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J. Biol. Chem., Vol. 275, Issue 35, 27021-27026, September 1, 2000

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Superoxide Reductase as a Unique Defense System against Superoxide Stress in the Microaerophile Treponema pallidum^*

Murielle Lombard, Danièle Touati^§, Marc Fontecave, and Vincent Nivière^¶

From the  Laboratoire de Chimie et Biochimie des Centres Redox Biologiques, DBMS-CEA/CNRS/Université Joseph Fourier, 17 Avenue des Martyrs, 38054 Grenoble, Cedex 9, France and the ^§ Institut Jacques Monod, CNRS/Universités Paris 6 et Paris 7, 2 place Jussieu, 75251 Paris, Cedex 05, France

Received for publication, May 17, 2000, and in revised form, June 21, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Aerobic life requires the presence of antioxidant enzymes, such as superoxide dismutase, catalase, and peroxidase to eliminate deleterious oxygen derivatives. Treponema pallidum, a microaerophilic bacterium responsible for venereal syphilis, is an interesting organism because it lacks all of the above-mentioned enzymes, as deduced from its recently sequenced genome. In this paper, we describe a gene in T. pallidum with sequence homologies to a new class of antioxidant systems, named superoxide reductases, recently isolated from sulfate-reducing bacteria (Lombard, M., Fontecave, M., Touati, D., and Nivière, V. (2000) J. Biol. Chem. 275, 115-121). We report that (i) expression of the T. pallidum gene fully restored to a superoxide dismutase-deficient Escherichia coli mutant the ability to grow under aerobic conditions; (ii) the corresponding protein displays a strong superoxide reductase activity; and (iii) the T. pallidum protein contains only one mononuclear nonheme ferrous center, able to reduce superoxide selectively and efficiently, whereas previously characterized superoxide reductase from Desulfoarculus baarsii contains an additional rubredoxin-like ferric center. These results suggest that T. pallidum antioxidant defenses rely on a new class of superoxide reductase and raise the question of the importance of superoxide reductases in mechanisms for detoxifying superoxide radicals.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Superoxide radical (O₂)¹ is the univalent reduction product of molecular oxygen. It belongs to the group of the so-called toxic oxygen derivatives, which also include hydrogen peroxide and hydroxyl radicals (1). For years, the only enzymatic system known to catalyze the elimination of superoxide was the superoxide dismutase (SOD), discovered in 1969 by McCord and Fridovich (2). Four classes of SOD have been characterized so far (3, 4), depending on the nature of the metal ion of their active sites. They all catalyze the same reaction, e.g. dismutation of superoxide radical anions to hydrogen peroxide and molecular oxygen as follows.

Very recently, a new concept in the field of the mechanisms of cellular defense against superoxide has emerged. It was discovered that elimination of O₂ could occur by reduction, a reaction catalyzed by an enzyme thus named superoxide reductase (SOR).

Up to now, two examples of superoxide reductase have been described (5, 6). The first one is a small protein found in anaerobic sulfate-reducing bacteria called desulfoferrodoxin (Dfx). Dfx is a homodimer of 2 × 14 kDa, which has been well studied (7-9) and structurally characterized (10). The monomer is organized in two protein domains, each with a specific mononuclear iron site, named center I and center II, respectively. Center I contains a mononuclear ferric iron coordinated by four cysteines in a distorted rubredoxin-type center. Center II has an oxygen-stable ferrous iron with square pyramidal coordination to four nitrogens from histidines as equatorial ligands and one sulfur from a cysteine as the axial ligand. We have shown that the iron center II of Dfx from Desulfoarculus baarsii is the active site for the SOR activity and that it reduces superoxide very efficiently, without significant SOD activity (5). That Dfx could act as a true SOR enzyme was further supported by the fact that Escherichia coli extracts contain NAD(P)H-dependent reductase activities able to provide electrons to Dfx, allowing then catalytic cycles for reduction of superoxide (5). Whether center I was participating in the electron transfer and therefore essential for a full SOR activity could not be concluded from this study. Although Dfx is not naturally present in E. coli, Pianzzola et al. demonstrated that expression of Dfx in this bacterium could totally replace the classical SOD enzymes to overcome a superoxide stress (11). That Dfx was also an antioxidant protein in sulfate-reducing bacteria was further shown when the dfx gene was deleted in the chromosome of Desulfovibrio vulgaris. This deletion increased the oxygen sensitivity of D. vulgaris during transient exposure to microaerophilic conditions (12).

Another example of SOR has been isolated from the anaerobic archaea, Pyrococcus furiosus (6). The protein presented strong homologies to neelaredoxin (Nlr), a small protein containing a single mononuclear center, earlier characterized from sulfate-reducing bacteria (13). Very recently, the three-dimensional structure of the P. furiosus SOR has been determined at high resolution (14). The protein fold and the unique mononuclear iron center are similar to those of the second domain of Dfx (containing center II), but the first protein domain, chelating the iron center I, in Dfx is missing, as expected from earlier studies of neelaredoxin (13). The protein is a homotetramer, in contrast with the dimeric structure reported for Dfx (10). In P. furiosus, an electron-transferring chain, including NADH, NADH rubredoxin oxidoreductase, and rubredoxin, was proposed to provide the electrons necessary for the reaction (6). However, there is no evidence that neelaredoxin functions as an antioxidant system in vivo, so far.

Whether SOR activity in anaerobic microorganisms, which have to face transitory exposure to air, would present a selective advantage with regard to SOD activity is still an open question. Although some hypotheses have been already proposed elsewhere (5, 6), careful analysis of bacterial genomes pointed out that several anaerobic bacteria possess both genes encoding for putative SORs and SODs, which makes the real physiological function of SOR puzzling. Analysis of the complete genome of the bacterium Treponema pallidum (15), the causative agent of venereal syphilis, a microaerophilic bacteria optimally growing at 5% oxygen tension (16), reveals that this organism does not possess the classical antioxidant enzymes, such as SODs, catalases, and peroxidases. However, a gene encoding a protein with strong sequence homology to Dfx, but lacking cysteine residues involved in the chelation of the iron center I, was found (Fig. 1).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Sequence comparison of the putative Dfx from T. pallidum with various Dfx sequences. From top to bottom are shown Dfxs from T. pallidum (Tp.), D. baarsii (Db.), D. desulfuricans (Dd.), and D. vulgaris Hildenborough (Dv.). The alignments were produced by Clustal W. Shading indicates the residues involved in the binding of the two mononuclear iron centers, center I and center II (10).

Consequently, we have overproduced, purified, and characterized this putative Dfx protein from T. pallidum. Here we report that this protein, despite the lack of iron center I, has powerful SOR activity and provides a protection from superoxide radicals comparable with SOD. T. pallidum is thus a unique microorganism in that its superoxide scavenging capacity might only rely on SOR.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Strains and Plasmid Constructs-- E. coli strain QC 2375 (sodA sodB recA) was described previously (17). For pVN10-2 construction, a 492-base pair DNA fragment containing the dfx gene of T. pallidum was amplified from pGTPEC10 (15) by polymerase chain reaction, using the oligonucleotides 5'-ACGGAATTCACGCGGAGGCACGACAG and 5'-CGCGGATCCCCCAATCTCCTGCTCC, with an EcoRI and a BamHI restriction site (underlined), respectively. The amplified fragment was digested with EcoRI and BamHI and inserted into the corresponding sites of pJF119EH (5) under ptac promoter control, and the resulting plasmid, pVN10-2, was transformed in DH5. The construct was verified by sequencing.

Biochemical and Chemical Reagents-- 1-2 mM KO₂ stock solutions were prepared in anhydrous Me₂SO as described in Ref. 5. Xanthine oxidase grade IV from milk (0.24 units/mg), catalase from Aspergillus niger (6600 units/mg), cytochrome c from bovine heart, and CuZn-SOD from bovine erythrocytes (5800 units/mg) were from Sigma.

Purification of the Recombinant Dfx and Analytical Determination-- E. coli DH5/pVN10-2 cells were grown aerobically at 37 °C in Luria-Bertani (LB) medium complemented with 0.1 mM FeCl₃ and 100 µg/ml ampicillin. 1 mM IPTG was added at A₆₀₀ = 0.3. At A₆₀₀ of about 2.2, cells were chilled and collected by centrifugation. All of the following operations were carried out at 4 °C and pH 7.6. The cell pellet (20 g, wet weight) was suspended in 60 ml of 0.1 M Tris/HCl and sonicated. After ultracentrifugation at 45,000 rpm during 90 min in a Beckman 50.2 Ti rotor, the supernatant was treated with streptomycin sulfate and then precipitated with ammonium sulfate (final concentration 80% (w/v)). The pellet was dissolved in 12 ml of 25 mM Tris/HCl and loaded onto an ACA 54 column (360 ml) equilibrated with 25 mM Tris/HCl. A fraction (100 mg) corresponding to the volume of elution of low molecular weight protein was collected. Protein fractions of 10 mg were further chromatographed using a Bio-Rad Biologic system equipped with an anion exchange column, Uno Q-1 (Bio-Rad), and equilibrated with 10 mM Tris/HCl. A linear gradient was applied (0-0.15 M NaCl) in 10 mM Tris/HCl, with a flow rate of 1 ml min¹ during 65 min. A fraction (7 mg), eluted with about 40 mM NaCl, contained only one polypeptide of about 16 kDa, as shown by SDS-polyacrylamide gel electrophoresis analysis (15% acrylamide). The native molecular mass of the protein was determined with a Superdex 75 gel filtration column (120 ml; Amersham Pharmacia Biotech), as described in Ref. 5. Protein concentration was determined using the Bio-Rad protein assay reagent (18). Protein-bound iron was determined by atomic absorption spectroscopy. EPR measurements were made on a Bruker EMX 081 spectrometer equipped with an Oxford Instrument continuous flow cryostat. N-terminal sequence and mass spectra were obtained as described in Ref. 5.

Kinetic Parameters Associated with Oxidation of the Iron Center by O₂-- The kinetics of the oxidation of Dfx by O₂, generated by the xanthine-xanthine oxidase system, was followed spectrophotometrically at 644 nm, in the absence or in the presence of different amounts of CuZn-SOD, as reported previously (5). In these conditions, the reciprocal of the initial rate of oxidation of Dfx (v_ox) should be linear versus CuZn-SOD concentrations, according to the equation,

(Eq. 1)

where k_XO is the rate constant of production of O₂ by xanthine oxidase (XO) and k_Dfx and k_SOD are the second order rate constants of the reaction of Dfx and SOD with O₂, respectively. At the concentration of CuZn-SOD that decreases by 50% the rate of oxidation of Dfx, one can write the following (5):

(Eq. 2)

Taking into account the known second order rate constant of the reaction of O₂ with CuZn-SOD at low [O₂], 2 × 10⁹ M¹ s¹ (19), the second order rate constant of the oxidation of Dfx by O₂, k_Dfx, was calculated using Equation 2.

Assays for SOD and Reductase(s) Activities-- The SOD activity was measured as described in Ref. 5 using the cytochrome c reduction assay modified from McCord and Fridovich (2). All kinetics, in the absence or presence of different amounts of the purified Dfx, were linear for at least 4 min. One unit of SOD is defined as the amount of protein that inhibits the rate of the reduction of ferricytochrome c by 50%. E. coli crude extracts were prepared as described previously (5). Reduction of Dfx was followed spectrophotometrically at 650 nm, in a cuvette (0.1-ml final volume) containing 110 µM of fully oxidized Dfx, 50 mM Tris/HCl, pH 7.6, and 600 µM of NADPH or NADH. The reaction was initiated by adding 5-20 µg of cell extract anaerobically at 17 °C. Initial velocities of reduction of the iron center were calculated from the decrease of absorption at 650 nm. One unit of activity is defined as the amount of cell extract catalyzing the reduction of 1 nmol of the iron center per min.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Product of the dfx Gene from T. Pallidum Contains Only One Mononuclear Iron Center-- The gene encoding for the putative Dfx from T. pallidum was cloned under the control of the ptac promoter of the expression vector pJF119EH and overexpressed in E. coli. The gene product was identified as a 16-kDa protein on SDS-polyacrylamide gel electrophoresis analysis and purified using a two-step purification protocol (gel filtration and anion exchange chromatographies). The 16-kDa polypeptide had a GRELSFFLQK N-terminal amino acid sequence, identical to the N-terminal translated sequence of the T. pallidum dfx gene (15) but lacking the N-terminal Met residue. A minor amount of the polypeptide with the N-terminal Met residue was also detected. Electrospray mass spectrometry analysis of the solution showed two ionic species, a minor one at 13,801 Da and a major one at 13,671 Da, corresponding to the molecular masses expected from the dfx gene sequence with and without the N-terminal Met residue, respectively (15). These data show that the purified 16-kDa protein is the product of the dfx gene. Gel filtration experiments on a Superdex 75 column with the purified protein gave an apparent molecular mass of 27,800 Da (data not shown), showing that the Dfx from T. pallidum is a homodimer.

The iron content of Dfx was determined by atomic absorption spectroscopy. A value of 0.8 iron/polypeptide chain (13,801 Da) was found. No evidence for the presence of zinc or manganese atoms were found. Fig. 2 shows the UV-visible spectrum of the as-isolated Dfx, with weak absorption bands centered at 644 and 330 nm. No contributions at 370 and 503 nm, characteristic for iron center I in Dfx from Desulfovibrio desulfuricans (7) or D. baarsii (5), could be detected, suggesting that Dfx from T. pallidum is missing iron center I. When the protein was treated with potassium ferricyanide, the intensity of the bands at 644 and 330 nm greatly increased, and a value of 2300 M¹ cm¹ was determined for the molar extinction coefficient at 644 nm in the fully oxidized protein. Furthermore, the 4 K EPR spectrum of the isolated protein displays only a weak resonance at g = 4.3, which strongly increased during the treatment with ferricyanide (Fig. 3). This spectrum is similar to that reported for the ferric form of Dfx from D. desulfuricans (9) and from D. vulgaris (8) and was attributed to the oxidized center II. The iron center of the as isolated T. pallidum Dfx was thus essentially in the ferrous state and could be fully oxidized by ferricyanide.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Absorption spectra of the recombinant T. pallidum Dfx. A 23.6 µM protein containing 0.72 iron/polypeptide chain suspended in 50 mM Tris/HCl, pH 7.6 was used. Spectra of Dfx as isolated (lower trace) and as treated with 25 µM potassium ferricyanide (upper trace) are shown. The inset shows an enlargement of the 400-800-nm region.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   EPR spectra of the Dfx from T. pallidum. A, spectrum of the as-isolated Dfx. B, spectrum of the Dfx oxidized with 200 µM potassium ferricyanide. Experimental conditions were as follows: protein concentration, 200 µM; microwave power, 0.2 milliwatts; frequency, 9.44 Ghz; modulation amplitude, 20 G; receiver gain, 5.02 × 105. Spectra were recorded at 4 K.

Collectively, these data show that Dfx from T. pallidum contains only one iron center, equivalent to center II from well characterized Dfxs from sulfate-reducing bacteria, and is missing a second iron center, equivalent to center I, present in the other characterized Dfxs (7-10). These data are in agreement with the absence of three cysteine ligands in the T. pallidum Dfx sequence, replaced by a Gln, Ser, and Ala (Fig. 1).

Dfx from T. pallidum Functionally Complements E. coli SOD-deficient Mutants-- The capability of the dfx gene product from T. pallidum to complement E. coli SOD deficiency was tested. In fact, the E. coli sodA sodB recA mutant cannot grow in the presence of oxygen because of the combined lack of superoxide dismutase activity (sodA sodB) and the DNA strand break repair activity (recA), which results in lethal DNA oxidative damage (17, 20). As shown in Table I, in the presence of 1 mM IPTG, the plasmid pVN10-2, which encodes the structural T. pallidum dfx gene under the control of a tac promoter, fully restores aerobic growth to the sodA sodB recA mutant, whereas the parental plasmid pJF119EH did not. This clearly showed that production of Dfx from T. pallidum efficiently suppresses the deleterious effects due to the lack of SOD in E. coli and consequently fully protects against superoxide stress.

Reduction of Superoxide by T. pallidum Dfx-- That T. pallidum Dfx could catalyze the elimination of superoxide by reduction and then act as a superoxide reductase was further investigated. First, we have verified that Dfx from T. pallidum did not exhibit any significant SOD activity, assayed from its inhibitory effect on the reduction of cytochrome c by O₂ generated by the xanthine-xanthine oxidase system. The addition of 28 µg of purified Dfx was required to observe 50% inhibition of cytochrome c reduction, corresponding to a value for the specific SOD activity of 35 units mg¹ (data not shown). This value is only about 0.5% of a standard SOD enzyme specific activity and strongly suggested that Dfx from T. pallidum could not function as a SOD enzyme within the cell.

Successive additions of stoichiometric amounts of O₂ (KO₂ dissolved in Me₂SO) in the presence of catalase resulted in the oxidation of the iron center, as shown by the increase of the band at 644 nm of the visible spectrum of Dfx (Fig. 4). Spectral changes occurred during the mixing time. A 4-fold molar excess of O₂ was required for a complete oxidation of the iron center, and further addition of KO₂ did not promote additional changes (data not shown). Considering the very rapid spontaneous dismutation of superoxide (21), these data showed that superoxide efficiently oxidized the iron center of Dfx from T. pallidum.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effect of O2 on the visible spectra of T. pallidum Dfx. The microcuvette (100-µl final volume) contains 208 µM of Dfx (150 µM iron center) in 50 mM Tris/HCl, pH 7.6, 500 units/ml catalase. Successive additions of 150 µM KO2, from a 1.5 mM KO2 stock solution dissolved in 100% Me2SO (14 M), were performed. After each addition, a spectrum was recorded. From the bottom to the top, no addition, 1 eq, 2 eq, 3 eq, and 4 eq per iron center are shown.

This was confirmed by the determination of the rate constant for the oxidation of Dfx by O₂, using a methodology developed earlier (5). The kinetics of the oxidation of the iron center by O₂, generated by the xanthine-xanthine oxidase system in the presence of catalase, was followed spectrophotometrically at 644 nm, in the absence or in the presence of different amounts of CuZn-SOD. As shown in Fig. 5A, in the absence of SOD, oxidation of the iron center by O₂ was linear with time and was complete after about 2.5 min of reaction. In the presence of large amounts of CuZn-SOD, the rate of oxidation was decreased. Fig. 5B shows a linear plot of the reciprocal of the initial rate of oxidation of iron center (v_ox) as a function of CuZn-SOD concentration, according to Equation 1, as described under "Experimental Procedures." From this plot, the concentration of CuZn-SOD that decreases by 50% the rate of the iron center was determined to be 3.9 µM. The second order rate constant of the oxidation of the iron center by O₂ can be now calculated using Equation 2. A value of 1 10⁹ M¹ s¹ was obtained.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Kinetics of oxidation of the T. pallidum Dfx by O2. A, oxidation of the iron center was followed spectroscopically, at 25 °C, by the increase of absorbance at 644 nm. The cuvette contains (300-µl final volume) 10.3 µM Dfx (corresponding to 7.4 µM iron center), 50 mM Tris/HCl, pH 7.6, 400 µM xanthine, 500 units/ml catalase, and different amounts of CuZn-SOD. The oxidation was initiated by adding 0.013 units of xanthine oxidase. The following traces are presented: CuZn-SOD (, 0 µM; , 2 µM; , 3 µM) and 5 µM CuZn-SOD (). B, the reciprocal of the initial velocity of the oxidation of the iron center as a function of [CuZn-SOD].

The experiments presented above have been carried out in the presence of catalase in order to eliminate a possible effect of H₂O₂ that could be produced during spontaneous O₂ dismutation. The ability of H₂O₂ to oxidize Dfx was nevertheless tested. The kinetic of the oxidation of the iron center (22 µM Dfx in 50 mM Tris/HCl, pH 7.6) by 0.3, 0.5, 0.8, 1, and 1.5 mM H₂O₂ was followed spectrophotometrically at 644 nm, at 25 °C. In all cases, the reactions followed a pseudo-first order kinetic with a value for the second order rate constant equal to 120 M¹ s¹ (data not shown). This is almost negligible when compared with the value of the rate constant of the oxidation of the iron center by O₂.

Dfx from T. pallidum Can Act as a Superoxide Reductase-- In the experiments with the sodA sodB recA E. coli mutant strain (see above), Dfx was overexpressed. We thus could not a priori exclude a simple O₂ trapping effect (a noncatalytic elimination process) of an excess of Dfx, leading to complementation of the SOD deficiency. However, cytosolic E. coli extracts were able to reduce the oxidized form of the Dfx from T. pallidum with a specific activity of 22 nmol of iron center reduced/min/mg in the presence of either NADPH or NADH (data not shown). The membrane fractions presented also some Dfx reductase activities, with a specific activity of 10 nmol of iron center reduced/min/mg, in the presence of either NADH or NADPH (data not shown). These data demonstrated that both cytosolic and membrane E. coli extracts had the potential for catalytic reduction of Dfx from T. pallidum. This reaction regenerates the active ferrous center for new cycles of superoxide reduction. This result thus supports the notion that Dfx from T. pallidum is a superoxide reductase, which allows aerobic growth of E. coli sod mutant strains. It further indicates that, at least in E. coli, the presence of an iron center I is not required for providing Dfx with a functional SOR activity.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have isolated a protein from T. pallidum on the basis of its strong sequence homology with Dfxs from sulfate-reducing bacteria (Fig. 1). However, there is a major difference between this protein and the Dfxs previously described. Dfx from T. pallidum only chelates one iron center, which has all of the spectroscopic characteristics of the so-called ferrous center II in Dfx from D. vulgaris (8) and D. desulfuricans (9). Accordingly, all of the ligands chelating the iron center II in Dfxs are found strictly conserved in the sequence of T. pallidum, in addition to the residues surrounding these positions (Fig. 1). The second iron center (center I) is absent in Dfx from T. pallidum, in agreement with the absence of three cysteine ligands replaced by a Gln, Ser, and Ala (Fig. 1). In that respect, Dfx from T. pallidum shows interesting similarities to Nlr, a protein initially isolated from the sulfate-reducing bacteria Desulfovibrio gigas (13) and recently described as a SOR in Pyrococcus furiosus (6). Nlr also contains a single mononuclear iron center, with spectroscopic properties similar to those of the iron center II of Dfxs (13, 14). However, although Nlr presents a similar structural fold to the C-terminal domain of Dfxs (14), with conservation of the ligands of the iron center II, it lacks the whole protein domain corresponding to the N-terminal sequence of Dfxs from sulfate-reducing bacteria (Fig. 6). Instead, Dfx from T. pallidum can be aligned with the entire sequence of the other Dfxs, including the whole N-terminal domain (Figs. 1 and 6). In addition, Nlr sequences exhibit one major additional loop, which is not present in the C-terminal domain of classical Dfxs and in the sequence of the T. pallidum protein (Fig. 6). On the whole, it is correct to classify the protein from T. pallidum as a new type of Dfx rather than an Nlr.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Sequence comparison between Dfxs and Nlrs. From top to bottom, Nlr from P. furiosus (Pf.), Nlr from D. gigas (Dg.), Dfx from T. pallidum (Tp.), Dfx from D. baarsii (Db.), Dfx from D. desulfuricans (Dd.), and Dfx from D. vulgaris Hildenborough (Dv.). The alignments were produced by Clustal W. Shading indicates the residues involved in the binding of the two mononuclear iron centers (10, 14).

All of the data reported here strongly suggest that this new type of Dfx functions as an SOR. (i) Expression of Dfx from T. pallidum is able to fully protect an E. coli SOD mutant from oxidative stress (Table I). The data were comparable with the data reported for the Dfx from D. baarsii (5) and suggested that, in E. coli, the iron center I of Dfx is not important for a functional complementation. (ii) Dfx from T. pallidum can reduce O₂ very efficiently. The second order rate constant of the oxidation of the reduced Dfx from T. pallidum by O₂ has been determined to be 1 × 10⁹ M¹ s¹, a value even greater than that reported for the D. baarsii enzyme (6-7 × 10⁸ M¹ s¹) (5). The reaction is specific for O₂, since H₂O₂ did oxidize the iron center much more slowly (second order rate constant 120 M¹ s¹). Dfx from T. pallidum is also O₂-resistant, and the protein was isolated mainly in a stable ferrous iron state. (iii) That reduction of O₂ could be catalytic within the cell depends on the presence of a cellular system able to reduce the oxidized iron center for a complete catalytic cycle. We have found that cell extracts of E. coli contained NAD(P)H-dependent reductase activities, which may fulfill this function. Although these activities are smaller than those reported in the case of D. baarsii (5), it still demonstrated that E. coli extracts could catalytically reduce Dfx from T. pallidum. In addition, because the reductase activities are not specific to membrane or cytosol fractions and to the reduced pyridine nucleotides, it thus appears that E. coli extracts do not possess a single specific system to reduce the iron center of SOR. This is in line with the great accessibility of the active site of SORs (10, 14) and their high redox potential (9, 13), which make a large number of reducing agents and reductases potentially good candidates. Consequently, it is very likely that similar activities exist in T. pallidum as well.

A question remains as far as the role of iron center I in Dfxs from sulfate-reducing bacteria is concerned. The existence of SORs (Dfx from T. pallidum and Nlr from P. furiosus for example) containing only one iron center would suggest that center I in Dfx from D. baarsii does not participate in electron transfer/O₂ reduction during SOR activity and that this function resides only in iron center II. Further experiments are required to understand the function of center I.

Although SODs remain the most widespread defense mechanism against superoxide, several examples of another mechanism, SOR, have been reported. SORs primarily appeared as a simple and specific means to anaerobic bacteria to eliminate superoxide (5, 6, 11), possibly presenting advantage during transitory exposure to air (12). The benefit of an SOR, compared with a SOD, in these organisms may be in relation to the presence of large amounts of a variety of strongly autoxidizable redox proteins, such as redox carriers (cytochromes, ferredoxins, and flavodoxins, for example). As illustrated in Fig. 7, by shuttling the electrons from the autoxidizable redox proteins to superoxide, SOR could, in a single reaction, eliminate both superoxide and the source of its production. Such a reaction may allow the anaerobic bacteria to shut off transitory O₂ production from those redox carriers, with no need for sophisticated regulatory systems, such as are found in facultative anaerobes. Other authors have pointed out that reduction of superoxide does not produce molecular oxygen, as does the dismutation reaction, thus protecting O₂-sensitive cellular species from inactivation (4). However, this latter hypothesis is questionable, taking into account that from the genome and protein sequences available, it appears that several anaerobic microorganisms, like D. gigas (13, 24), D. desulfuricans (7, 25), D. vulgaris Hildenborough (22),² Methanobacterium thermoautotrophicum (23), or Clostridium acetobutylicum (Genome Therapeutics Corp., completed genome, not published; open reading frames CAC2865, CAC2999, CAC1647) contain both sor and sod genes. Further studies are necessary to determine the respective roles of each enzyme and why there is such an apparent redundancy in mechanisms for elimination of superoxide.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Scheme for the hypothesis of the detoxification activity of SOR. In the presence of O2, formed from the autoxidizable redox proteins in the presence of O2, SOR eliminates both O2 and its source of production. In the absence of O2/O2, SOR is not active, and the electrons are shuttled toward the cellular metabolisms.

In this respect, T. pallidum is a very interesting bacterium. It is a microaerophilic microorganism, with an optimal growth rate in the presence of 5% of molecular oxygen (16). This is the first example of an organism that can grow in the presence of oxygen without expressing a SOD enzyme (with the exception of Mn-SOD mimic complexes produced by lactic acid bacteria (26)). Here we have shown that T. pallidum relies on a simplified version of Dfx, with full SOR activity, as the only mechanism for elimination of superoxide and protection from oxidative stress. This makes T. pallidum a unique model for studying the link between superoxide reductase and oxidative stress.

	ACKNOWLEDGEMENTS

We are grateful to Dr. Eric Forest for mass spectrometry experiments, to Mathilde Louwagie for N-terminal amino acid sequence determination, and to Dr. Véronique Ducros for atomic absorption spectroscopy. We acknowledge Dr. Stéphane Ménage for helping in EPR experiments and Chantal Falco for technical assistance.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 33-4-76-88-91-09; Fax: 33-4-76-88-91-24; E-mail: vniviere@cea.fr.

Published, JBC Papers in Press, June 23, 2000, DOI 10.1074/jbc.M004201200

² N. V. Shenvi and D. M. Kurtz, GenBank^TM accession no. AF034841.

	ABBREVIATIONS

The abbreviations used are: O₂, superoxide; SOD, superoxide dismutase; SOR, superoxide reductase; Dfx, desulfoferrodoxin; Nlr, neelaredoxin; IPTG, isopropyl-1-thio--D-galactopyranoside.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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