Báo cáo khoa học: Synechocystis DrgA protein functioning as nitroreductase and ferric reductase is capable of catalyzing the Fenton reaction potx

Synechocystis DrgA protein functioning as nitroreductase and ferric reductase is capable of catalyzing the Fenton reaction Kouji Takeda 1 , Mayumi Iizuka 1 , Toshihiro Watanabe 2 , Junichi Nakagawa 1 , Shinji Kawasaki 1 and Youichi Niimura 1 1 Department of Bioscience, Tokyo University of Agriculture, Japan 2 Department of Food science and Technology, Tokyo University of Agriculture, Japan Oxygen is a double-edged sword in that it is essential for any aerobic organisms, but a part of it is conver- ted to reactive oxygen species (ROS), which could kill the cells. Among such ROS, the hydroxyl radical is the most cytotoxic agent, being generated via the Fen- ton reaction from hydrogen peroxide. The Fenton reaction is a collective designation for the reaction in which hydrogen peroxide is reduced univalently through the transfer of an electron in the presence of Fe 2+ to produce an hydroxyl radical. It is thought that most of intracellular iron exists as Fe 3+ in order not to trigger the Fenton reaction. Therefore, when the Fenton reaction occurs, the Fe 3+ must be reduced to Fe 2+ . In some in vitro Fenton systems, superoxide was shown to be capable of reducing free iron [1–3]. However, it is not likely that intracellular concentra- tion of superoxide is high enough to contribute in that way [4,5]. Other candidate reductants, such as thiols, a-ketoacids, and NAD(P)H, are all abundant inside cells, and each of these can reduce Fe 3+ in vitro [6–8]. However it is still impossible to conclude that these candidates would function as predominant reductants in vivo. Under exceptional pressure to the cells, Wood- mansee and Imlay [9] demonstrated that in Escheri- chia coli, the Fenton reaction takes place through reduction of Fe 3+ by the reduced free flavin generated Keywords DrgA; Fenton reaction; flavin reductase; iron(III) reductase; nitroreductase Correspondence K. Takeda, The Department of Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156–8502, Japan Fax ⁄ Tel: +81 3 54772764 E-mail: k2takeda@nodai.ac.jp (Received 19 November 2006, revised 31 December 2006, accepted 8 January 2007) doi:10.1111/j.1742-4658.2007.05680.x In order to identify an enzyme capable of Fenton reaction in Synechocystis , we purified an enzyme catalyzing one-electron reduction of t-butyl hydro- peroxide in the presence of FAD and Fe(III)-EDTA. The enzyme was a 26 kDa protein, and its N-terminal amino acid sequencing revealed it to be DrgA protein previously reported as quinone reductase [Matsuo M, Endo T and Asada K (1998) Plant Cell Physiol 39, 751–755]. The DrgA protein exhibited potent quinone reductase activity and, furthermore, we newly found that it contained FMN and highly catalyzed nitroreductase, flavin reductase and ferric reductase activities. This is the first demonstration of nitroreductase activity of DrgA protein previously identified by a drgA mutant phenotype. DrgA protein strongly catalyzed the Fenton reaction in the presence of synthetic chelate compounds, but did so poorly in the pres- ence of natural chelate compounds. Its ferric reductase activity was observed with both natural and synthetic chelate compounds with a better efficiency with the latter. In addition to small molecular-weight chemical chelators, an iron transporter protein, transferrin, and an iron storage pro- tein, ferritin, turned out to be substrates of the DrgA protein, suggesting it might play a role in iron metabolism under physiological conditions and possibly catalyze the Fenton reaction under hyper-reductive conditions in this microorganism. Abbreviations ROS, reactive oxygen species. 1318 FEBS Journal 274 (2007) 1318–1327 ª 2007 Tokyo University of Agriculture Journal compilation ª 2007 FEBS by flavin reductase in a hyper-reductive environment when respiration is blocked in the bacteria. In this process, electrons are transferred from the enlarged NADH pools to FAD, from FADH 2 to iron, and finally from iron to H 2 O 2 . In photosynthetic organisms, excess light energy over the utilizing capacity leads to generation of ROS. Especially under high-intensity light and other stresses, intercellular ROS accumulation tends to occur, but an antioxidant protection system usually exists to counter- act it [10–15]. Whether the Fenton reaction is involved in the production of ROS during photosynthesis as demonstrated in E. coli is an open question. In this study we investigated the Fenton reaction in Synechocystis sp. PCC6803, a prokaryote capable of photosynthesis and categorized as an oxygenic photo- synthetic bacterium. From cell-free extracts, we puri- fied an enzyme catalyzing one-electron reduction of t-butyl hydroperoxide in the presence of FAD and Fe(III)-EDTA. The enzyme turned out to be DrgA protein and its catalytic activities for ferric reductase, nitroreductase and flavin reductase were demonstrated. Enzyme characterization and its possible involvement in the Fenton reaction will be presented. Results Cell free NAD(P)H oxidoreductase activity driving the Fenton reaction We examined the Fenton reaction by measuring t-butyl hydroperoxide reducing activity using cell-fee extracts after dialysis, NADH or NADPH (as electron donor), FAD or FMN (as free flavin), and FeCl 3 or Fe(III)- EDTA (as iron compounds). In Fenton reactions using the cell-free extracts prepared after dialysis supplemen- ted with NADH and NADPH, we detected lower activity with FeCl 3 than with Fe(III)-EDTA in the presence or absence of free flavin. We detected the highest Fenton activity with NADPH and Fe(III)- EDTA, while a marked potentiation by flavin was observed when using NADH and Fe(III)-EDTA (Table 1). Although the enzyme system in E. coli proposed by Woodmansee and Imlay [9] required free flavin for activation, in Synechocystis, there are flavin-dependent and flavin-independent systems. In the Fenton reaction with NADH using Synechocystis cell-free extracts pre- pared prior to dialysis, we detected high activity in the presence of Fe(III)-EDTA, but this was not further po- tentiated by addition of free flavin (supplementary Table S1). We attributed this to free flavin contained in the cell-free extracts. Purification of the NAD(P)H oxidoreductase responsible for the Fenton reaction associated with free flavin In an attempt to identify the presumed enzyme in the presence of the Fenton reaction, we purified an enzyme catalyzing flavin-dependent peroxide-reducing activity using t-butyl hydroperoxide. The purification proce- dure was described in Experimental procedures. The purified protein showed a single protein band of 26 kDa on a SDS ⁄ PAGE gel (supplementary Fig. S1). The N-terminal amino acid sequence was determined to be MDTFDAIYQRRSVKHFDPDH, and it turned out to be identical to that of DrgA protein [16]. The purification procedure, as described in the Experimental procedures section, gave a yield of 51% in the terms of t-butyl hydroperoxide reducing activity (Table 2). Characterization of DrgA protein Identification of FMN contained in DrgA protein The amino acid sequence predicted from the DNA sequence of the drgA gene displayed sequence homolo- gies to several bacterial flavoproteins [17–22]. Several highly FMN-binding regions (at positions 10–14 and 148–151) have been identified in the amino acid sequences of DrgA. Both endogenous and recombinant DrgA protein exhibited an absorption spectrum typical of a flavoprotein (Fig. 1 at 459 nm, arrow). Further- more, by HPLC analysis, the flavin coenzyme released from native DrgA protein by hot methanol treatment [23] was identified as FMN (data not shown). The Table 1. NAD(P)H oxidoreductase activities responsible for Fenton reaction in the dialyzed cell-free extracts. The activity was deter- mined following absorbance of NAD(P)H oxidation at 340 nm in a 50 m M sodium phosphate buffer (pH 7.0) at 30 °C. The reaction mixture contained 100 l M Fe(III)-EDTA, 15 lM flavin and 1 mM t-bu- tyl hydroperoxide. Specific activity is expressed as enzyme activity per mg of total protein. ND, not detected. NAD(P)H oxidoreductase activities responsible for Fenton reaction (mU ⁄ mg protein) FAD FMN No addition NADH No addition ND ND ND FeCl 3 ND ND ND Fe(III)-EDTA 67.9 ± 13.9 59.1 ± 14.3 10.7 ± 3.0 NADPH No addition ND ND ND FeCl 3 16.8 ± 1.6 11.3 ± 0.6 ND Fe(III)-EDTA 119.2 ± 18.4 87.2 ± 0.7 76.2 ± 13.7 K. Takeda et al. DrgA protein catalyzing the Fenton reaction FEBS Journal 274 (2007) 1318–1327 ª 2007 Tokyo University of Agriculture Journal compilation ª 2007 FEBS 1319 recombinant DrgA protein preparation also showed a similar absorption maximum, confirming the associ- ation of FMN with DrgA protein. The absorption maximum at 459 nm disappeared upon addition of 0.3 mm NADH under anaerobic conditions indicating reduction of protein-bound FMN (Fig. 1). The ratio of absorbance at 280 and 459 nm was 4.32 : 1 for native DrgA protein, and 4.36 : 1 for recombinant DrgA protein. Substrate specificity As summarized in Table 3, the Synechocystis DrgA protein showed significant substrate preference to qui- nones as previously reported by Matsuo et al. [24]. We measured quinone reductase activity using ubiquinone 0 as substrate. In the presence of NADH, the specific activities of endogenous Synechocystis DrgA protein and recombinant DrgA protein were 7.03 UÆmg )1 pro- tein and 7.67 UÆmg )1 protein. Those in the presence of NADPH were 11.33 UÆmg )1 protein and 11.98 UÆmg )1 protein, respectively, indicating endogenous and recombinant DrgA protein were equally potent as qui- none reductase. Moreover, the recombinant DrgA protein showed substrate specificity similar to that of endogenous Syn- echocystis DrgA protein (data not shown). Therefore, we used recombinant DrgA protein in subsequent experiments. DrgA protein showed nitroreductase activity for nitrobenzene, dinoseb and nitrofurazone, with the highest activity for nitrofurazone. The flavin reductase activities of DrgA protein for FAD and FMN were 7.41 and 6.95 UÆmg )1 protein, respectively, in the presence of NADPH. Ferric reductase activities and peroxide reducing activities responsible for Fenton reaction Reduction of iron is known to require reduced flavins provided by flavin reductase. In the presence of free FAD, we found ferric reductase activity of recombin- ant DrgA protein using various iron compounds (Table 4). The specific activity of ferric reductase of DrgA protein for natural chelators varied between 0.1 and 2.0 UÆmg )1 protein, and that for synthetic chela- tors varied between 1.7 and 5.2 UÆmg )1 protein Surprisingly, as well as being active with small molecular weight chemicals, DrgA protein was also active with the iron transport protein transferrin (1.06 UÆmg )1 protein), and the iron storage protein ferritin (1.74 UÆmg )1 protein). For measurement of the Fenton reaction we used peroxide as substrate, and the specific activities for natural chelators were 0.5–3.5 UÆmg )1 protein and those for synthetic chelators were 12.3–39.0 UÆmg )1 protein. Thus, the activity for the Fenton reaction was about 10 times higher for synthetic chelators than for natural chelators. Chemical stoichiometry of the Fenton reaction The chemical stoichiometry of hydrogen peroxide reducing activity of DrgA protein in the presence of NADH and Fe(III)-EDTA was investigated. From a mass balance, we estimated that in this enzymatic reac- tion, 148 lm of hydrogen peroxide were reduced by Table 2. Purification of NADH-dependent t-butyl hydroperoxide- reducing activity responsible for the Fenton reaction. The activity was determined following absorbance of NAD(P)H oxidation at 340 nm in a 50 m M sodium phosphate buffer (pH 7.0) at 30 °C. The reaction mixture contained cell-free extracts, 150 l M NADH, 100 l M Fe(III)-EDTA, 15 lM FAD and 1 mM t-butyl hydroperoxide. Specific activity is expressed as enzyme activity per mg of total protein. The cell-free extracts were prepared starting from 10 g wet cells Total protein (mg) Total activity (U) Specific activity (U ⁄ mg protein) Purification index Yield (%) Cell-free extracts 695.4 7.0 0.01 1.0 100 Butyl toyopearl 50.8 17.0 0.33 33 242.9 DEAE Sepharose 2.0 6.2 3.1 310 88.6 HQ-H 0.2 3.6 18.0 1800 51.4 0.15 0.10 0.05 0.00 300 400 500 600 Wavelength (nm) Absorbance Fig. 1. Absorption spectra of native, recombinant DrgA protein and recombinant DrgA protein reduced by NADH. Absorption spectra of the purified native (16.8 l M; ——), recombinant DrgA protein (23.6 l M; ) and recombinant DrgA protein after anaerobic reduc- tion with 0.3 m M NADH (– – – –) in a 50 mM sodium phosphate buf- fer, pH 7.0, at 25 °C. DrgA protein catalyzing the Fenton reaction K. Takeda et al. 1320 FEBS Journal 274 (2007) 1318–1327 ª 2007 Tokyo University of Agriculture Journal compilation ª 2007 FEBS consuming 84 lm of NADH, generating 148 lm of hydroxyl radical as final product. Collectively, the chemical stoichiometry of the reac- tion can be formulated as follows (a one-electron reduction): 2H 2 O 2 þ NADH þ H þ ! 2OH  þ NAD þ þ 2H 2 O In addition, when DNA degradation was measured using pBR322 plasmid as substrate (in the absence of FAD), the reaction resulted in complete degradation of the DNA (no band was detected in Fig. 2, lane V). A partial DNA degradation was observed in the absence of iron compound (Fig. 2, lane III). Kinetic parameters for substrates DrgA protein catalyzed activity for nitroreductase, fla- vin reductase and ferric reductase. In the presence of saturated concentration of the substrates for these activities, namely, 50 lm nitrofurazone (nitroreduc- tase), 30 lm FAD or FMN (flavin reductase), and 50 lm Fe(III)-EDTA in the presence and absence of 30 lm FAD (ferric reductase), we measured the K m values of NADH and NADPH (supplementary Table S2). As the K m values for NADPH were much lower using any substrates than those for NADH, we meas- ured the K m and k cat values of these reactions with a Table 3. Substrate specificity of purified DrgA protein in the pres- ence of either NADH or NADPH. Experimental details are described in the Experimental procedures section. Oxidation of 150 l M NADH or NADPH was measured in the presence of an electron acceptor. Specific activity is expressed as enzyme activity per milligram of purified native or recombinant DrgA protein. ND, not detected. Electron acceptor Enzyme activity (UÆmg protein )1 ) NADH NADPH Quinone reductase Ubiquinone 0 7.03 11.33 Duroquinone 7.44 10.60 Flavin reductase FAD 4.70 7.41 FMN 4.60 6.95 Other related enzyme activity Ferricyanide 0.72 0.54 Oxygen ND ND Cytochrome C 0.14 0.13 Nitroreductase Nitrobenzene 0.32 0.36 Dinoseb 1.08 5.77 Nitrofurazone 10.24 14.68 Table 4. Effect of different iron compounds on the ferric reductase activities and NAD(P)H oxidoreductase activities responsible for the Fenton reaction. Experimental details are described in the Experi- mental procedures section. Oxidation of 150 l M NADPH was meas- ured at 340 nm in a reaction mixture containing Fe(III) complexes, 15 l M FAD and recombinant DrgA protein for ferric reductase activ- ity, and the same reaction mixture was used the addition of 200 l M H 2 O 2 for the Fenton reaction. The final concentration of the Fe(III) complexes was 10 l M except for Ferritin, where the reaction mix- ture contained 382.5 lg ferritin. Specific activity is expressed as enzyme activity per milligram of purified recombinant DrgA protein Iron compounds Enzyme activity (UÆmg protein )1 ) Ferric reductase activity Fenton reaction Natural chelate iron compounds FeCl 3 0.91 ± 0 1.03 ± 0.09 Fe(III) citrate 1.41 ± 0 1.83 ± 0.26 Fe(III) ammonium citrate 1.98 ± 0.06 2.82 ± 0.18 Fe(III)-deoxymugineic acid 0.13 ± 0.05 0.53 ± 0.16 Fe(III)-nicotianamine 0.58 ± 0.25 3.46 ± 0.11 Fe(III)-ferrichrome 1.48 ± 0.09 2.38 ± 0.14 Fe(III)-deferoxamine 0.51 ± 0.18 1.33 ± 0.03 Synthetic chelate iron compounds Fe(III)-nitrilotriacetic acid 1.74 ± 0.3 12.33 ± 0.12 Fe(III)-EDTA 3.28 + 0.31 38.98+1.66 Fe(III)-DTPA a 5.22 ± 0.18 25.65 ± 0.27 Natural iron transporter protein Transferrin from bovine 1.06 ± 0 8.16 ± 0.05 Natural iron storage protein Ferritin from horse spleen 1.74 ± 0.03 0.95 ± 0.06 a Diethylenetriamine-N,N,N ¢,N ¢¢,N ¢¢-pentaacetic acid. M I II III IV V Fig. 2. DNA degradation. Experimental details are described in the Experimental procedures section. The reaction mixture contained 3.2 lg pBR322 (lane I), 3.2 lg pBR322 plus 300 l M H 2 O 2 (lane II), 3.2 lg pBR322 plus recombinant DrgA protein (lane III), 3.2 lg pBR322 plus Fe(III)-EDTA plus 300 l M H 2 O 2 (lane IV), 3.2 lg pBR322 plus Fe(III)-EDTA plus 300 l M H 2 O 2 plus recombinant DrgA protein (lane V). K. Takeda et al. DrgA protein catalyzing the Fenton reaction FEBS Journal 274 (2007) 1318–1327 ª 2007 Tokyo University of Agriculture Journal compilation ª 2007 FEBS 1321 saturated concentration of NADPH (150 lm). Table 5 summarizes the values of K m , k cat and k cat ⁄ K m for these reactions. The k cat ⁄ K m value of DrgA protein for nitrofura- zone was 8.57 ± 0.67 · 10 5 m )1 Æs )1 , a value similar to those reported for nitrofurazone of E. coli nitro- reductase NfsA and NfsB (6.5 · 10 6 m )1 Æs )1 and 8.3 · 10 4 m )1 Æs )1 , respectively) [25,26]. The k cat ⁄ K m value for FMN reductase activity of DrgA protein was 2.65 ± 0.14 · 10 5 m )1 Æs )1 . It is relatively low com- pared with the corresponding value of Vibrio harveyi NADPH-flavin oxidoreductase, which is 5.5 · 10 6 m )1 Æs )1 [27–29]. Many flavin reductases display ferric reductase activ- ity [30–34]. In parallel, iron compounds, such as FeCl 3 or Fe(III)-EDTA have been used as model substrates to study ferric reductase, and such effort has yielded in identification of several ferric reductases from various organisms [35–39]. The k cat ⁄ K m value for Fe(III)- EDTA of DrgA protein was 10.9 ± 0.21 · 10 6 m )1 Æs )1 in the presence of free FAD. In the absence of free FAD, it was 3.67 ± 0.05 · 10 4 m )1 Æs )1 , indicating that this activity is markedly stimulated by addition of free flavin. Discussion The Fenton reaction generates compounds that are toxic to cells and presumably plays a role in restrain- ing bacterial growth under severe environmental pressure. In E. coli, the Fenton reaction takes place when the respiratory chain is blocked, as shown by Woodmansee and Imlay [9]. The photosynthetic bacterium Synechocystis would be under stress when exposed to strong light due to overproduction of ROS, and the enzyme responsible for the Fenton reaction is identified here as DrgA protein. DrgA protein was first purified by Matsuo et al. [24], who showed that its reductase activity worked best towards quinone, among other substrates tested; weak activity for nitro- benzene was also demonstrated. However, upon homology search for DrgA protein using BLAST, the amino acid sequence of the DrgA protein deduced from its DNA sequence was found to be similar to that of several nitroreductase-like proteins [17–22]. The highest sequence homology (67% identity) was assigned to a nitroreductase from Trichodesmium ery- thraeum IMS101. Furthermore, by examining DrgA mutant strains, Elanskaya and co-workers demonstrated that the pro- tein could be involved not only in quinone reduction [40,41], but also in the reduction of nitroaromatic com- pounds [40,42]. In the present study, nitroreductase activity of purified DrgA protein was first demonstrated by using nitrobenzene, dinoseb and nitrofrazone as sub- strate, with the highest activity for nitrofurazone. The k cat ⁄ K m value of DrgA protein for nitrofurazone was 8.57 ± 0.67 · 10 5 m )1 Æs )1 . Together, our data indicate that DrgA protein functions as nitroreduc- tase in vitro. The two crystallized nitroreductases of E. coli and Enterobacter cloacae which are homologous to DrgA protein were reported to contain FMN [43–47] and their highly conserved FMN binding sites (NCBI database, Conserved domains cd02149.2) are also found in the DrgA protein sequence at positions 10– 14 and 148–151. Indeed our DrgA protein was also shown to contain FMN, although this was not so in the previous report by Matsuo et al. [24]. As pro- tein-bound FMN is known to be readily released by dialysis and gel filtration, we kept these procedures at a minimum. Therefore, it is likely that the differ- ence in the FMN content in the two DrgA protein preparations is due to the difference in the purifica- tion scheme. Table 5. Kinetic parameters of DrgA protein (recombinant DrgA protein was used). Experimental details are described in the Experimental procedures section. Oxidation of 150 l M NADPH (saturated concentration) was measured in the presence of an electron acceptor. Substrate K m value for substrate (l M) k cat (s )1 ) k cat ⁄ K m (M )1 Æs )1 ) Nitroreductase Nitrofurazone 4.96 ± 0.52 4.22 ± 0.12 8.57 ± 0.67 x 10 5 Flavin reductase FAD 8.35 ± 0.66 2.02 ± 0.1 2.43 ± 0.08 x 10 5 FMN 8.49 ± 0.68 2.24 ± 0.07 2.65 ± 0.14 x 10 5 Ferric reductase Fe(III)-EDTA (in the presence of FAD) 0.31 ± 0.01 3.38 ± 0.05 10.9 ± 0.21 x 10 6 Fe(III)-EDTA (in the absence of FAD) 0.45 ± 0.02 0.0165 ± 0.0005 3.67 ± 0.05 x 10 4 DrgA protein catalyzing the Fenton reaction K. Takeda et al. 1322 FEBS Journal 274 (2007) 1318–1327 ª 2007 Tokyo University of Agriculture Journal compilation ª 2007 FEBS An amino acid sequence homology search of DrgA protein picked up flavin reductase as the second high- est score after nitroreductase. Indeed in this study it was demonstrated that DrgA protein has a reductase activity to flavin as well as to nitroaromatic com- pounds. Flavin reductases are known to be capable of ferric reduction [30–34] and, recently, Woodmansee and Imlay [9] proposed that this reaction can be involved in the Fenton reaction both in vivo and in vitro. Our DrgA protein also catalyzed the Fenton reaction as well as iron(III) reduction in vitro. There are two types of ferric reductase reactions: namely, a reaction using flavin, and a reaction inde- pendent of flavin. While ferric reductase observed in E. coli required flavin, it was not essential for iron(III) reduction by DrgA protein, though addition of flavin stimulated the reaction. The k cat ⁄ K m values of DrgA protein for Fe(III)-EDTA were 10.9 ± 0.21 · 10 6 m )1 Æs )1 in the presence of FAD and 3.67 ± 0.05 · 10 4 m )1 Æs )1 in its absence, much higher than the reported k cat ⁄ K m value of a ferric reductase, FerB, of Paracoccus denitrificans, which is only 1 · 10 2 m )1 Æs )1 [39]. Although variation caused by technical differences in the measurement of the two experiments should be considered, these results support the idea that DrgA protein probably functions as a fer- ric reductase using free flavin rather than functioning simply as a flavin reductase. We have showed here that DrgA protein utilizes both a synthetic iron chelator, such as EDTA, and natural chelators such as citric acid. In addition to these small molecular weight chemical chelators (natural chelate compounds and synthetic chelate compounds), transfer- rin and ferritin could be substrates of the ferric reduc- tase activity of DrgA protein. These observations indicate that DrgA protein might function in iron meta- bolism under physiological conditions. Collectively, DrgA protein is an oxidoreductase util- izing NADH or NADPH as electron donors, and qui- none, nitroaromatic compounds, flavin and iron chelated compounds as electron acceptors. Enzyme kinetic studies indicate that DrgA protein exerts an efficient reductase reaction to iron in the presence of flavin. The driving force of the Fenton reaction is a diva- lent iron generated from the ferric reductase reaction. In a hyper-reductive environment, possibly caused by exposure to strong light, this enzyme system might trigger the Fenton reaction. It would now be interest- ing to compare wild-type strains and drgA gene dele- tion mutant strains for growth rate and the regulation of DrgA protein expression under environmental stresses such as iron depletion. Experimental procedures Cell culture and preparation of cell-free extracts Synechocystis sp. PCC6803 cell culture and preparation of cell-free extracts were carried out as described previously [48]. Enzyme purification All purification steps were carried out below 4 °C. The cell-free extracts from 10 g wet cells were ultracentrifuged at 100 000 g for 2 h (XL-100K centrifuge, Beckman, rotor type 45 Ti) and the supernatant (38 mL) was treated with streptomycin (final concentration 2%) to remove nucleic acids and stirred for 30 min on ice. After centrifugation at 17 400 g for 20 min (Avanti HP-25 centrifuge, Beckman, rotor type JA 25.5), the supernatant (47 mL) was supplied with 1.14 m ammonium sulfate and the pH of the cell-free extracts was adjusted to 7.0 with 2.8% ammonium solu- tion, followed by stirring for 30 min. After centrifugation at 17 400 g for 15 min (Avanti HP-25 centrifuge, Beck- man, rotor type JA 25.5), the supernatant (49 mL) was subjected to a butyl toyopearl (Tosoh, Tokyo, Japan) col- umn (3.5 · 22.0 cm) equilibrated with a 50 mm sodium phosphate buffer, pH 7.0, containing 1.14 m ammonium sulfate. The column was washed with four column vol- umes of the same buffer, and the protein was eluted with a linear gradient of 1.14 m ammonium sulfate to 0 m. The pooled fraction (100 mL) was dialyzed twice against 5 L of a 10 mm sodium phosphate buffer, pH 8.0. The dialy- sate was subjected to a DEAE Sepharose Fast Flow (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) column (3.3 · 23.5 cm) equilibrated with a 10 mm sodium phos- phate buffer, pH 8.0. The column was washed with three column volumes of the same buffer, and the enzyme was eluted with a linear gradient of NaCl (0–250 mm). The active fractions (62 mL) were pooled, concentrated and dialyzed against 10 mm sodium phosphate buffer, pH 8.0, by an Apollo membrane (cut-off size 10 kDa, Orbital Bio- science, Topsfield, MA, USA). Pooled fractions (6.2 mL) were put on a POROS HQ-H (Applied Biosystems, Tokyo, Japan) column (1.0 · 10.0 cm) equilibrated with the same buffer. The column was washed with five column volumes of the same buffer, and the enzyme was eluted with a linear gradient of NaCl (0–250 mm). The active fractions (120 mL) were pooled, concentrated and dialyzed against a 50 mm sodium phosphate buffer, pH 7.0, by an Apolo membrane (cut-off size 10 kDa, Orbital Bioscience). The purity and molecular mass of the enzyme were deter- mined by SDS ⁄ PAGE by the method of Laemmli [49]. The proteins were electro-transferred to a polyvinylidene difluoride membrane and the N-terminal sequence was determined by a protein sequencer (model 492, Applied Biosystems). K. Takeda et al. DrgA protein catalyzing the Fenton reaction FEBS Journal 274 (2007) 1318–1327 ª 2007 Tokyo University of Agriculture Journal compilation ª 2007 FEBS 1323 Enzyme assay Fenton reaction activity Enzyme activities were measured anaerobically. Enzyme solu- tions containing cell-free extracts (0.14–0.48 mg protein), or 1 lg purified enzyme in the presence or absence of flavin in a 50 mm sodium phosphate buffer (pH 7.0) were loaded into a Tunberg tube. After anaerobiosis was established by repeated evacuation and equilibration with oxygen-free argon at 30 °C, the reaction was initiated by addition of enzyme solu- tion to mixtures of iron(III) compounds and NADH solution. The reaction was monitored at 340 nm in a spectrophotome- ter (Hitachi U-3000). The iron(III) compounds and 150 lm NADH solution in a 50 mm sodium phosphate buffer (pH 7.0), in the presence or absence of 1 mm t-butyl hydro- peroxide, were made anaerobic by bubbling with oxygen-free argon at 30 °C. Fenton reaction activity was determined by measuring the difference of NAD(P)H consumption in the presence and absence of t-butyl hydroperoxide. The absorbance coefficient of NADH and NADPH were set to be 6.22 and 6.20 m m )1 Æcm )1 , respectively. One unit activity of the Fenton reaction is defined as the amount of enzyme that oxidizes 1 lmole of NAD(P)H per minute. Ferric reductase activity Ferric reductase activity was measured anaerobically in the same reaction mixture as for the Fenton reaction, but without t-butyl hydroperoxide, at 30 °C. The activity was determined by measuring the difference of NAD(P)H consumption at 340 nm in the presence and absence of iron(III) compounds. Flavin reductase activity Flavin reductase activity was measured anaerobically using the same reaction mixture as for ferric reductase, but with- out iron(III) compounds, at 30 °C. Flavin reductase activity was determined by measuring the difference in NAD(P)H consumption at 340 nm in the presence and absence of the enzyme. Nitroreductase activity The nitroreductase activity was measured aerobically at 30 °C. The reaction mixture contained 50 mm sodium phos- phate buffer (pH 7.0), 150 lm NAD(P)H, nitro compounds and enzyme. Nitroreductase activity was determined by measuring NAD(P)H consumption at 340 nm in the presence and absence of an enzyme. Substrate specificity for NAD(P)H oxidation Substrate specificity was examined under aerobic conditions because purified DrgA protein does not react with oxygen. NAD(P)H solution (final concentration 150 lm, in a 20 mm Tris ⁄ HCl buffer, pH 7.5) was prewarmed to 30 °C and placed in a micro black-cell and set into a spectrophoto- meter (Hitachi U-3000). NAD(P)H oxidation measurement was immediately started at 340 nm, and substrates were added to the mixture. After baseline equilibrium was reached, DrgA protein was added to the mixture 2,3- dimethoxy-5-methyl-1, 4-benzoquinone (ubiquinone 0), duroquinone, ferricyanide, FAD, FMN, nitrobenzene, di- noseb and nitrofurazone were used as substrates at a final concentration of 100 lm each. In the case of cytochrome C, the concentration was set to 50 lm and absorbance was measured at 550 nm. The absorbance coefficient of NADH and NADPH was set as described above. Stoichiometry of the Fenton reaction Stoichiometry and confirmation of the product of the Fen- ton reaction were carried out under anaerobic conditions. DrgA protein (140.8 lg), deoxyribose (final concentration 0.6 mm) and Fe(III)-EDTA (final concentration 5 lm) were mixed in a 15 mm sodium phosphate buffer, pH 7.0, in a Tunberg tube (final volume, 1.6 mL), then the air was sub- stituted with argon for 15 min. NADH (final concentration 100 lm) and hydrogen peroxide (final concentration 300 lm) were added in a 15 mm sodium phosphate buffer, pH 7.0, in another aerobic cuvette, and air was substituted with argon for 15 min. The anaerobic cuvette and the tube were warmed at 30 °C for 5 min, and the initial concentra- tion of NADH was determined on site by measuring its absorption at 340 nm. Then, the content of the Tunberg tube was transferred to an anaerobic cuvette using a syringe and the solution was mixed well. The reaction was monit- ored by measuring the consumption of NADH at 340 nm. In parallel, the amount of hydrogen peroxide and hydroxyl radicals was measured before and after the reaction. The quantitation of hydrogen peroxide and hydroxyl radicals was carried out as described previously [50,51]. DNA degradation in the Fenton reaction DNA degradation was measured under anaerobic condi- tions. The recombinant DrgA protein (140.8 lg), Fe(III)- EDTA (final concentration 5 lm) and 3.2 lg pBR322 DNA were mixed in a 15 mm sodium phosphate buffer, pH 7.0, in a Tunberg tube (final volume 1.6 mL), and the air was substituted with argon for 15 min. In another aerobic cu- vette NADH (final concentration 100 lm) and hydrogen peroxide (final concentration 300 lm) were added in a 15 mm sodium phosphate buffer, pH 7.0, and the tubes were warmed at 30 °C for 5 min. Following confirmation of the initial concentration of NADH by measuring its absorption at 340 nm, the content of the Tunberg tube was transferred to an anaerobic cuvette using a syringe, mixed well and incubated for 5 min. The reaction was monitored DrgA protein catalyzing the Fenton reaction K. Takeda et al. 1324 FEBS Journal 274 (2007) 1318–1327 ª 2007 Tokyo University of Agriculture Journal compilation ª 2007 FEBS by measuring the decrease of absorption of NADH at 340 nm. Each 20 lL of reaction mixture was subjected to agarose gel electrophoresis and DNA bands were visualized on the gel by staining with ethidium bromide. Steady-state kinetics The values of K m and k cat for Fe(III)-EDTA, FAD, FMN and nitrofurazone was determined from Lineweaver)Burk plots of the kinetic data obtained at 30 °C at various sub- strate concentrations in a 50 mm sodium phosphate buffer, pH 7.0, containing 150 lm NADPH. The consumption of NADPH was monitored with a spectrophotometer at 340 nm (Hitachi U-3000). Cloning, expression, and purification of DrgA from Synechocystis sp. PCC6803 We cloned the gene of drgA from Synechocystis sp. PCC6803. A Synechocystis DNA fragment containing the open reading frame, slr 1719, was amplified by the PCR using the forward primer, 5’-ac g aat tcc acc acc acc acc acc aca tgg aca cct ttg acg cta tt-3’ and the reverse primer, 5’-tag ctc gag tta ggc aaa gga gtt ttc cca-3’. The forward primer was designed to introduce six His Tags following an EcoR I site, and the reverse primer contained a Xho I site as underlined. Amplified DNA fragments were subcloned into the pTrc99A vector for transformation of E. coli strain JM109. IPTG-induced recombinant protein was purified. All steps of the purification procedure of recombinant Synechocystis DrgA were carried out at 4 °C and monit- ored by SDS ⁄ PAGE. Cells (23 g wet weight) were suspen- ded in 92 mL of 50 mm sodium phosphate buffer, pH 7.0. The suspension was stirred at 4 °C for 20 min. Cells were thawed and passed through a French pressure cell (Thermo IEC, Needham, Heights, MA, USA) twice at 88.99 kgÆcm )2 and then sonicated for 3 min. Phenylmethylsulfonyl fluoride (final concentration, 2 mm) was added to the suspension three times, i.e. immediately before and after the passage through the French pressure cell, and after sonication. The resultant suspension was centrifuged at 64 000 g for 20 min (Avanti HP-25 centrifuge, Beckman, JA 25.5 rotor) to remove unbroken cells. The supernatant was treated with streptomycin to remove nucleic acids and was stirred at 4 °C for 30 min. After centrifugation at 64 000 g for 20 min (Avanti HP-25 centrifuge, Beckman, JA 25.5 rotor), the supernatant (112 mL) was dialyzed twice against 5 L of the 50 mm sodium phosphate buffer, pH 7.0, containing 300 mm NaCl. The dialysate (120 mL) was subjected to a Talon (Takara, Tokyo, Japan) column (2.2 · 5.3 cm) equili- brated with 50 mm sodium phosphate buffer, pH 7.0, con- taining 300 mm NaCl. The column was washed with five volumes of the same buffer. The enzyme was eluted step- wise with 50, 100 and 150 mm imidazole from the column. The pooled fraction from 50 to 100 mm imidazole elution was dialyzed three times against 5 L of a 10 mm sodium phosphate buffer, pH 8.0. The dialysate (35.5 mL) was sub- jected to a DEAE Sepharose Fast Flow (GE Healthcare Bio-Sciences) column (3.3 · 23.5 cm) equilibrated with a 10 mm sodium phosphate buffer, pH 8.0. The column was washed with five column volumes of the same buffer, and the enzyme was eluted with a linear gradient of NaCl (0– 250 mm). Active fractions were pooled, concentrated and dialyzed against 45 mL of 50 mm sodium phosphate buffer, pH 7.0, by an Apollo membrane (cut-off size 10 kDa, Orbi- tal Bioscience). 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Nature 227, 680–685. 50 Sedewitz B, Schleifer KH & Go ¨ tz F (1984) Purification and biochemical characterization of pyruvate oxidase from Lactobacillus plantarum. J Bacteriol 160, 273–278. 51 Gutteridge JMC (1987) Ferrous-salt-promoted damage to deoxyribose and benzoate. The increased effectiveness of hydroxyl-radical scavengers in the presence of EDTA. Biochem J 243, 709–714. 52 Ohnishi K, Niimura Y, Hidaka M, Masaki H, Suzuki H, Uozumi T & Nishino T (1995) Role of cysteine 337 and cysteine 340 in flavoprotein that functions as NADH oxidase from Amphibacillus xylanus studied by site-directed mutagenesis. J Biol Chem 270, 5812–5817. Supplementary material The following supplementary material is available online: Fig. S1. SDS ⁄ PAGE of the purified t-butyl hydro- peroxide reducing enzyme. SDS ⁄ PAGE was carried out as described in Experimental procedures using 15% polyacrylamide gels. Table S1. NAD(P)H oxidoreductase activities responsible for the Fenton reaction in the pre-dialysis cell-free extracts. The activity was determined follow- ing absorbance of NAD(P)H oxidation at 340 nm in a 50 mm sodium phosphate buffer (pH 7.0) at 30 °C. The reaction mixture contained 100 lm Fe(III)-EDTA, 15 lm flavin and 1 mm t-butyl hydroperoxide. Specific activity is expressed as enzyme activity per milligram of total protein. Table S2. Km for NADH or NADPH. Experimen- tal details are described in the Experimental proce- dures section. Oxidation of 150 lm NADH or NADPH was measured in the presence of an electron acceptor. This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corres- ponding author for the article. K. Takeda et al. DrgA protein catalyzing the Fenton reaction FEBS Journal 274 (2007) 1318–1327 ª 2007 Tokyo University of Agriculture Journal compilation ª 2007 FEBS 1327 . FMN and highly catalyzed nitroreductase, flavin reductase and ferric reductase activities. This is the first demonstration of nitroreductase activity of DrgA protein previously identified by a drgA mutant. Synechocystis DrgA protein functioning as nitroreductase and ferric reductase is capable of catalyzing the Fenton reaction Kouji Takeda 1 , Mayumi Iizuka 1 ,. FAD and recombinant DrgA protein for ferric reductase activ- ity, and the same reaction mixture was used the addition of 200 l M H 2 O 2 for the Fenton reaction. The final concentration of the