicating that both had identical polypeptide chains.



Optical Absorbance Spectra of Recombinant Desulforedoxin

Desulforedoxin fractions 1 and 2 exhibit absorbance in the ultraviolet region due to the single tyrosine residue and absorbance in the visible region resulting from ligand-to-iron charge transfer bands (Fig. 2). Both fractions have identical absorbance maxima at 278.1, 370.1, and 506.4 nm with distinct shoulders at 284, 300, and 570 nm. In addition, both fractions have nearly identical A/A ratios of approx1.7. Desulforedoxin fractions 1 and 2 differ, however, in the ratio of optical absorbance at 278 nm versus either 506 or 370 nm. The ratio A/A for fraction 1 is 1.07 versus 1.50 for fraction 2 while the A/A ratio is 0.63 for fraction 1 and 0.86 for fraction 2, indicating a difference in protein-to-iron stoichiometry for these fractions.


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.



Electron Paramagnetic Resonance Spectra of Recombinant Desulforedoxin

Low temperature EPR spectra of desulforedoxin fractions 1 and 2 are essentially the same with g values at 7.7, 5.7, 4.1, and 1.8 (data not shown). The spectra are identical to those obtained from desulforedoxin isolated from D. gigas(2, 4) and are characteristic of a high-spin ferric ion with S = 5/2. These resonances can be described by the spin Hamiltonian:

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.

Ultracentrifugation of Desulforedoxin

Analytical sedimentation equilibrium of desulforedoxin fraction 1 yielded a native molecular mass of 8,670 ± 154 Da, indicative of an alpha(2) homodimer. A value of 9,890 ± 210 Da was obtained for desulforedoxin fraction 2, a value also consistent with a dimeric tertiary structure. These values was determined using a partial specific volume calculated from the amino acid composition of 0.73 g/ml(23) .

Metal Ion Analysis of Desulforedoxin Fractions 1 and 2

Both fractions of desulforedoxin were analyzed for the presence of iron, zinc, magnesium, copper, and calcium. While the latter three metals were either absent or present only in trace amounts, both samples contained iron while only fraction 2 contained zinc (Table 2). Using a protein concentration determined from amino acid analysis, fraction 1 had an iron/subunit stoichiometry of 0.97 while fraction 2 had iron/subunit and zinc/subunit stoichiometries of 0.56 and 0.52, respectively. These results indicate that fraction 1 is an alpha(2) homodimer fully reconstituted with iron while fraction 2 is a dimer containing 1 mol each of iron and zinc.



ESI-MS of Desulforedoxin Fractions 1 and 2

Mass spectrometry of desulforedoxin fractions 1 and 2 indicated the presence of both apo- and metal-bound forms of desulforedoxin. Fraction 1 exhibited ions with 3805, 3859, 7611, and 7,718 Da, corresponding to the apo-monomer (calculated mass, 3804 Da), iron-bound monomer (calculated mass = 3860 Da), apo-dimer, (calculated mass = 7609 Da), and Fe(2) dimer (calculated mass = 7720 Da), respectively (Fig. 3A). ESI-MS of fraction 2 also revealed molecular ions corresponding to monomeric and dimeric forms of apo-desulforedoxin at 3805 and 7611 Da, respectively (Fig. 3B). Additional species were evident at 3859, 3869, 7675, and 7729 Da corresponding to the iron-bound monomer, zinc-bound monomer (calculated mass = 3870 Da), a desulforedoxin dimer containing 1 zinc atom (calculated mass = 7674 Da), and a desulforedoxin dimer containing one atom each of iron and zinc (calculated mass = 7730 Da), respectively. The molecular ion at 7718 Da in desulforedoxin fraction 2 is assigned to a desulforedoxin dimer containing two iron atoms, presumably formed during preinjection or ionization processes.


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.''




DISCUSSION

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(2) 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 (max) 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(2) 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 alpha(2) 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. (^2)

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM46865 (to F. R.), Junta Nacional de Investigaçäo Cient&ıacute;fica e Tecnológica Grants STRDA/BIO/359 and PBIC/QUI/616 (to I. M.), STRDA/C/CEN/538 and PBIC/BIO/1668 (to J. J. G. M.), Grant ERBCHRXCT920014 from the European Community, and for an instrument grant from Finnigan MAT for the ESI source (to S. N. and A. J. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 507-284-2289; Fax: 507-284-8286.

(^1)
The abbreviations used are: dsr, gene for desulforedoxin; ESI, electrospray ionization; IPTG, isopropyl-beta-thio-D-galactoside; MS, mass spectrometry; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; Tricine, N-tris(hydroxymethyl) methylglycine; MOPS, 4-morpholinepropanesulfonic acid.

(^2)
C. Czaja and F. Rusnak, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Linda Benson for her assistance with mass spectrometry, and Dr. Whyte Owen for assistance with analytical ultracentrifugation.


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