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J. Biol. Chem., Vol. 275, Issue 37, 28439-28448, September 15, 2000

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Neelaredoxin, an Iron-binding Protein from the Syphilis Spirochete, Treponema pallidum, Is a Superoxide Reductase^*

Tijana Jovanovi^§, Carla Ascenso^¶, Karsten R. O. Hazlett, Robert Sikkink, Carsten Krebs^**, Robert Litwiller, Linda M. Benson, Isabel Moura^¶, Jose J. G. Moura^¶, Justin D. Radolf, Boi Hanh Huynh^**, Stephen Naylor^§, and Frank Rusnak^§§§

From the  Section of Hematology Research, ^§ Department of Biochemistry and Molecular Biology, and  Biomedical Mass Spectrometry and Functional Proteomics Facility, Mayo Clinic, Rochester, Minnesota 55905, the ^¶ Departmento de Química and Centro de Química Fina e Biotecnologia, Faculdade de Ciéncias e Tecnologia, Universidade Nova de Lisboa, 2825 Monte de Caparica, Portugal, the  Department of Medicine and the Center for Microbial Pathogenesis, University of Connecticut Health Center, MC3710, Farmington, Connecticut 06030, and the ^** Department of Physics, Emory University, Atlanta, Georgia 30322

Received for publication, April 18, 2000, and in revised form, June 20, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Treponema pallidum, the causative agent of venereal syphilis, is a microaerophilic obligate pathogen of humans. As it disseminates hematogenously and invades a wide range of tissues, T. pallidum presumably must tolerate substantial oxidative stress. Analysis of the T. pallidum genome indicates that the syphilis spirochete lacks most of the iron-binding proteins present in many other bacterial pathogens, including the oxidative defense enzymes superoxide dismutase, catalase, and peroxidase, but does possess an orthologue (TP0823) for neelaredoxin, an enzyme of hyperthermophilic and sulfate-reducing anaerobes shown to possess superoxide reductase activity. To analyze the potential role of neelaredoxin in treponemal oxidative defense, we examined the biochemical, spectroscopic, and antioxidant properties of recombinant T. pallidum neelaredoxin. Neelaredoxin was shown to be expressed in T. pallidum by reverse transcriptase-polymerase chain reaction and Western blot analysis. Recombinant neelaredoxin is a 26-kDa ₂ homodimer containing, on average, 0.7 iron atoms/subunit. Mössbauer and EPR analysis of the purified protein indicates that the iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states. The fully oxidized form, obtained by the addition of K₃(Fe(CN)₆), exhibits an optical spectrum with absorbances at 280, 320, and 656 nm; the last feature is responsible for the protein's blue color, which disappears upon ascorbate reduction. The fully oxidized protein has a A₂₈₀/A₆₅₆ ratio of 10.3. Enzymatic studies revealed that T. pallidum neelaredoxin is able to catalyze a redox equilibrium between superoxide and hydrogen peroxide, a result consistent with it being a superoxide reductase. This finding, the first description of a T. pallidum iron-binding protein, indicates that the syphilis spirochete copes with oxidative stress via a primitive mechanism, which, thus far, has not been described in pathogenic bacteria.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Syphilis is a sexually transmitted disease caused by the noncultivable human pathogen Treponema pallidum. Despite the availability of effective antimicrobial therapy since the late 1940s, syphilis remains a significant threat to global health by virtue of the morbidity and mortality it inflicts as well as its ability to facilitate the transmission of human immunodeficiency virus-1 (1-3). The disease begins as an ulcer (chancre) at the site of inoculation of T. pallidum, usually in the genital area, and, when untreated, may progress through secondary (disseminated), late, and tertiary (recrudescent) stages (4). The protean nature of syphilitic infection underscores T. pallidum's remarkable ability to thrive in diverse nutritional milieus within the human host while, at the same time, evading the robust cellular and humoral immune responses it provokes. Paradoxically, T. pallidum has long been known to lack numerous biosynthetic and catabolic capabilities (5), and the sequence of its 1.138 megabase chromosome has further underscored the bacterium's metabolic limitations (6).

T. pallidum is currently classified as a microaerophile because of its limited tolerance for the toxic effects of oxygen during in vitro cultivation (7, 8). This presents something of a paradox, however, given that the bacterium readily disseminates hematogenously and thrives in well oxygenated tissues. An examination of the mechanism(s) by which T. pallidum copes with oxidative stress would appear to be a logical starting point for resolution of the apparent contradiction between the syphilis spirochete's susceptibility to oxygen toxicity and its frequent exposure to environments with high redox potential.

It has recently been hypothesized that T. pallidum has a limited requirement for iron (9-11), and an analysis of the T. pallidum genome indicates that the syphilis spirochete lacks most of the iron-binding proteins present in other bacterial pathogens, including the oxidative defense enzymes superoxide dismutase, catalase, and peroxidase. Nevertheless, it does possess an orthologue (TP0823) for neelaredoxin, a superoxide reductase of hyperthermophilic and sulfate-reducing anaerobes. Neelaredoxin was first purified from Desulfovibrio gigas and shown to bind an iron atom in a unique coordination environment (12). Neelaredoxin is homologous to the C-terminal domain of desulfoferrodoxin, a superoxide reductase that binds two distinct iron atoms (centers I and II) (12-14) (Fig. 1).

The N-terminal domain of desulfoferrodoxin binds an iron atom in a distorted tetrahedral environment coordinated by four cysteinyl sulfur atoms (center I), while the C-terminal domain accommodates an iron atom in a unique (N)₄S coordination environment of four histidines and one cysteine residue (center II) (13, 15). Center II of desulfoferrodoxin has a relatively high redox potential of +240 mV, exhibits a blue color in the oxidized state (_max = 656 nm), and becomes colorless upon reduction (13, 16). The recent x-ray structure of neelaredoxin from Pyrococcus furiosus reveals the same (N)₄S coordination (17), and indeed neelaredoxin from D. gigas has spectroscopic features comparable with center II of desulfoferrodoxin (12, 18). The four histidine and cysteine residues that coordinate the iron atom of center II is uniformly conserved in all known enzymes of this family, including the T. pallidum homologue (Fig. 1).

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Multiple sequence alignment of the neelaredoxin and desulfoferrodoxin family of enzymes. The multiple sequence alignment was done using the Wisconsin Package Version 10.0 (Genetics Computer Group, Madison, WI). DX, D. gigas desulforedoxin (45); Ds, D. desulfuricans desulfoferrodoxin (46); Dv, Desulfovibrio vulgaris desulfoferrodoxin (47); Db, D. baarsii desulfoferrodoxin (35); Af1, Archaeoglobus fulgidus desulfoferrodoxin (48); Mt, Methanobacterium thermoautotrophicum desulfoferrodoxin (49); Pa, Pyrococcus abyssi neelaredoxin; Pf, P. furiosus superoxide reductase (37); Af2, A. fulgidus neelaredoxin (48); Tm, Thermotoga maritima neelaredoxin (50); Mj, Methanococcus jannaschii neelaredoxin (51); Dg, D. gigas neelaredoxin (18); Ph, Pyrococcus horikoshii neelaredoxin; Tp, T. pallidum neelaredoxin (6). The dots indicate cysteine residues that provide the four sulfur ligands to the iron ion of center I in desulfoferrodoxins and D. gigas desulforedoxin. Residues highlighted in boldface type represent the residues that coordinate the iron ion of center II in D. desulfuricans ATCC 27774 desulfoferrodoxin (15) and P. furiosus superoxide reductase (17); a glutamate residue (E14) in P. furiosus superoxide reductase is also a ligand in two out of four subunits in the unit cell (indicated by ). The consensus sequence denotes residues conserved in at least 10 family members.

Here we demonstrate that the treponemal neelaredoxin orthologue is an iron-binding protein with superoxide reductase activity. This finding, the first description of an iron-binding protein in T. pallidum, indicates that the syphilis spirochete copes with oxidative stress via a primitive mechanism, which, thus far, has not been identified in pathogenic bacteria.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

Spectrophotometric grade guanidine·HCl was purchased from Pierce. Powdered LB¹ medium was obtained from Fisher. Isopropyl--thio-D-galactoside was purchased from Bachem (Torrance, CA). Ampicillin, duroquinone, horse heart cytochrome c, 2-hydroxy-1,4-napthoquinone, phenazine methosulfate, potassium ferricyanide, potassium superoxide, xanthine, and xanthine oxidase were purchased from Sigma. Phenazine, 5-hydroxy-1,4-napthoquinone, 1,2-napthoquinone, 1,4-napthoquinone, and sodium ascorbate were purchased from Aldrich. Sodium dithionite (Na₂S₂O₄) was purchased from VWR.

Methods

Iron, zinc, magnesium, copper, and calcium concentrations were determined by the Mayo Metals Laboratory using inductively coupled plasma emission spectrometry (19, 20).

Protein concentrations for metal stoichiometry determinations were determined by amino acid analysis. It was found that protein concentration determined by use of the Bradford assay using bovine serum albumin as a standard yielded a concentration within ~±15% of the concentration determined by amino acid analysis. The molar extinction coefficient of the protein at 280 nm was determined by dividing the absorbance at 280 nm of the oxidized protein by the protein concentration determined by amino acid analysis. The value obtained, 17,000 ± 6000, is about 25% higher than the value calculated for the apoprotein (13,940 M¹·cm¹) based on the presence of two tryptophan and two tyrosine residues in the primary sequence according to the method of Gill and von Hippel (21), indicating contributions to the UV spectrum from the metal center.

ESI-MS measurements of T. pallidum neelaredoxin were done on a Finnigan (Bremen, Germany) MAT 900 mass spectrometer of EB geometry. ESI measurement of neelaredoxin was done in the positive mode. The ESI source used was a modified Finnigan MAT source modified for optimal operation at microflow delivery rates as described previously (22). Sulfur hexafluoride was used to prevent formation of ESI source corona discharge. Prepared protein solutions were introduced into the source at a flow rate of 0.3 µl/min through an emitter of 20-µm inner diameter fused silica. The electrospray source voltage used was set at 2.8 (3.6) kV and the heated capillary temperature at 180 °C. The instrument was scanned from mass to charge (m/z) 1000 to 5000 at a rate of 10 s/decade using an instrument resolution of 1000. A position- and time-resolved ion counter array detector was used for ion detection. Multiple scans were collected and summed, and the multiply charged spectra were transformed into the molecular mass (M_r) scale using instrument software. Protein solutions were buffer-exchanged into 20 mM ammonium acetate and then diluted 1:1 with a solution of H₂O/CH₃CN/isopropyl alcohol/trifluoroacetic acid (50:40:10:0.1, v/v/v/v) to enhance denaturation of the dimer to obtain an accurate monomeric molecular mass. In order to analyze the native multimeric composition of the protein, the protein solution was sprayed in H₂O/methanol/acetic acid (50:50:1, v/v/v).

Propagation of T. pallidum-- T. pallidum (Nichols) was propagated by intratesticular inoculation of adult New Zealand White rabbits as described previously (23). Spirochetes were separated from testicular tissue by low speed centrifugation (350 × g for 10 min). Genomic DNA was isolated using the Stratagene (La Jolla, CA) DNA extraction kit.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)-- RNA was isolated from freshly harvested T. pallidum using the Ultraspec RNA isolation reagent (Biotecx, Houston, TX) following the manufacturer's recommendations. Isolated RNA was treated with RQ1 RNase-free DNase (Promega, Madison, WI), phenol/chloroform-extracted, ethanol-precipitated, and resuspended in diethyl pyrocarbonate-treated water along with RNase inhibitor (Promega). RT-PCR analysis was carried out using the Titan One-Tube RT-PCR system (Roche Molecular Biochemicals) and primers specific for neelaredoxin (5'-AGGCAGTAGTGTCGCGTGCGG-3' and 5'-AAAGGTCACCTCAGGCGCTCC-3') and flaA (5'-TGAATTATCCTCATGGTTTGTACGTG-3' and 5'-TCAGCACCGCCTTATCATAGATAATC -3'). For each primer set, four reactions were performed: (i) RT-PCR with 50 ng of RNA template, (ii) PCR with 50 ng of RNA template, (iii) RT-PCR with water only, and (iv) PCR with 3 ng of DNA. In reactions without reverse transcription, the RT-DNA polymerase mixture was replaced by the Expand High Fidelity DNA polymerase (Roche Molecular Biochemicals). Following the RT reaction (45 °C for 30 min), PCR was performed in a Perkin-Elmer 9700 thermocycler (PE Applied Biosystems, Foster City, CA) using the following parameters: 98 °C for 2 min followed by 40 cycles of 98 °C for 10 s, 60 °C for 10 s, and 68 °C for 30 s followed by a single terminal extension for 2 min at 68 °C. One-fifth (5 µl) of each reaction was electrophoresed through a 2% agarose gel containing ethidium bromide prior to photography.

Generation of Anti-Neelaredoxin Antiserum-- Two 4-week-old male Harlan Sprague-Dawley rats were primed intraperitoneally with 20 µg of purified recombinant protein emulsified in a 1:1 mixture with complete Freund's adjuvant and phosphate-buffered saline (PBS; pH 7.4). Booster immunizations consisting of 20 µg of protein in a 1:1 mixture with incomplete Freund's adjuvant and PBS were administered by the same route on weeks 4 and 6.

Immunoblot Analysis-- Samples consisting of whole T. pallidum (5 × 10⁷) or purified recombinant neelaredoxin were diluted 1:2 in Tricine sample buffer consisting of 200 mM Tris-HCl (pH 6.8), 40% glycerol (v/v), 2% SDS (v/v), 2% 2-mercaptoethanol (v/v), and 0.04% Coomassie G-250 (w/v) and boiled for 5 min prior to electrophoresis through 16.5% polyacrylamide Tris-Tricine gels (Bio-Rad). Resolved proteins were electrophoretically transferred to 0.2-µm pore size nitrocellulose (Schleicher and Schuell) and blocked in PBS containing 5% skim milk, 5% fetal calf serum, and 0.05% Tween 20 for 1 h prior to immunoblotting. Immunoblots were incubated with 1:1000 dilutions of antisera in blocking buffer for 1 h, after which the membranes were extensively washed with PBS and then incubated with a 1:75,000 dilution of goat anti-rat Ig(H + L)-horseradish peroxidase conjugate (Southern Biotechnology Associates, Birmingham, AL) in blocking buffer for 1 h. Immunoblots were developed using the SuperSignal Femto chemiluminescence substrate (Peirce) followed by exposure to chemiluminescence film (Amersham Pharmacia Biotech).

Cloning of the Neelaredoxin Gene from T. pallidum-- The gene encoding neelaredoxin was amplified by PCR using T. pallidum genomic DNA isolated as described above and a pair of oligonucleotides, 5'-CCATGGCATATGGGACGGGAGTTGTCG-3' and 5'-TTAAGCTTGGATCCCTACTTACCTGACCACAC-3', homologous to the 5'- and 3'-ends of the gene (6), respectively. Following PCR amplification, the PCR product was purified by electrophoresis in an 8% polyacrylamide gel in 89 mM Tris-HCl, 89 mM boric acid, 2.5 mM EDTA buffer. The 410-base pair fragment was excised from the gel, and the DNA fragment was electroeluted using an IBI model UEA Unidirectional Electroeluter according to the manufacturer's instructions. After ethanol precipitation, the purified fragment was digested with NdeI and BamHI and subsequently ligated into the plasmid pT7-7 (24) digested with the same restriction enzymes. The resulting ligation mix was transformed into the Escherichia coli strain DH5- and plated on LB agar containing 0.1 mg/ml ampicillin. From the plate, individual colonies were transferred to 5 ml of LB medium containing 0.1 mg/ml ampicillin and grown overnight, after which plasmid DNA was isolated using the Promega Wizard^® Plus Miniprep DNA Purification System. Positive clones were identified by restriction digest analysis of plasmid DNA with NdeI and BamHI and subsequently confirmed by DNA sequencing of the entire gene. The plasmid pTpNeelT77-8/19 was subsequently used for protein overexpression in E. coli.

Overexpression and Purification of Recombinant T. pallidum Neelaredoxin-- Competent E. coli BL21(DE3) cells (Novagen, Madison, WI) were transformed with pTpNeelT77-8/19 and plated onto LB/agar plates containing 0.1 mg/ml ampicillin, after which a colony was used to inoculate 10 ml of LB/amp (0.1 mg/ml) medium. This culture was grown at 37 °C overnight, and the next morning it was used to inoculate 1.5 liters of LB medium containing 0.1 mg/ml ampicillin in a 6-liter Erlenmeyer flask. This culture was grown at 37 °C with shaking until the absorbance of the culture at 595 nm reached 0.8. At this point, isopropyl--thio-D-galactoside was added to a final concentration of 1 mM. After an additional 6 h, the cells were isolated by centrifugation for 30 min at 2500 × g at 4 °C. The cell pellet was resuspended in 50 mM Tris-HCl, pH 7.8, and lysed by three passages through a French pressure cell operating at 15,000 p.s.i. at the orifice. The resulting cell lysate was centrifuged for 60 min at 39,100 × g, and the supernatant representing the crude cell extract was applied to a column (2.6 × 17.5 cm) containing DEAE-Sepharose CL6B anion exchange resin equilibrated with 20 mM Tris-HCl, pH 7.8. After loading the crude extract, the column was washed with the same buffer to remove contaminating protein. At this point, the presence of the recombinant protein became evident by the appearance of a blue band adsorbed at the top of the column. The protein was eluted from the column by use of a 0-1.0 M NaCl linear gradient in the same buffer, with the majority of protein eluting as a blue fraction at a NaCl concentration of ~0.1 M. Fractions were analyzed by SDS-PAGE, and the best fractions in terms of purity were pooled, concentrated to 10 ml, and applied to a column containing Sephadex G75 gel filtration resin equilibrated with 50 mM Tris-HCl, 0.3 M NaCl, pH 7.8. The protein was eluted from this column with the same buffer and appeared homogeneously pure as judged by SDS-PAGE.

For preparation of ⁵⁷Fe-enriched protein, the cells were grown in M9 medium containing 0.4% glucose, 0.1 mg/ml ampicillin, 5 µM ⁵⁷FeCl₃, and a 20-µl aliquot per liter of trace metals solution. The trace metals solution was prepared by dissolving 184 mg of CaCl₂·2H₂O, 64 mg of H₃BO₃, 40 mg of MnCl₂·4H₂O, 18 mg of CoCl₂·6H₂O, 4 mg of CuCl₂, 340 mg of ZnCl₂, and 605 mg of Na₂MoO₄·2H₂O, in 8 ml of concentrated HCl, after which the volume was brought up to 100 ml with distilled water. A 10-ml volume of defined medium was inoculated with a colony grown on LB/ampicillin plates and incubated overnight at 37 °C. The next morning it was used to inoculate a 6-liter Erlenmeyer flask containing the same defined medium. Expression and purification of the protein proceeded as described above.

Native Molecular Weight Determination-- The molecular mass of recombinant T. pallidum neelaredoxin was determined by size exclusion chromatography using a 300 × 7.8-mm Bio-Sil SEC-125 HPLC Gel Filtration Column from Bio-Rad. The mobile phase was 25 mM Tris-HCl, pH 7.5, 0.15 M NaCl. The column was calibrated using gel filtration chromatography standards from Bio-Rad, consisting of thyroglobulin (670 kDa), bovine -globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa), and vitamin B₁₂ (1.35 kDa). The void volume was defined as the volume at which thyroglobulin eluted.

UV-visible Spectrophotometry of Oxidized and Reduced Recombinant T. pallidum Neelaredoxin-- A sample of the oxidized protein for obtaining the UV-visible spectrum was obtained by treating a sample of the purified protein with a slight excess of 15 mM K₃(Fe(CN)₆) in 50 mM Tris-HCl, pH 7.8, incubating for 10 min at room temperature, followed by removal of the excess K₃(Fe(CN)₆) by passage of the sample over a NAP-25 column equilibrated in 50 mM Tris-HCl, pH 7.8, buffer. A slight excess of K₃(Fe(CN)₆) is defined as the amount of K₃(Fe(CN)₆) added that no longer produces any further increase in the absorbance at 656 nm. Blue-colored fractions containing the protein were pooled, and the optical spectrum was recorded immediately using a Cary 1 UV-visible spectrophotometer and 1-cm cuvettes.

In order to estimate the ₆₅₆ of the oxidized protein, a sample of neelaredoxin was analyzed for the iron content by inductively coupled plasma emission spectrometry as described above, after which it was titrated with a slight excess of K₃(Fe(CN)₆). The optical spectrum was recorded immediately, and ₆₅₆ was determined by dividing the A₆₅₆ by the iron concentration.

Optical spectra of the reduced protein were obtain by treating a sample of the protein with a freshly prepared solution of 15 mM sodium ascorbate in 50 mM Tris-HCl, pH 7.8.

Enzyme Assays-- The ability of recombinant T. pallidum neelaredoxin to catalyze the dismutation of superoxide was followed by monitoring the inhibition of cytochrome c reduction by superoxide generated by xanthine/xanthine oxidase as described (25). The SOD assay was performed at 25 °C in 1 ml of 50 mM potassium phosphate, pH 7.8, 100 µM EDTA, 10 µM horse heart cytochrome c, 7.5 mM xanthine, varying amounts of neelaredoxin, and an amount of xanthine oxidase that gives an initial rate of A₅₅₀ of 0.025 absorbance units/min. One unit of SOD activity is defined as the amount of protein that inhibits the rate of cytochrome c reduction by 50%.

The reaction of neelaredoxin with superoxide was followed using two different methods. In the first experiment, the xanthine/xanthine oxidase system was used to generate superoxide in situ. A solution of neelaredoxin (~100 µM) was completely reduced by the addition of 1 eq of ascorbic acid from a 15 mM stock solution in 50 mM Tris-HCl, pH 7.8, followed by buffer exchange to remove the excess ascorbate by passage over a NAP 25 (Amersham Pharmacia Biotech) gel filtration column equilibrated in 50 mM Tris-HCl, pH 7.8. An initial optical spectrum was obtained to verify the reduced state of the protein and to estimate the protein concentration using ₂₈₀, which indicated a protein concentration of 70 µM. Xanthine was added to a final concentration of 7.5 mM, the reaction to generate superoxide was initiated by the addition of 0.0028 units of xanthine oxidase, and optical spectra were subsequently recorded at various times.

In the second experiment, optical spectra of 67 µM ascorbate-reduced neelaredoxin (obtained as described above) in 50 mM Tris-HCl, pH 7.8, were obtained following successive additions of superoxide from a KO₂ stock solution, prepared by dissolving KO₂ to ~1.2 mM in Me₂SO that had been dried over 3-Å molecular sieves. The exact concentration of superoxide was determined using ₂₆₀ = 2086 M¹ cm¹. An optical spectrum was obtained 2-3 min after each addition of KO₂.

The second order rate constant for the reaction of reduced neelaredoxin with superoxide was determined in the presence of various concentrations of SOD as described by Lombard et al. (14). Superoxide was generated by the xanthine/xanthine oxidase system, and neelaredoxin oxidation was followed at 25 °C spectrophotometrically at 656 nm. A solution of neelaredoxin (~100 µM) was completely reduced by the addition of 1 eq of ascorbic acid from a 15 mM stock solution in 50 mM Tris-HCl, pH 7.8, followed by buffer exchange over a NAP 25 column equilibrated in 50 mM Tris-HCl, pH 7.8. An initial optical spectrum was obtained to verify the reduced state of the protein and to estimate the protein concentration using ₂₈₀, which indicated a protein concentration of 50 µM. Oxidation was initiated upon the addition of 7.5 mM xanthine and 0.028 units of xanthine oxidase. CuZn-SOD concentrations were varied from 0 to 8 µM.

Redox Titration of Neelaredoxin-- Redox titrations of T. pallidum neelaredoxin were carried out by diluting a solution of the protein to 270 µM into 50 mM Tris-HCl, pH 7.8, containing 5 µM of the following mediators: 1,2-napthoquinone, phenazine, 1,4-napthoquinone, 5-hydroxy-1,4-napthoquinone, duroquinone, phenazine methosulfate, and 2-hydroxy-1,4-napthoquinone. The solution was transferred to a 3-ml optical grade glass cuvette fitted with a rubber septum. The potential was measured between a working gold electrode and a reference Ag/AgCl electrode (Microelectrodes Inc., Londonderry, NH), which were inserted through the septum. The contents were made anaerobic by continuous purging with oxygen-free argon. The protein was first reduced by the addition of 200 µl of an anaerobic solution of 15 mM sodium dithionite. After the potential had stabilized, the solution was titrated with aliquots of K₃(Fe(CN)₆) to give potential changes on the order of 10-15 mV/aliquot. After each addition, the potential was measured using a voltmeter, and the optical spectrum was recorded. The data were fit to the Nernst equation using a least squares approach. Reduction potentials reported with reference to the normal hydrogen electrode were obtained by adding 199 mV to the measured potential (26).

Mössbauer and Electron Paramagnetic Resonance Characterization of Recombinant T. pallidum Neelaredoxin-- Samples of purified ⁵⁷Fe-labeled neelaredoxin were concentrated to approximately 1 mM using a Centricon 3 microconcentrator and transferred to delrin Mössbauer cuvettes or quartz EPR cuvettes. Mössbauer spectra were recorded in a weak field spectrometer equipped with a Janis 8DT variable temperature cryostat operating in a constant acceleration mode in a transmission geometry. The zero velocity of the spectra refers to the centroid of a room temperature spectrum of a metallic iron foil. Low temperature electron paramagnetic resonance (EPR) spectroscopy was performed using a Bruker ESP300E spectrometer operating at X-band (9-GHz) frequencies and equipped with an Oxford Instruments cryostat for temperature regulation from 3.0 to 300 K.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of Neelaredoxin by T. pallidum-- To analyze the expression of neelaredoxin in T. pallidum, RT-PCR and immunoblot analyses were performed. As shown in Fig. 2A, the neelaredoxin transcript was detected in freshly harvested spirochetes although at lower levels than the abundantly expressed flagelin subunit. To confirm that these RT-PCR data were indicative of expression at the protein level, T. pallidum lysates were probed with anti-neelaredoxin serum in Western analysis. A protein migrating with the same apparent molecular mass as recombinant neelaredoxin was readily detected in freshly harvested treponemes as shown in Fig. 2B. Using densitometry (11 ng of neelaredoxin detected in 5 × 10⁷ cells) and the molecular weight of neelaredoxin as determined by ESI-MS, we estimate that each T. pallidum cell contains 9500 neelaredoxin monomers.

View larger version (42K): [in this window] [in a new window]   Fig. 2.   Expression of neelaredoxin in T. pallidum (Tp). A, detection of transcripts for neelaredoxin and flaA by RT-PCR (+RT). Controls consisted of PCR performed on 50 ng of RNA template (RT), RT-PCR of water (H2O), and PCR of 3 ng of DNA. B, detection of protein expression by immunoblot analysis of T. pallidum (5 × 107) and purified recombinant protein using rat antiserum generated against purified recombinant protein. Numbers above the neelaredoxin lanes indicate the amount of protein in ng. Numbers to the left of each panel indicate standards in base pairs (A) and kilodaltons (B).

Cloning, Overexpression, and Purification of Recombinant T. pallidum Neelaredoxin-- The neelaredoxin gene was cloned from genomic T. pallidum DNA using the polymerase chain reaction, overexpressed in E. coli, and purified to homogeneity using anion exchange and gel filtration chromatographies. The presence of recombinant iron-containing protein was immediately evident as a blue-colored band adsorbed at the top of the resin during the first anion exchange chromatography step. At various stages during purification, the protein appeared to undergo reversible redox reactions, and the color of the protein varied between blue (oxidized) and colorless (reduced) redox states (see Fig. 3). When colorless, the presence of the protein in various column fractions could be followed by SDS-PAGE. The protein eluting from the first anion exchange column was at least 80% pure as judged by SDS-PAGE and could be purified to homogeneity (95%) by passage over a gel filtration column.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   UV-visible spectra of T. pallidum neelaredoxin. A, the optical spectrum of a 41 µM solution of T. pallidum neelaredoxin in 1.0 ml of 25 mM Tris-HCl, pH 7.8, is shown following oxidation with K3(Fe(CN)6) as described under "Methods." The A280/A656 ratio is 10.3. B, the UV-visible spectrum of the same sample following the addition of 5.3 µl of a 15 mM solution of sodium ascorbate to reduce the sample.

Characterization of Recombinant T. pallidum Neelaredoxin-- The molecular mass of the protein as determined using Tris-glycine buffered 15% SDS-PAGE was ~14 kDa. The molecular mass of the protein was also determined using ESI-MS. Using protein denatured in acetonitrile/isopropyl alcohol/trifluoroacetic acid, two molecular ions were evident with molecular masses of 13,800 and 13,670. These correspond to the full-length polypeptide and the polypeptide missing the N-terminal methionine residue (calculated values are 13,802 and 13,671, respectively). ESI-MS analysis of the undenatured protein solution spraying in H₂O/methanol/acetic acid revealed a complex ion signal corresponding to the homodimers consisting of monomer units M_r = 13,800 and M_r = 13,670. There was also evidence for the presence of the mixed dimer consisting of both M_r = 13,800 and M_r = 13,670 (mass accuracy ±0.01%). Using gel filtration chromatography, it was also noted that the native protein eluted with a molecular mass of 26 ± 2 kDa, confirming the presence of the ₂ quaternary structure.

Samples of recombinant T. pallidum neelaredoxin from four separate preparations were analyzed for iron, zinc, copper, magnesium, and calcium using inductively coupled plasma emission spectrometry, and the protein concentration was determined by amino acid analysis. These results and the corresponding metal stoichiometries are listed in Table I.

View this table: [in this window] [in a new window]   Table I Metal ion stoichiometries of recombinant T. pallidum neelaredoxin For copper, calcium, and magnesium, 0.1 eq is shown.

Using protein concentration determined by amino acid analysis, an average stoichiometry of 0.67 iron atoms/subunit was obtained. Zinc was also present and amounted to approximately 0.25 atoms per subunit. Copper, calcium, and magnesium were present only in trace quantities (0.1 eq). Although the average iron content of four independent preparations is slightly less than 1 eq expected for a fully occupied center II-like iron site, the iron stoichiometry varied between preparations. Furthermore, spectroscopic evidence presented below suggests that some of the iron may be bound in sites other than the (N)₄S-center II site. Thus, there is a possibility that apoprotein may be present at least in some preparations. Alternatively, some of the center II-like sites could be occupied with zinc, a situation that often occurs during overexpression of iron-containing proteins in E. coli (27-29). However, at least with one preparation (preparation IV, Table I), a significant quantity of zinc was present (0.28 eq) despite the presence of stoichiometric iron, suggesting that the small amount of zinc detected in each preparation is probably adventitious.

UV-visible Spectra of T. pallidum Neelaredoxin-- The optical spectra of recombinant neelaredoxin in the oxidized and reduced states are shown in Fig. 3.

As isolated, T. pallidum neelaredoxin exhibits a pale blue color, because the iron atom exists as a mixture of two different oxidation states, ferric and ferrous (see below). The blue color results from the oxidized form, which exhibits a broad peak in the visible region of the optical spectrum from 550 to 800 nm with _max = 656 nm (Fig. 3A). Complete oxidation of the as-isolated sample was afforded by treatment with K₃(Fe(CN)₆), causing the color to intensify and the intensity of the 656-nm feature to increase. The extinction coefficient of this feature based on the iron content is 2600 ± 200 M¹·cm¹. This value may represent a lower limit if some of the iron is bound in sites other than the center II-like site (discussed below). The fully reduced (Fe²⁺) state could be produced by treatment with ascorbate and resulted in the disappearance of color and loss of absorbance at 656 nm, with only a weak feature remaining at 319 nm (Fig. 3B).

Mössbauer and EPR Spectroscopy of T. pallidum Neelaredoxin-- As with the optical studies described above, the Mössbauer data also indicate that the iron center in the as-isolated neelaredoxin exists in equilibrium between its oxidized ferric and reduced ferrous states. Fig. 4A shows the Mössbauer spectrum of an ⁵⁷Fe-enriched sample of as-isolated neelaredoxin recorded at 4.2 K in a magnetic field of 50 milliTeslas applied parallel to the -rays.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Mössbauer spectrum of the as-isolated (A) and ascorbate-reduced (B) 57Fe-enriched recombinant neelaredoxin. The spectra are recorded at 4.2 K in a parallel field of 50 milliTeslas. The bracket indicates the positions of the quadrupole doublet, and the arrows mark the resolved absorption peaks of the magnetic component. The solid line is a theoretical simulation of oxidized center II from D. desulfuricans desulfoferrodoxin, using the parameters published in Ref. 16.

Three components are observed: (i) a sharp quadrupole doublet, marked by a bracket, (ii) a magnetic hyperfine split spectrum showing resolved absorption peaks at 7.6, 4.4, 5, and 7 mm/s, marked by arrows, and (iii) a broad and undefined component having absorptions in the central region of the spectrum. The quadrupole doublet accounts for 42 ± 5% of the total iron absorption. Its parameters, E_Q = 2.80 ± 0.05 mm/s and  = 1.02 ± 0.03 mm/s, are typical for high spin Fe(II) with octahedral coordination and are very similar to that of the reduced center II in desulfoferrodoxin from Desulfovibrio desulfuricans (16). The isomer shift of 1.02 mm/s is significantly lower than the ~1.3 mm/s usually observed for octahedral N/O coordinate Fe(II) and suggests the ligation of a more covalent cysteinyl sulfur (30) as detected in center II of desulfoferrodoxin (15).

The magnetic component accounts for 30 ± 5% of the total iron absorption and is very similar to that of the oxidized center II in desulfoferrodoxin. To illustrate this point, a simulated theoretical spectrum for the oxidized center II in desulfoferrodoxin (16) is plotted in Fig. 4 as a solid line above spectrum A for direct comparison. The similarities between the magnetic component of spectrum A and the simulated spectrum of center II are obvious and are further demonstrated by low temperature EPR spectroscopy. The EPR spectrum of K₃(Fe(CN)₆) oxidized neelaredoxin (Fig. 5) is similar to the 4.4 K EPR spectrum of oxidized center II of desulfoferrodoxin (16). Both proteins exhibit EPR spectra with g values of 9.5-9.8 as well as a pronounced feature at g = 4.3. These features are typical of a rhombic high spin ferric species.

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Low temperature electron paramagnetic resonance spectroscopy of T. pallidum neelaredoxin. A sample of the as-isolated enzyme was oxidized with K3(Fe(CN)6) and excess oxidant removed by passage over a NAP-25 column. EPR conditions were as follows: temperature, 3.6 K; microwave frequency, 9.4555 GHz; microwave power, 0.1 milliwatts; modulation field, 10 G and 100 kHz; gain, 1.25 × 105; time constant, 164 ms.

Treatment of the EPR sample with dithionite resulted in disappearance of the EPR signal, consistent with reduction to the ferrous oxidation state. Reduction of the Mössbauer sample also resulted in the disappearance of the magnetic component and its conversion to the quadrupole doublet described above (Fig. 4B). Thus, the Mössbauer and EPR results confirm unambiguously that neelaredoxin expressed in E. coli contains an iron center in a ligand environment similar to center II of D. desulfuricans desulfoferrodoxin.

The central broad component observed in the Mössbauer spectrum of the as-isolated neelaredoxin (Fig. 4A) represents most probably adventitiously bound ferric ions, the origin of which is currently not clear. This component is not reduced by ascorbate, as indicated by the presence of this same broad component in the Mössbauer spectrum of the reduced sample (Fig. 4B) and therefore does not contribute to the absorbance at 656 nm of the oxidized sample (Fig. 3). Thus, the extinction coefficient at 656 nm calculated using the iron stoichiometry may be underestimated. The Mössbauer spectrum of this adventitious iron is broad and featureless, indicating a distribution of environments that should result in a difficult-to-detect and broad EPR signal that nevertheless may also contribute to the spectrum of Fig. 5.

Redox Titrations of T. pallidum Neelaredoxin-- A redox titration of neelaredoxin was carried out in order to determine the midpoint potential of the iron center. A sample of the protein was made anaerobic by flushing with oxygen-free argon, reduced by the addition of sodium dithionite, and the potential was measured following reoxidation with aliquots of K₃(Fe(CN)₆). The extent of oxidation was determined by following the appearance of the _max = 656 nm feature of the oxidized center. Fig. 6 shows the results that indicate that this feature disappears when the potential (E) falls below +100 mV (versus normal hydrogen electrode) and reaches maximum intensity for E  +300 mV.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Redox titration of T. pallidum neelaredoxin. The relative absorbance at 656 nm is plotted as a function of redox potential as described under "Methods." The circles represent data obtained during an oxidative titration with K3(Fe(CN)6), while triangles represent data from a reductive titration with Na2S2O4. Several data points from both oxidative and reductive titrations were collected as low as 349 mV. The optical spectra at these low potentials are identical to spectra for E  +50 mV. Data points from these potentials are omitted for clarity.

The data resulting from an oxidative titration with K₃(Fe(CN)₆) can be fit to the Nernst equation for an n = 1 electron process to give E_o = 209 ± 4 mV (Fig. 6). The process is reversible; lowering the potential by back titration with dithionite eliminated the _max = 656 nm feature according to a Nernst-type process with an E_o = 192 ± 1 mV. A second titration carried out in this manner gave E_o values of +215 and +188 for oxidative and reductive titrations, respectively.

Superoxide Dismutase and Superoxide Reductase Activity of T. pallidum Neelaredoxin-- The ability of T. pallidum neelaredoxin to catalyze the dismutation of superoxide was determined by following its effect on the rate of cytochrome c reduction by superoxide. In the presence of neelaredoxin, the reduction of cytochrome c by superoxide occurred in two distinct kinetic phases (Fig. 7).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   The effect of T. pallidum neelaredoxin on cytochrome c reduction by superoxide. The absorbance at 550 nm due to cytochrome c reduction by superoxide generated using xanthine/xanthine oxidase is followed as a function of time. At t = 0, xanthine oxidase is added to initiate the reaction. Each curve is offset by a small amount on the abscissa to facilitate a comparison between curves. From top to bottom, the amount of neelaredoxin added in each experiment is 0, 15, 30, 61, 91, and 121 µg, respectively. Inset, a plot of the lag time corresponding to the initial phase of the reaction (see text) as a function of the amount of neelaredoxin added to the reaction.

Initially, a lag period occurs in which no change in A₅₅₀ occurred. Since the increase in A₅₅₀ is due to cytochrome c reduction, the lag period must reflect a reaction of superoxide with neelaredoxin. Indeed, the length of the lag was proportional to the amount of neelaredoxin added (Fig. 7, inset). A similar lag has been observed in the same assay in the presence of Desulfoarculus baarsii desulfoferrodoxin (14). The second kinetic phase was approximately linear with time, and samples that contained 15 µg or more of protein showed a slight inhibition of cytochrome c reduction as evidenced by a decreased slope of the A₅₅₀ versus time curve compared with the control reaction (Fig. 7). The observed change in the slope yielded an activity of 10 ± 5 units·mg¹ and suggests that T. pallidum neelaredoxin is not a SOD.

Weak SOD activity was also observed using either fully oxidized or fully reduced neelaredoxin. However, the initial lag phase was only observed with the reduced enzyme, suggesting that superoxide reacted only with reduced neelaredoxin. To further explore this, we followed the reaction between reduced neelaredoxin and superoxide by UV-visible spectrophotometry. Fig. 8 shows that superoxide, produced either in situ using the xanthine/xanthine oxidase system (Fig. 8A) or by the addition of a potassium superoxide solution dissolved in Me₂SO (Fig. 8B), can reoxidize ferrous neelaredoxin as evidenced by the increase in absorbance at the 656-nm band.

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Oxidation of T. pallidum neelaredoxin by superoxide. A sample of reduced neelaredoxin was generated as described under "Methods," and superoxide was either generated in situ using xanthine/xanthine oxidase (A) or added directly from a stock solution of KO2 in dry Me2SO (B). Each spectrum was obtained by subtracting the spectrum of the fully reduced (t = 0) sample. The curves in A represent (from bottom to top) the optical spectrum of the enzyme from 500 to 800 nm at t = 2, 8, 10, and 20 min, respectively, following the addition of xanthine oxidase to initiate the reaction. In B, the curves represent (from bottom to top) the optical spectrum of neelaredoxin after 2-3 min following the addition of 1, 2, 3, and 4 eq of KO2, respectively.

Oxidation of ferrous neelaredoxin with superoxide produced by the xanthine/xanthine oxidase system showed a linear dependence, with the maximum change of absorbance occurring after ~20 min (data not shown). Using KO₂, complete oxidation required ~4 eq. Careful examination of Fig. 8 shows that each spectrum has slightly different _max values. At present, it is not known whether these are significant or whether they are an artifact of spectral subtractions and/or a kinetic process that varies during data acquisition.

The second order rate constant for the reaction of reduced neelaredoxin with superoxide was determined by measuring the rate of oxidation (at 656 nm) in the presence of various concentrations of SOD. SOD competes with neelaredoxin for superoxide, resulting in a slower rate of oxidation in the presence of increasing amounts of CuZn-SOD (data not shown). A plot of the reciprocal velocity as a function of CuZn-SOD concentration was linear and yielded a second order rate constant of 8.94 × 10⁷ M¹ s¹.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The inability to culture T. pallidum on artificial medium has impeded the study of trace metal utilization and homeostasis by this organism. With the determination of its complete genomic sequence, several proteins that utilize trace metals have been identified based on homology to proteins of known function, thus resurrecting an interest in metal ion metabolism in this organism (6). Of the proteins encoded within the genomic sequence of T. pallidum, those that can utilize iron as an inorganic cofactor include rubredoxin (TP0991), ribonucleoside diphosphate reductase (TP0053 and TP1008), pyruvate oxidoreductase (TP0939), quinoline 2-oxidoreductase (TP0080), methionine aminopeptidase (TP0842), bacterioferrin (TP1038), and neelaredoxin (TP0823). Since it is technically unfeasible to isolate any of these T. pallidum proteins in amounts necessary for detailed biochemical and spectroscopic studies, we have resorted to overexpressing the recombinant forms in the heterologous host E. coli in order to determine their biochemical and metal binding characteristics.

We have demonstrated in the present report that T. pallidum neelaredoxin is a competent iron-binding protein. Neelaredoxin was first identified as a novel iron-binding protein from the sulfate-reducing bacterium D. gigas (12). A comparison of the first 28 residues of D. gigas neelaredoxin with the C terminus of the protein desulfoferrodoxin indicated a number of identities (Fig. 1), and in fact, the two polypeptides share considerable spectroscopic properties due to a conserved iron-binding domain (12, 13, 16, 31). X-ray structures of desulfoferrodoxin from D. desulfuricans and superoxide reductase from P. furiosus indicate that both proteins bind an iron atom coordinated by four equitorial nitrogen atoms from histidine residues and a cysteine thiolate as an axial fifth ligand (15, 17). Interestingly, in the P. furiosus structure, two out of the four protein molecules in the asymmetric unit had a glutamate as a sixth ligand, indicating flexibility in the coordination environment of this center.

T. pallidum neelaredoxin exhibits spectroscopic and biochemical features demonstrated for neelaredoxin and center II of desulfoferrodoxin proteins. In the oxidized form, neelaredoxin exhibits a blue color associated with a charge transfer band in the visible region of the optical spectrum (_max = 656 nm). The Mössbauer and EPR spectra indicate a rhombic high spin ferric center quite similar to the iron atom in desulfoferrodoxin, indicating similar coordination environments. Reduction of the protein by one electron results in bleaching of the color, elimination of the EPR spectrum, and a Mössbauer spectrum very similar to that of the reduced center II in desulfoferrodoxin (16).

Whether these proteins utilize iron within the metabolic confines of T. pallidum will depend, of course, on the availability of trace metal ions and whether or not some other competent metal ion cofactor is able to substitute for iron as an active site cofactor. For example, although the E. coli methionine aminopeptidase can utilize Fe²⁺ as a cofactor, Co²⁺, Mn²⁺, and Zn²⁺ are all able to sustain activity with Co²⁺  Fe²⁺ > Mn²⁺ > Zn²⁺ (32). Furthermore, although rubredoxin isolated from several native sources has been characterized as an iron-binding protein (33), overexpression in E. coli results in the production of both iron- and zinc-bound forms (27, 28). Although the purification of zinc-containing rubredoxin has never been reported from native sources, the question has been raised of whether its presence may have escaped detection (34).

It has recently been reported that the Lyme disease spirochete, Borrelia burgdorferi has no iron requirement (11). The authors found that in vitro growth of B. burgdorferi is iron-independent and that these spirochetes contain less than 10 atoms of iron per cell. On the basis of these results and the fact that T. pallidum, like B. burgdorferi, has a limited genome, the authors speculated that T. pallidum may also have no iron requirement. While the paucity of predicted iron-binding proteins in the T. pallidum genome (6) supports the notion that the syphilis spirochete has a markedly reduced iron requirement, our findings indicate that, unlike B. burgdorferi, T. pallidum does not entirely forgo the need for iron. We feel that these findings highlight an important difference in the metal utilization by these pathogens that could have meaningful implications with respect to their distinct modes of host-pathogen interaction.

Until recently, the function(s) of neelaredoxin and desulfoferrodoxin were unknown. The first report that these proteins may have some role in superoxide metabolism was made by Touati and colleagues (35), who attempted to isolate an SOD from D. baarsii by complementing SOD-deficient E. coli. From a positive clone, they isolated a DNA fragment encoding a gene with homology to desulfoferrodoxin. Although having desulfoferrodoxin functioning as an SOD was one mechanism discussed by the authors that could account for the rescue of SOD-deficient strains, the authors concluded that desulfoferrodoxin was unlikely to be an SOD analog in these sulfate-reducing bacteria. Subsequently, Liochev and Fridovich (36) found little SOD activity in extracts of E. coli sodA sodB mutants overexpressing D. baarsii desulfoferrodoxin and concluded instead that it catalyzed the reduction of superoxide at the expense of cellular reductants such as NAD(P)H. Independently, Jenney et al. (37), in attempting to purify a putative SOD from the hyperthermophile P. furiosus, purified a protein 40% identical to the C terminus of desulfoferrodoxin and 50% identical to neelaredoxin. These authors showed that this protein exhibited a fundamental difference from classical SODs in that oxygen was not a product of the reaction. Rather, neelaredoxin was postulated to function as a superoxide reductase, accepting electrons from NAD(P)H, via rubredoxin and NAD(P)H-rubredoxin oxidoreductase, to reduce superoxide to hydrogen peroxide. A reaction between superoxide and D. baarsii desulfoferrodoxin has also recently been reported (14), consistent with a role for both neelaredoxin and desulfoferrodoxin to function as superoxide reductases.

The redox potential measured for T. pallidum neelaredoxin, ~+200 mV is slightly lower than the potential determined for center II of D. desulfuricans desulfoferrodoxin center II (+240 mV at pH 7.6) (16). Nevertheless, the redox potential is high enough to require a strong oxidant such as K₃(Fe(CN)₆) or superoxide (for the one-electron reduction to H₂O₂), which have E_o = +360 and +890 mV at pH 7.0 (38), respectively, to afford reoxidation of this center. Indeed, superoxide can reoxidize the reduced enzyme, presumably forming hydrogen peroxide according to the reaction recently proposed for the superoxide reductase (SOR) from P. furiosus,

Thus, T. pallidum neelaredoxin appears to exhibit superoxide reductase activity, in agreement with recent reports for P. furiosus neelaredoxin and D. baarsii desulfoferrodoxin (14, 37). As an organism lacking genes for classical SODs, the importance of this activity is emphasized by the fact that although T. pallidum is sensitive to atmospheric oxygen, it is a microaerophile requiring low levels of oxygen for survival and metabolic activity (7, 8, 39). Thus, cellular machinery for detoxifying byproducts of oxidative metabolism such as superoxide and hydrogen peroxide are likely to be necessary.

If neelaredoxin functions as a superoxide reductase, of interest is the fate of the product of this reaction, hydrogen peroxide, a strong oxidant with equally detrimental consequences. Indeed, T. pallidum is known to be sensitive to low doses of hydrogen peroxide (40, 41). In most organisms, catalases and peroxidases detoxify hydrogen peroxide. T. pallidum, however, lacks genes encoding these enzymes (10). A potential mechanism by which T. pallidum eliminates hydrogen peroxide may involve the treponemal homologues of the Salmonella typhimurium alkyl hydroperoxide reductase enzyme system, AhpC/AhpF. In addition to alkyl hydroperoxide reductase activity, the S. typhimurium AhpC-F enzyme complex has been shown to reduce hydrogen peroxide to water at the expense of NAD(P)H (42). While the treponemal AhpC homologue is denoted as such within the T. pallidum genomic sequence (6), a search of the Institute for Genomic Research data base with the S. typhimurium AhpF gave significant hits for genes designated thioredoxin reductase (32.1% identity over 305 amino acids) and NADH oxidase (29.2% identity over 144 amino acids). Notably, both thioredoxin reductase and NADH oxidase homologues have been shown to function as heterologous surrogates of AhpF in the reduction of AhpC and the resulting catalysis of hydrogen peroxide to water (43, 44). Moreover, both thioredoxin reductase and AhpF are members of the flavoprotein pyridine nucleotide-disulfide oxidoreductase enzyme family (42). While the hydrogen peroxide reductase activity of the treponemal AhpC/AhpF homologue(s) remains to be verified, our finding that the T. pallidum neelaredoxin is an iron-binding superoxide reductase leads us to speculate that these flavoproteins may fulfill a role as antioxidant defense enzymes.

The findings reported here indicate that T. pallidum copes with oxidative stress via a mechanism that appears to be associated exclusively with microorganisms with limited oxygen tolerance (37). While reliance upon this primitive pathway is consistent with the bacterium's microaerophilic nature in vitro (39), it appears to be at odds with the organism's ability to thrive in well oxygenated environments within its obligate human host. Assuming that this conception of the bacterium's exposure to environmental oxygen in vivo is correct, one must conclude that T. pallidum is protected from oxygen toxicity during infection by one or more mechanisms that are not accurately reproduced by current in vitro cultivation systems. If so, then identification of these factors could be instrumental for achieving continuous in vitro propagation, a goal that has eluded syphilis researchers for nearly a century.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants AI 26756 (to J. D. R.) and GM58778 (to B. H. H.), by PRAXIS XXI (to C. A., I. M., and J. J. G. M.), and by support from the Mayo Foundation.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: Section of Hematology Research, Dept. of Biochemistry and Molecular Biology, Mayo Clinic, 200 First St., S.W., Rochester, MN 55905. Tel.: 507-284-4743; Fax: 507-284-8286; E-mail: rusnak@mayo.edu.

Published, JBC Papers in Press, June 26, 2000, DOI 10.1074/jbc.M003314200

	ABBREVIATIONS

The abbreviations used are: LB, Luria-Bertani; ES, electrospray ionization; MS, mass spectrometry; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RT, reverse transcriptase; PAGE, polyacrylamide gel electrophoresis; SOD, superoxide dismutase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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