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FEBS Letters 581 (2007) 5605–5610

Desulfoferrodoxin of Clostridium acetobutylicum functions as a reductase

Oliver Riebe, Ralf-Jo¨rg Fischer, Hubert Bahl* University of Rostock, Institute of Biological Sciences, Division of Microbiology, Albert-Einstein-Strasse 3, D-18051 Rostock, Germany

Received 10 October 2007; revised 1 November 2007; accepted 2 November 2007

Available online 19 November 2007

Edited by David Lambeth

Desulfoferrodoxin (Dfx) which has been discovered in sul- Abstract Desulfoferrodoxin (cac2450)ofClostridium aceto- butylicum was purified after overexpression in E. coli.Inan phate-reducing bacteria was shown to possess the ability to in vitro assay the exhibited superoxide reductase activity complement SOD deficient Escherichia coli strains [5,6]. This with rubredoxin (cac2778)ofC. acetobutylicum as the proximal complementation was proposed to be a result of a NAD(P)H electron donor. Rubredoxin was reduced by ferredoxin:NADP+ dependent SOR activity of Dfx. In turn, a desulfoferrodoxin reductase from spinach and NADPH. The superoxide anions, knock out mutant of Desulfovibrio vulgaris showed increased generated from dissolved oxygen using Xanthine and Xanthine oxygen sensitivity [7]. It has also been shown, that the two iron oxidase, were reduced to . Thus, we assume centre containing Dfx from D. vulgaris, D. desulfuricans and that desulfoferrodoxin is the key factor in the superoxide reduc- Desulfoarculus baarsii possess SOR activity in vitro [5,8,9].In tase dependent part of an alternative pathway for detoxification clostridia, with SOR activity in general or Dfxs in of reactive oxygen species in this obligate anaerobic bacterium. particular have not yet been analyzed. According to sequence 2007 Federation of European Biochemical Societies. Pub- lished by Elsevier B.V. All rights reserved. data, C. acetobutylicum Dfx contains only one iron [Fe(NHis)4(SCys)] and thus belongs to the 1Fe-SOR subclass Keywords: Oxidative stress; Superoxide reductase; Anaerobic [1]. bacteria; Clostridium In this study, the SOR activity of C. acetobutylicum Dfx rep- resenting a 1Fe-SOR was analyzed. Our in vitro results clearly demonstrate the SOR function of this enzyme. Thus, we assume that Dfx has an important role in the first part of a 1. Introduction ROS-detoxification pathway in C. acetobutylicum.

Aerobic microorganisms defeat reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide via 2. Materials and methods the catalase/ (SOD) system. In anaerobes the function of this system is restricted or incomplete. Thus, in 2.1. Reagents, proteins, and general procedures the genome of Clostridium acetobutylicum no catalase is anno- Reagents and buffers were at least analytical grade. Xanthine + tated [1] and a function of the three annotated SODs could not oxidase, bovine liver catalase, ferredoxin:NADP reductase (FNR), horseradish peroxidase (HRP), Escherichia coli superoxide dismutase yet been demonstrated. Furthermore, a disadvantage for (Fe-SOD) and Clostridium pasteurianum rubredoxin were purchased anaerobes is also the production of oxygen by both enzymes. from Sigma. Concentrations were assumed to be those provided by Nevertheless, C. acetobutylicum survives short periods of aera- Sigma. 10-Acetyl-3,7-dihydroxyphenoxazine (Amplex Red, AR) was tion [2]. Therefore, an alternative detoxification system must from Synchem OHG. Reagents were used without further purification. exist. Interestingly, all genes for a superoxide reductase (SOR) and peroxidase dependent detoxification pathway for 2.2. Cloning, expression and purification Clostridium acetobutylicum ATCC824 genomic DNA was isolated reactive oxygen species proposed for Pyrococcus furiosus [3] according to Bertram and Du¨rre [10]. The rubredoxin- (cac2778) and are also present in C. acetobutylicum and other clostridia desulfoferrodoxin genes (cac2450) were amplified using the following (Table 1). In P. furiosus, a SOR reduces superoxide to hydro- oligonucleotides: Rbo_Fw_BamHI 50-CATTGGATCCATGGTAT- gen peroxide with electrons from NAD(P)H involving rubre- ATCCCAATAAAAGG-30, Rbo_Rev_PstI 50-AATTTCTGCAGTT- 0 0 doxin (Rbo) and an . Hydrogen peroxide is CTTCAGATGGCTCAAA-3 , Dfx_Fw_KpnI 5 -GGTAGGTACCA- TGAATAACGATTTATCAA-30 and Dfx_Rev_BamHI 50-GCTCA- then further reduced to water by the peroxidase activity of GGATCCTATATCTGCTTTCC-30 introducing BamHI, PstI or rubrerythrin. KpnI restriction sites. The resulting fragments were cloned into a SORs are a class of non-heme iron enzymes and are subclas- pASK-IBA 3 vector (http://www.IBA-GO.com). The resulting plas- sified into 1Fe-SORs and 2Fe-SORs. In the latter, the mono- mids pI3rbo and pI3dfx were transformed into E. coli DH5a. The re- combinant strains were grown in 500 ml Luria–Bertani broth nuclear iron active site [Fe(NHis)4(SCys)] present in 1Fe- supplemented with 100 lgml1 ampicillin at 37 C under continuous SORs is complemented by an additional N-terminal shaking to an OD600 of 0.4–0.5. Gene expression was induced by addi- 1 [Fe(SCys)4] centre [4]. The function of the second iron site is tion of 0.2 lgml anhydrotetracycline. A better incorporation of iron 1 not clear. into the proteins was achieved after addition of 40 lgml FeSO4Æ7- H2O to the cultures. After 4 h cultures were harvested at 8000 · g for 10 min and pellets were suspended in 5 ml buffer W (100 mM Tris–HCl, 150 mM NaCl) without EDTA. The disruption of the cells *Corresponding author. Fax: +49 381 498 6152. was achieved by ultrasonication using a MedLab Disintegrator Sono- E-mail address: [email protected] (H. Bahl). puls HD60 apparatus for 1 min at a power of 30% and 50 pulses per

0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.11.008 5606 O. Riebe et al. / FEBS Letters 581 (2007) 5605–5610

Table 1 Genes of different Clostridia potentially involved in the SOR pathway Organism Rbo Dfx NROR Normal Rbr Reverse Rbr C. acetobutylicum cac2778 cac2450 cac2448 cac2575 cac3597 ATCC 824 cac3018 cac3598

C. difficile cd0828 cd0827 cd1623 cd2845 cd1524 Strain 630 cd1157 cd1474

C. perfringens cpe0777 GI:18311480 cpf1269 cpe0135 cpe0689 ATCC 13124 cpe0780 cpe1331 cpe0082 cpe0855 cpe2618

C. tetani E88 ctc01387 ctc02454 ctc02521 ctc02638 ctc00826 ctc01182 min. The lysate was centrifuged for at least 30 min at 12000 · g and lowed by a subsequent addition of the missing reaction partners 4 C. The resulting crude extract was loaded onto columns containing FNR, Rbo and Dfx at a concentration of 1 lM every 30 s. Xanthine a Strep-Tactin-Sepharose-Matrix. Further purification steps were car- oxidase (3.5 lgml1) was added to start the SOR-reaction by superox- ried out as described in the IBA standard protocol (www.IBA-GO. ide generation. Production of resorufin was followed by an increasing com). The protein content of the elution fractions was determined absorbance at 560 nm. using the Bradford assay [11]. Purity of the elution fractions was ana- lyzed by SDS–PAGE [12]. 2.6. Gel staining for SOD activity of Dfx Non-denaturating 10% PAGE gels were prepared as described else- 2.3. Determination of SOR activity of Dfx in a cytochrome c assay where [12] replacing ammonium persulphate by 28 lM riboflavin. Gels This method was based upon a superoxide dismutase assay [13]. The were then photopolymerized for 1 h under exposure to a 40 W lamp in assay was performed in 1 ml cuvettes under aerobic conditions. All a distance of 25 cm. Gel separation of the proteins was followed by solutions contained a buffer of 100 mM Tris–HCl and 150 mM NaCl determination of SOD activity according to Beauchamp and Fridovich at pH 8.0. Cytochrome c (cyt c,20lM) was reduced in aerobic solu- [18]. After a shaking in 2.45 mM nitroblue tetrazolium for 20 min the tions resulting in an increasing absorbance at 550 nm. Generation of gels were transferred to a solution containing 28 mM tetramethylene- superoxide anions by addition of xanthine (0.2 mM) and xanthine oxi- diamine, 36 mM potassium phosphate and 28 lm riboflavin at pH dase (3.5 lgml1) to the reaction mixture strongly enhanced this effect. 7.8 for 15 min in complete darkness. The gels were washed with water SOD inhibits superoxide dependent reduction of cyt c. Enzymes and again illuminated until clear bands were visible. (E. coli Fe-SOD or Dfx) were added after a constant baseline reaction was obtained. The tested Dfx enzyme concentrations were as follows: 0, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, and 10 lM. SOD was taken as a control for the functionality of the assay. 1 U of SOD activity was de- 3. Results fined as the amount of SOD causing 50% inhibition of reduction of cyt c under superoxide generation. 3.1. Purification of Dfx and Rbo from C. acetobutylicum C. acetobutylicum Rbo (cac2778) and Dfx (cac2450) were 2.4. Reaction of Dfx in presence of Rbo, FNR and NADPH expressed in E. coli DH5a labelled with a Strep-Tag and puri- SOR activity of Dfx was monitored essentially as described by Emerson [9]. A calibrated flux of superoxide generated by the xan- fied via Strep-Tactin column chromatography. This resulted in thine/ reaction as described for the standard SOD as- highly pure Rbo and Dfx protein fractions as shown exempl- say was used [13]. C. acetobutylicum Rbo was tested as the possible arily for Dfx in Fig. 1. Absorption spectra of the purified pro- electron donor transferring the electrons to C. acetobutylicum Dfx. It teins are illustrated in Fig. 2. The spectrometric analysis of Dfx was shown that NADH:rubredoxin oxidoreductase (NROR) can transfer electrons to Rbo and in turn regenerates the reduced form revealed a spectrum without any remarkable features between of this protein [14–16]. In this assay, the NROR was displaced by 400 and 700 nm (graph c). A peak with a maximum at 330 nm the NADPH dependent protein FNR from spinach. Heterologously could be observed as it was also present in spectra of SOR expressed NROR is likely to be toxic for E. coli and could not be puri- from other bacteria [4]. The spectrum did not change under fied so far [14]. The reaction was monitored by measuring the NADPH aerobic conditions for several hours indicating an extreme sta- consumption in a 1 ml cuvette as a decrease in absorbance at 340 nm 1 1 bility of the enzyme. In contrast, the spectrum of Rbo exhib- (e340 = 6220 M cm ). All reagents and proteins were added from aerobic stock solutions at room temperature. The reaction mix con- ited absorbance peaks at 350, 375, 492 and 565 nm (graph tained 500 lM xanthine, 100 lM NADPH, 200 U ml1 catalase, a). The peak at 492 nm is characteristic for an oxidized iron 1 lM FNR in a buffer of 50 mM sodium phosphate and 100 mM sulphur cluster of the [Fe(SCys) ] type as it has been described EDTA at pH 7.5. After 30 s C. acetobutylicum Rbo (1 lM) and after 4 further 30 s either Dfx (1 lM) or Fe-SOD (250 U ml1) were added for Rbos of other bacteria [19,20]. Thus, we assumed that in to this premix. When a constant baseline NADPH consumption rate the purified Rbo of C. acetobutylicum this centre is occupied was obtained 3.5 lgml1 of xanthine oxidase were added to start the with iron and therefore this heterologously expressed protein reaction by superoxide generation. should be functional. The purified Rbo was completely re- duced by addition of an equimolar amount of sodium dithio- 2.5. Detection of H2O2 nite under anaerobic conditions (graph b). Reoxidation of In presence of hydrogen peroxide, horseradish peroxidase (HRP) oxidizes AR to the fluorescent resorufin. Assays were per- Rbo (reappearance of the peak at 492 nm) occurred slowly formed according to Seaver and Imlay [17] except that 100 llofa within a few hours under aerobic conditions. 200 lM stock solution of AR in 50 mM potassium phosphate buffer pH 7.8 were added to 250 ll of the sample mix. The reaction mix con- 3.2. SOR activity of C. acetobutylicum Dfx tained 500 lM xanthine and 100 lM NADPH in a 50 lM MOPS buf- According to its proposed role as a SOR in ROS detoxifica- fer suspended with 0.1 mM EDTA at a pH of 7.8. H2O2 detection was started by addition of 10 ll of the HRP stock solution. This was fol- tion pathway C. acetobutylicum Dfx should be capable to act O. Riebe et al. / FEBS Letters 581 (2007) 5605–5610 5607

Fig. 1. Purification of Dfx from C. acetobutylicum. Dfx overexpression Fig. 3. SOR activity of C. acetobutylicum Dfx. The reaction was was induced in E. coli DH5a (pI3dfx) by the addition of 0.2 lgml1 monitored by measuring the NADPH consumption in a 1 ml cuvette as anhydrotetracycline. The overexpressed protein bound specifically to a decrease in absorbance at 340 nm. All reagents and proteins were Strep-Tactin-Sepharose Matrix and was eluted as described in the IBA added from aerobic stock solutions at room temperature at indicated standard protocol (www.IBA-GO.com): (1) marker proteins; (2) E. coli time points. Absorbance spikes caused by mixing and addition of the DH5a (pI3dfx), crude extract, not induced; (3) E. coli DH5a (pI3dfx) different substances are omitted from the traces: (a) superoxide crude extract, induced; (4) column discharge; (5–7) elution fractions. generation was started by addition of X-O; (b) FNR was added to The proteins were separated in a 12.5% SDS–polyacrylamide gel and the cuvette containing all other components; (c) reaction of Fe-SOD visualized by staining with Coomassie brilliant blue. from E. coli was taken as a control. Inset: Effect of addition of SOD (40 U) to an activity assay of Dfx (compare graphs a and b) on NADPH consumption. Rbo, rubredoxin; Dfx, desulfoferrodoxin; FNR, ferre- doxin:NADH reductase; X-O, xanthine oxidase; Fe-SOD, superoxide dismutase from E. coli.

cially available C. pasteurianum Rbo without a loss of func- tionality of the pathway. The activities of both Rbos were comparable (data not shown). Different concentrations of Dfx and Rbo influenced the NADPH consumption rate (Fig. 4). The turnover rate was calculated to be 5.75 lM NADPH per lM Rbo per min with an excess of Dfx (Fig. 4a) and 4.65 lM NADPH per lM Dfx per min with an excess of Rbo (Fig. 4b).

3.3. Generation of H2O2 by Dfx mediated SOR reaction Hydrogen peroxide, the product of the SOR reaction exerted Fig. 2. Absorption spectra of C. acetobutylicum Rbo and Dfx: (a) Rbo by Dfx of C. acetobutylicum was detected by determining the (air oxidized); (b) Rbo (dithionite reduced); (c) Dfx. amount of resorufin at 560 nm, produced by the Amplex Red/HRP reaction subject to the presence of H2O2 ([17], Fig. 5). In this assay, H2O2 is also produced by xanthine oxi- as the terminal component transferring electrons from dase (graph a), which is needed to generate superoxide. Never- NAD(P)H to superoxide. We have reconstituted this pathway theless, in the presence of Dfx, H2O2 production due to the in vitro using heterologously expressed and purified C. acetobu- reduction of superoxide is significantly enhanced (graph b). tylicum Dfx as a SOR and reduced Rbo as electron donor. Rbo of C. acetobutylicum was reduced by FNR from spinach using 3.4. Dfx oxidizes cytochrome c NADPH as an electron donor. As shown in Fig. 3 NADPH The SOR activity of Dfx of C. acetobutylicum was confirmed consumption could be observed after the addition of xanthine in a second independent test. For this purpose the cytochrome oxidase to generate superoxide when all the above mentioned c dependent superoxide dismutase assay [13] was adapted for protein components were present (graph a). No consumption the detection of a superoxide reductase activity. This assay is of NADPH occurred when one protein, e.g. FNR, was omitted independent from electron donors like NAD(P)H. Cyt c exhib- from the assay mixture. Complementation of the assay with its a reduction due to its reduction potential resulting in an this protein resumed SOR activity (graph b). Substitution of increasing absorption at 550 nm (Fig. 6, graph a). The genera- Dfx by a classical superoxide dismutase, the Fe-SOD from tion of superoxide in the buffer by the xanthine/xanthine oxi- E. coli, did not result in any activity above background levels, dase reaction increased the reduction of cyt c drastically indicating that this enzyme cannot accept electrons efficiently (Fig. 6, graph b). Both, SOD and SOR can inhibit this reduc- from the components present in the assay (graph c). However, tion of cyt c in a concentration dependent manner. The differ- when SOD was added to the activity assay of Dfx, both ence is that a SOD can only inhibit the reduction, even in high enzymes competed for the superoxide, which resulted in a amounts, whereas an excess of a SOR can also oxidize cyt c. reduced NADPH consumption of Dfx (inset in Fig. 3). It This was also observed for Dfx of C. acetobutylicum (Fig. 6, was possible to replace C. acetobutylicum Rbo by commer- graphs c and d). Dfx (0.2 lM) inhibited the reduction of cyt 5608 O. Riebe et al. / FEBS Letters 581 (2007) 5605–5610

Fig. 6. Effect of Dfx from C. acetobutylicum on cytochrome c reduction/oxidation. The assay was performed in 1 ml cuvettes under aerobic conditions. All solutions contained a buffer of 100 mM Tris– HCl and 150 mM NaCl at pH 8.0. Enzymes were added after a constant baseline reaction was obtained: (a) cyt c (20 lM) was reduced in aerobic solutions resulting in an increasing absorbance at 550 nm. (b) Generation of superoxide anions by addition of xanthine (0.2 mM) and xanthine oxidase (3.5 lgml1) to the reaction mixture strongly enhanced this effect. (c) Dfx inhibits superoxide-dependent reduction of cyt c (0.2 lM Dfx). (d) High amounts of Dfx can oxidize cyt c (10 lM Dfx). (e) High amounts of SOD can only inhibit the reduction of cyt c (40 U SOD). The same effect was achieved using 0.875 lM Dfx. E. coli Fe-SOD was used as a control for the functionality of the assay.

Fig. 4. Rbo (a) and Dfx (b) dependent NADPH consumption in the Dfx (Fig. 6, graph e). The observation that Dfx was able to SOR activity assay SOR activities were measured as in Fig. 3 using oxidize cyt c is in agreement with its proposed SOR activity. different concentrations of Rbo (50 nM–10 lM) at constant concen- trations of Dfx (1 lM) and FNR (1 lM) or using different concentra- tions of Dfx (9 nM–9 lM) at constant concentrations of Rbo (1 lM) 3.5. Potential SOD activity of Dfx and FNR (1 lM).The NADPH consumption rates were corrected for A low SOD activity was reported (200 U mg1 compared to the blind reaction rates prior addition of xanthine oxidase. 1800 U mg1 bovine SOD) for the SOR of P. furiosus [21].A SOD activity of Dfx of C. acetobutylicum was not detectable using the standard photospectrometric NBT dependent SOD assay (data not shown). Nevertheless, in a gel staining assay evidence for a slight SOD activity of Dfx was found (Fig. 7). The purified Dfx (10 lg) clearly showed an activity band in the zymogram (lane 4), which corresponded to a band in the Dfx-overexpressing E. coli extract (lane 3). However, com-

Fig. 5. Generation of H2O2 in the SOR activity assay. The determi- nation of H2O2 as a product of the SOR-reaction was achieved by measuring the increasing absorption at 560 nm due to the generation of resorufin by the Amplex Red/HRP reaction in presence of H2O2. All reagents and proteins were added from aerobic stock solutions at room temperature at indicated time points. Absorbance spikes caused by mixing and addition of the different substances are omitted from the traces. (a) H2O2 production by xanthine oxidase. (b) H2O2 production by xanthine oxidase and Dfx. Rbo, rubredoxin; Dfx, desulfoferrodox- in; FNR, ferredoxin:NADP+ reductase; X-O, xanthine oxidase; HRP, horseradish peroxidase. Fig. 7. SOD activity of Dfx from C. acetobutylicum. The gel staining assay was performed as described [18]: (a) 12.5% native PAGE; Coomassie brilliant blue stained; (1) 10 lg protein from E. coli DH5a; c by 50% and high amounts (10 lM) oxidized cyt c. An excess (2) 2.5 lg Fe-SOD E. coli; (3) 40 lg protein E. coli DH5a (pI3dfx) of the Fe-SOD of E. coli only inhibited the reduction of cyt c to induced; (4) 10 lg purified C. acetobutylicum Dfx. (b) Gel stained for ground level extent. This could also be achieved by 0.875 lM SOD activity, lanes as in (a). O. Riebe et al. / FEBS Letters 581 (2007) 5605–5610 5609 pared to the Fe-SOD the observed activity is very low. Only of that [Fe(SCys)4] site. The function of this rudimentary site 2.5 lg of the E. coli enzyme resulted in a considerable activity in 1Fe-SORs and also of the complete [Fe(SCys)4] site in band (lane 2) compared to the faint band produced by 10 lgof 2Fe-SORs is not clear. The engineered 2Fe-SOR of D. vulgaris Dfx. containing an altered [Fe(SCys)4] site retained its catalytic properties [4]. Activity of a SOD in crude extracts of C. acetobutylicum could 4. Discussion not been demonstrated using different methods. Thus, the anno- tated SODs in the genome of this obligate anaerobic bacterium On the basis of the experiments reported here, we conclude [1] seem to be inactive, even under oxidative stress. Whether the that Dfx of C. acetobutylicum encoded by cac2450 functions very low SOD activity of C. acetobutylicum Dfx observed in the as a superoxide reductase. Thus, this enzyme contributes to zymogram is of any significance remains to be elucidated. Nev- the defence mechanism of this obligate anaerobic bacterium ertheless, C. acetobutylicum obviously employs a pathway for against oxidative stress, e.g., superoxide stress. In agreement the detoxification of ROS, favouring superoxide reduction via with this proposed function of Dfx Northern blot analysis re- NAD(P)H consumption over SOD-dependent disproportion- vealed increased transcript levels of the Dfx encoding gene ation. The first part of this pathway was reconstructed here cac2450 after exposure of C. acetobutylicum to low levels of in vitro. In addition to the identification of the proteins involved oxygen [22]. Furthermore, we observed a nearly twofold in- in the second part it will be of interest whether and how this bac- crease of Dfx at the protein level after oxygen stress (unpub- terium is able to lower also the dioxygen concentration. lished results). In our in vitro system which represents the first part of the alternative pathway for the detoxification of Acknowledgement: O.R. was supported by a fellowship of the Gradu- ROS in anaerobic bacteria [14,21,23] NADPH consumption iertenfo¨rderung of the Federal State of Mecklenburg-Vorpommern. was only obtained when Rbo and FNR were also present in the reaction mixture. In turn, no NADPH consumption was obtained when Dfx was missing. This indicates an electron References flow from NADPH via these two proteins to the reaction cen- tre of Dfx. In our assay we had to replace the putative [1] Noelling, J., Breton, G., Omelchenko, M.V., Makarova, K.S., NADH:rubredoxin oxidoreductase (NROR) from C. acetobu- Zeng, Q., Gibson, R., Lee, H.M., Dubois, J., Qiu, D., Hitti, J., tylicum with the FNR from spinach [9]. Our attempts to isolate Wolf, Y.I., Tatusov, R.L., Sabathe, F., Doucette-Stamm, L., Soucaille, P., Daly, M.J., Bennett, G.N., Koonin, E.V. and Smith, an active enzyme after overexpression of cac2448 in E. coli D.R. (2001) Genome sequence and comparative analysis of the failed. The gene cac2448 is annotated as NROR and is located solvent-producing bacterium Clostridium acetobutylicum. J. Bac- in an operon together with Dfx and other redox proteins [22]. teriol. 183 (16), 4823–4838. The observed consumption rates of NADPH of the C. acetobu- [2] O’Brien, R.W. and Morris, J.G. (1971) Oxygen and the growth and metabolism of Clostridium acetobutylicum. J. Gen. Microbiol. tylicum proteins (5.75 lM NADPH per lM Rbo per min with 68 (3), 307–318. an excess of Dfx and 4.65 lM NADPH per lM Dfx per min [3] Grunden, A.M., Jenney Jr., F.E., Ma, K., Ji, M., Weinberg, M.V. with an excess of Rbo) in our SOR assay were in the same or- and Adams, M.W. (2005) In vitro reconstitution of an NADPH- der of magnitude as reported for other bacteria. Nevertheless, dependent superoxide reduction pathway from Pyrococcus furio- the consumption rate of D. vulgaris Dfx is about three times sus. Appl. Environ. Microbiol. 71 (3), 1522–1530. [4] Emerson, J.P., Cabelli, D.E. and Kurtz Jr., D.M. (2003) An higher (12 lM NADPH min1) than for C. acetobutylicum engineered two-iron superoxide reductase lacking the [Fe(SCys)4] Dfx [9]. This might reflect the ability of D. vulgaris to live in site retains its catalytic properties in vitro and in vivo. Proc. Natl. microaerophilic habitats and to cope with higher ROS concen- Acad. Sci. USA 100 (7), 3802–3807. trations for a longer time than C. acetobutylicum [2,24,25]. [5] Romao, C.V., Liu, M.Y., Le Gall, J., Gomes, C.M., Braga, V., Pacheco, I., Xavier, A.V. and Teixeira, M. (1999) The superoxide Thus, the activity of the ROS detoxification system plays an dismutase activity of desulfoferrodoxin from Desulfovibrio desul- important role to the ecological fitness of anaerobic bacteria, furicans ATCC 27774. Eur. J. Biochem. 261 (2), 438–443. either to survive short exposures to oxidative stress or during [6] Pianzzola, M.J., Soubes, M. and Touati, D. (1996) Overproduc- life at the border line between anaerobic and aerobic habitats. tion of the rbo gene product from Desulfovibrio species suppresses The near UV–visible absorption spectra of purified Dfx re- all deleterious effects of lack of superoxide dismutase in Esche- richia coli. J. Bacteriol. 178 (23), 6736–6742. vealed no absorption between 400 and 700 nm in contrast to [7] Voordouw, J.K. and Voordouw, G. (1998) Deletion of the rbo Dfx of D. vulgaris, a 2Fe-SOR [4]. Interestingly, an engineered gene increases the oxygen sensitivity of the sulfate-reducing version of the latter enzyme lacking the [Fe(SCys)4] site shows bacterium Desulfovibrio vulgaris Hildenborough. Appl. Environ. a very similar spectrum as Dfx of C. acetobutylicum. These Microbiol. 64 (8), 2882–2887. [8] Lombard, M., Le Touati, D., Fontecave, M. and Nivie`re, V. data are in agreement with the sequence data which indicate (2000) Reaction of the desulfoferrodoxin from Desulfoarculus only one iron centre [Fe(NHis)4(SCys)] in the clostridial en- baarsii with superoxide anion. J. Biol. Chem. 275 (1), 115–121. zyme. Therefore, Dfx from C. acetobutylicum belongs to the [9] Emerson, J.P., Coulter, E.D., Phillips, R.S. and Kurtz Jr., D.M. 1Fe-SOR subclass. The observed superoxide reactivity proper- (2003) Kinetics of the superoxide reductase catalytic cycle. J. 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