Hexameric ring structure of a thermophilic archaeon NADH oxidase that produces predominantly H 2 O Baolei Jia 1, *, Seong-Cheol Park 1, * , , Sangmin Lee 1 , Bang P. Pham 1 , Rui Yu 1 , Thuy L. Le 1 , Sang Woo Han 2,3 , Jae-Kyung Yang 4 , Myung-Suk Choi 4 , Wolfgang Baumeister 5 and Gang-Won Cheong 1,3 1 Division of Applied Life Sciences (BK21 Program), Gyeongsang National University, Jinju, Korea 2 Department of Chemistry, Research Institute of Natural Science, Gyeongsang National University, Jinju, Korea 3 Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju, Korea 4 Division of Environmental Forest Science and Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Korea 5 Department of Molecular Structural Biology, Max-Planck-Institute for Biochemistry, Martinsried, Germany Thermococcus profundus is a thermophilic anaerobic archaeon belonging to the Thermococcaceae family that also includes Thermococcus kodakaraensis KOD1, a model thermophilic organism whose whole genome sequence has been reported [1]. As an anaerobe living in deep-vent environments, it seems likely that Thermo- coccus encounters high levels of oxygen stress in the water surrounding the vent [2]. In anaerobes, flavin- dependent NAD(P)H oxidases play an important role protecting organisms from oxidative stress [3]. NADH oxidase (NOX) is a member of the flavo- protein disulfide reductase family that catalyzes the pyridine-nucleotide-dependent reduction of various substrates, including O 2 ,H 2 O 2 and thioredoxin [4]. There are two types of NOX: those that catalyze the two-electron reduction of O 2 to H 2 O 2 and those that catalyze the four-electron reduction of O 2 to H 2 O [5]. The physiological role of NOX is diverse, depending on its substrates and products in different organisms. In anaerobic mesophiles, NOX enzymes, such as those Keywords electron microscopy; H 2 O-producing; hexameric ring structure; NADH oxidase; thermophilic archaeon Correspondence G W. Cheong, Division of Applied Life Sciences, Gyeongsang National University, Jinju 660-701, Korea Fax: +82 55 752 7062 Tel: +82 55 751 5962 E-mail: gwcheong@gnu.ac.kr Present address Research Center for Proteineous Materials, Chosun University, Kwangju 501-759, Korea *These authors contributed equally to this work (Received 7 August 2008, revised 31 August 2008, accepted 3 September 2008) doi:10.1111/j.1742-4658.2008.06665.x An NADH oxidase (NOX) was cloned from the genome of Thermococ- cus profundus (NOXtp) by genome walking, and the encoded protein was purified to homogeneity after expression in Escherichia coli. Subsequent analyses showed that it is an FAD-containing protein with a subunit molecular mass of 49 kDa that exists as a hexamer with a native molecular mass of 300 kDa. A ring-shaped hexameric form was revealed by electron microscopic and image processing analyses. NOXtp catalyzed the oxidiza- tion of NADH and NADPH and predominantly converted O 2 to H 2 O, but not to H 2 O 2 , as in the case of most other NOX enzymess. To our knowl- edge, this is the first example of a NOX that can produce H 2 O predomi- nantly in a thermophilic organism. As an enzyme with two cysteine residues, NOXtp contains a cysteinyl redox center at Cys45 in addition to FAD. Mutant analysis suggests that Cys45 in NOXtp plays a key role in the four-electron reduction of O 2 to H 2 O, but not in the two-electron reduction of O 2 to H 2 O 2 . Abbreviations CoADR, coenzyme A disulfide reductase; GR, glutathione reductase; Nbs 2 , 5,5¢-dithiobis-(2-nitrobenzoic acid); NOX, NADH oxidase (EC 1.6.99.3); NOXtp, Thermococcus profundus NADH oxidase. FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS 5355 of Clostridium aminovalericum [6], Enterococcus (Strep- tococcus) and Lactococcus [3], are considered to be important enzymes in protecting against oxidative stress and in regenerating oxidized pyridine nucleo- tides through their capacity to reduce O 2 to H 2 O without the formation of harmful reactive oxygen spe- cies. Some NOX proteins have also been purified and studied in (hyper)thermophilic organisms. NOX from Archaeoglobus fulgidus may be involved in electron transfer in sulfate respiration [7]. An H 2 O 2 -forming NOX functions as an alkyl hydroperoxide reductase in Amphibacillus xylanus [8]. Some NOX enzymes, such as those of Pyrococcus furiosus [9] and Thermotoga maritima [10], have been proposed to protect anaer- obes from oxidative stress. In (hyper)thermophiles, the roles of some NOX enzymes remain to be elucidated [11]. NADH oxidase varies with the organism; however, these proteins generally share similar secondary struc- tural folding [4,12]. An NOX from Thermus thermophi- lus is a homodimer as determined by X-ray crystallography [13]. Gel filtration chromatography indicated that NADH:flavin oxidoreductase from Eubacterium is composed of three identical subunits [14]; NOX in Clostridium thermohydrosulfuricum is probably made up of six subunits, as demonstrated by gel filtration [15]. In contrast, a heterogeneous NOX from Eubacterium ramulus is proposed to have an a 8 b 4 assembly, as revealed by gel filtration and PAGE [16]. Two NOX enzymes from the Thermococcaceae fam- ily have been described. One is a novel enzyme in P. furiosus that produces both H 2 O 2 (77%) and H 2 O (23%) [9]. The other NOX, in Pyrococcus horikoshii OT3, may function as a CoA disulfide reductase (CoA- DR) [17]; however, the function and structure of NOX in Thermococcus, a genus of the Thermococcaceae fam- ily, has not been clarified. In this study, we have cloned, overexpressed and purified a NOX that is com- posed of two cysteine moieties from T. profundus.We report its biochemical characterization and structure, and also used mutants to analyze its catalytic mecha- nism. Results Cloning and sequencing of the nox gene from T. profundus In order to clone the T. profundus nox gene (NOXtp), we utilized a PCR-based DNA-walking method using the ClonTech genome-walker cloning kit, as described in Experimental procedures; the resulting DNA sequence comprised an ORF of 1329 bp, predicting a protein composed of 442 amino acids with a molecular mass of 48 611 Da. Figure 1 shows the nucleotide sequence of NOXtp and its flanking regions, together with the translated amino acid sequence. The 5¢-flank- ing region of NOXtp contained a putative Archaea promoter with a TATA box and ribosome-binding site. The 3¢-flanking region did not match with other Archaea genes, as judged by homolog searches in the NCBI database. Unlike NOX, which has only one conserved cysteine residue (Cys45) in its N-terminus [4], the amino acid composition of NOXtp revealed the presence of two cysteine residues, Cys45 and Cys122. Additionally, two conserved cofactor-binding domains were also identified in NOXtp. One was a FAD-binding domain containing the AMP-binding and FMN-binding motifs observed in enzymes belong- ing to the glutathione reductase (GR) family [18]. The other domain was a glycine-rich NAD-binding motif located between the AMP-binding and FMN-binding motifs (two FAD-binding domains) (Fig. 1). We pro- pose that NOXtp belongs to the GR family, because of the high sequence identity of the cofactor-binding domains described above. Multiple sequence alignment (Fig. S1) revealed that Cys45 is located at a similar position to that of the cysteine residue in the conserved active site of NOX from P. horikoshii (also called CoADR) [17] and NOX and NADH peroxidase from Enterococcus faecalis [19,20]. Sequence analysis by clustal w showed that NOXtp shared a significant level of identity with NOX (CoADR) from P. horikoshii (80%) [17], NADH per- oxidase from E. faecalis (28%) [20], and NOX from P. furiosus (36%) [9], Lactococcus lactis (30%) [21], Lactococcus sanfranciscensis (26%) [11] and E. faecalis (27%) [19] (Fig. S1). These proteins are generally com- posed of two identical subunits related by two-fold symmetry. Each subunit can be divided into a C-termi- nal dimerization domain and an N-terminal pyridine nucleotide disulfide oxidoreductase domain, which is actually a small NADH-binding domain with a large FAD-binding domain [4,12]. NOXtp has similar primary structure architecture to these proteins as determined by NCBI protein blast analysis. Purification of native and recombinant NOXtp In order to understand the oxygen detoxification mecha- nism of anaerobic microbes, we purified NOXtp from T. profundus by several chromatographic methods. The purified protein revealed a subunit with a molecular mass of approximately 50 kDa (Fig. S2). The N-termi- nal amino acid sequence of purified NOXtp from T. pro- fundus was determined to be MERKRVVIIGGGAAG, Hexameric thermophilic NADH oxidase-producing H 2 O B. Jia et al. 5356 FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS which is highly similar to that of NOX in T. kodakaren- sis KOD1, P. furiosus, Pyrococcus abyssi and P. horiko- shii OT3, belonging to the pyridine nucleotide disulfide oxidoreductase family. The purification of recombinant NOXtp from Escherichia coli was performed by ion exchange chromatography as described in Experimental procedures. SDS ⁄ PAGE analysis of recombinant NOXtp revealed a molecular mass of approximately 50 kDa, which is close to that of the purified protein from T. profundus (Fig. S2). However, gel filtration analysis under nondenaturing conditions showed that the purified NOXtp had a molecular mass of approximately 300 kDa (Fig. 2). These results indicated that NOXtp is a hexamer of 50 kDa subunits, in contrast to NOX proteins from thermophilic archaeans, which have been reported to be dimers or tetramers [13,17]. Structure of NOXtp Gel filtration analysis under nondenaturing conditions revealed that purified NOXtp has a molecular mass of  300 kDa, corresponding to a hexamer with 50 kDa subunits (Fig. 2). This structure is different from that of other homologous NOX proteins, which consist of dimers or tetramers as revealed by X-ray crystallographic studies [12,13,22]. In order to clarify the oligomeric structure of NOXtp, we performed electron microscopy using purified NOXtp. The elec- tron micrographs of the negatively stained NOXtp oligomers showed a uniform distribution of the ring- shaped structure in the top-view orientation (Fig. 3A). In total, 939 well-stained particles were translationally aligned, and were subjected to multi- variate statistical analysis [23]. The eigenimages Fig. 1. Nucleotide sequence of the noxtp gene and predicted amino acid sequence of the gene product from Thermo- coccus profundus. The putative TATA-box and ribosome-binding site are underlined and in bold letters, respectively. The resi- dues involved in FAD binding are shadowed in gray. The NAD-binding site is boxed. The cysteine residues are in bold italic. B. Jia et al. Hexameric thermophilic NADH oxidase-producing H 2 O FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS 5357 obtained from the translationally, but not rotation- ally, aligned images revealed a six-fold rotational symmetry (Fig. 3Ba). Using the 10 most significant eigenvectors, nine classes were discriminated on the basis of similarity of features after rotational align- ment without symmetrization. Most class averages showed a star-shaped structure with six-fold symme- try with heavy stain accumulation in its center (Fig. 3Bb). In particular, the two class averages shown in panels 3 and 6 of Fig. 3Bb exhibited an obvious deviation from the star-like structure. This could result from incomplete stain embedding of the particle or from an unintentional inclination during preparation or microscopy. In order to analyze further the rotational symmetry of the top-on-view images, the same dataset was separated into many classes (10–30) using different eigenimages (10–20). The dataset was also aligned with an arbitrarily chosen reference and separated according to the simi- larity of features in the eigenimages. The resulting class averages revealed no other statistically signifi- cant symmetry (data not shown). We found no Fig. 2. Gel filtration chromatography profile of NOXtp purified from Es. coli. The purified protein was subjected to Superdex-200 gel fil- tration chromatography. Absorbance was measured at 280 nm. The x-axis shows the elution time. The standard proteins are ferritin (440 kDa), catalase (232 kDa), albumin (67 kDa) and ovalbumin (43 kDa). A B a C D b Fig. 3. Electron micrograph and structural analysis of NOXtp. (A) Purified NOXtp was absorbed onto the grids as described in Experimental procedures. The electron micrograph of the protein was then obtained by negative staining with 2% uranyl acetate. (B) Multivariate statistical analysis of NOXtp. (a) The average (AV) of 939 translationally, but not rotationally, aligned particles with end-on orientation and the 10 most signifi- cant eigenimages (numbers 1–10) are shown. In (b), the nonsymmetrized class averages (numbers 1–9) were derived from rotationally aligned images using the 10 most significant eigenvectors. The numerals shown in the top right corner of the class averages are the number of particles seen in each class. (C) The average of the side-on view of NOXtp (939 particles). (D) A sche- matic model for the assembly of NOXtp complexes. The diameters of the cavity, middle ring and outer ring are 4, 15 and 19 nm, respectively. Hexameric thermophilic NADH oxidase-producing H 2 O B. Jia et al. 5358 FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS evidence for the existence of a NOXtp protein with intrinsically lower symmetry, at least at the resolution employed. The average of the 939 top-on views revealed a star- shaped structure (Fig. 3C), which contained a middle region of high density with heavy stain accumulation in its center. The average view also revealed that the density of the complex was not homogeneous; the den- sity increased towards the middle, such as seen in the valosine-containing protein-like ATPase from Thermo- plasma acidophilum complex, which is composed of two stacked ring structures of different diameters [24]. The upper (or middle) ring of the NOXtp complex has a region that is denser than that of the outer ring, indi- cating the presence of a cavity in the complex with a width of approximately 4 nm. The diameters of the outer ring and the middle ring (Fig. 3D) were approxi- mately 19 and 15 nm, respectively. These projected images as well as the gel filtration analysis indicated that NOXtp predominantly exhibits a hexameric star- shaped structure, in contrast to the structure recently reported by Kuzu et al. [22], which suggested a tetra- meric structure for NOX from Lactobacilluus brevis. NOXtp is an FAD-dependent NADH and NADPH oxidase On the basis of the amino acid sequence, NOXtp con- tains two FAD-binding domains. The isoalloxazine ring system in FAD has been suggested to induce light absorbance in the UV and visible spectral range, giving rise to the yellow appearance of flavin and flavopro- teins [25]. We performed light absorbance analysis to confirm NOXtp binding to FAD. Purified NOXtp from Es. coli has absorption maxima at 378 and 456 nm, with a shoulder at 480 nm, which are charac- teristic spectral features of proteins with bound flavin cofactors (Fig. 4A). The absorbance behavior also allowed the determination of the number of flavin mol- ecules bound per mole of NOXtp subunit [17,25,26]. A stoichiometry of 0.7–0.9 mol FAD per mol NOXtp subunit was determined from the absorbance at 460 nm. As NOXtp contains FAD as a prosthetic group, apo-NOXtp was prepared by hydrophobic interaction chromatography under acidic conditions (pH 3.5) with saturated NaBr buffer [26,27], in order to 0 0.04 0.08 0.12 0.16 0.2 300 400 500 600 700 800 (nm) Absorbance 0 10 20 30 40 50 60 70 80 90 100 0 2 4 6 8 10 12 (min) NAD(P) amount (μM) 0 1 2 3 4 5 6 7 8 20 30 40 50 60 70 80 90 100 (ºC) Specific activity (U·mg –1 ) 0 1 2 3 4 5 6 7 8 Specific activity (U·mg –1 ) 5678910 (pH) A B C D Fig. 4. Activity assays of NADH and NADPH oxidase. (A) Visible spectra of NOXtp (solid line), apo-NOXtp (dashed line) and the C45A mutant (dotted line). The absorbance was measured in 50 m M sodium phosphate buffer (pH 7.2) at 25 °C. (B) FAD effect on NAD(P)H oxidase activity. An activity assay was performed as described in Experimental procedures. The solid line shows the NADH oxidase activity of NOXtp purified from Es. coli (h), reconsti- tuted NOXtp (s), and apo-NOXtp (4). The dashed line shows the NADPH oxidase activity of NOXtp from Es. coli (h), reconstituted NOXtp (s), and apo-NOXtp (4). (C) Optimal temperature of NAD(P)H oxidase activity. The assay was performed at the indicated temper- atures in 50 m M potassium phosphate buffer (pH 7.2). NADH and NADPH oxidase activity are shown by a solid line and a dashed line, respectively. The squares show the measured temperature points. (D) Optimal pH of NAD(P)H oxidase activity. Different buffers were used in this assay. Sodium phosphate was used at pH 6.0, 6.6, 7.2 and 7.7; Hepes buffer and Tris buffer were used at pH 8.0 and 8.5; sodium borate buffer was used at pH 9.0. These buffers were used at a concentration of 50 m M. NADH and NADPH oxi- dase activity are shown by a solid line and a dashed line, respec- tively. The squares show the measured pH points. B. Jia et al. Hexameric thermophilic NADH oxidase-producing H 2 O FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS 5359 confirm the function of FAD. The absorption spec- trum of apo-NOXtp did not show any significant absorbance in the visible region, revealing that FAD was indeed absent (Fig. 4A). To determine whether FAD was required for the enzymatic activity of NOXtp, holoprotein and apoprotein activities were assayed. The NADPH oxidase activity of NOXtp was also measured, as described previously for NOX (CoADR) from P. horikoshii [17] and NOX from L. sanfranciscensis [12], which show high similarity to NOXtp and accept both NADH and NADPH as cofactors. The activity of the reconstituted enzyme, which was accomplished by incubating equimolar concentrations of apomonomers and FAD at room temperature for 5 min [26], was also measured. These assays revealed that NADH oxidase activity was slightly higher than that of NADPH oxidase, and FAD significantly restored the oxidase activity of apo-NOXtp (Fig. 4B). These results clearly indicated that NOXtp is an FAD-dependent NADH and NADPH oxidase, in contrast to NOX enzymes from other thermophilic archaeons, which only exhibit activity towards NADH [9–11]. To further determine the function of NOXtp, the steady-state kinetic parameters of NOXtp with either NADH or NADPH as the reducing substrate were measured at pH 7.2. NOXtp could catalyze NADH and NADPH oxidization with k cat values of 6.2 ± 0.5 ⁄ s and 2.5 ± 0.3 ⁄ s, respectively. The steady- state kinetic parameters of NOXtp were similar to those of NOX (CoADR) from P. horikoshii (Table 1). On the basis of the K m , both enzymes preferred NADPH as the substrate for oxidase activity, indicat- ing that NOX (CoADR) from P. horikoshii and NOX- tp belong to similar enzyme families. The optimal temperature for the NADH and NADPH oxidase activity of NOXtp was near 70 °C (Fig. 4C), which is lower than the optimal growth temperature (80 °C) of this organism. The optimal pH was between 7.5 and 8.0 for both NADH and NADPH oxidase activity (Fig. 4D). NOXtp preferentially produces H 2 O The product of O 2 reduction is an important factor in evaluating the physiological function of NOXs [10]. For instance, NOX from P. furiosus, which may pro- tect anaerobic thermophiles against oxidative stress, can produce both H 2 O 2 and H 2 O [9]. In order to determine the product of the NAD(P)H oxidase activ- ity of NOXtp, reactions containing 100 lm NAD(P)H were performed [all NAD(P)H consumed] according to the published method [9], and H 2 O 2 was quantified using a peroxi-DETECT kit from Sigma (St Louis, MO, USA). When NADPH oxidation was performed at 80 °C, approximately 7% of the NADPH supplied was used to produce H 2 O 2 , and 2% of the NADH was recovered as H 2 O 2 under the same conditions (Fig. 5D). These results demonstrated that NOXtp produces predominantly H 2 O using NADH and NADPH as electron donors. Cys45 but not Cys122 functions as the nonflavin redox center NADH oxidase in members of the Thermococcaceaee family, such as T. kodakaraensis KOD1, P. horrikoshii, P. abyssi and P. furiosus, have only one conserved cys- teine residue, Cys45; however, the sequence of NOXtp (Fig. 1) revealed that it contains two cysteine residues, Cys45 and Cys122. As cysteines are important residues for NOX enzyme activity, we replaced Cys45 and Cys122 with alanines to analyze the function of these two residues. After purification using the same method as that used for the wild-type enzyme, the number of cysteines in the three mutant enzymes (NOXtpC45A, NOXtpC122A and NOXtpC45A⁄ C122A) was exam- ined using Ellman’s method (Table 2). The single mutants, NOXtpC45A and NOXtpC122A, contained about one cysteine, and the double mutant contained no cysteines. These data confirmed the identity of the mutants and also indicated that the nonmutated cyste- ine remained in its native state. The visible absorption spectra showed that the three mutants contained tightly bound FAD (Fig. 4A, NOXtpC45A only shown – the other two mutants produced similar absorbance spectra). Electron microscopy and native PAGE showed no significant difference between wild- type NOXtp and its mutants (Fig. 5A,B). All of the data indicated that the disulfide bond was not respon- sible for hexameric oligomerization and that substitu- tion of Cys45 and Cys122 with alanine did not result in major changes in NOXtp quaternary structure. In order to determine the catalytic mechanism of NOXtp, NAD(P)H oxidase assays were performed Table 1. Steady-state kinetic parameters of NOXtp and NOX (CoA- DR) from Pyrococcus horikoshii (50 m M potassium phosphate buf- fer, pH 7.2, 75 °C). Data shown are means of triplicate determinations ± SD. Parameter NOXtp- NADH oxidase NOXtp- NADPH oxidase CoADR- NADH oxidase CoADR- NADPH oxidase K m (lM) 53.1 ± 2.8 12.1 ± 1.1 73 a 13 a k cat (s )1 ) 6.2 ± 0.5 2.5 ± 0.3 8.2 a 2.0 a a From reference [17]. Hexameric thermophilic NADH oxidase-producing H 2 O B. Jia et al. 5360 FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS with the three mutants under the same conditions as used for the wild-type. The results showed that the C122A mutant had similar NADH and NADPH oxi- dase activity to that of the wild-type protein; however, the C45A mutant and the double C45A ⁄ C122A mutant had < 10% of the NAD(P)H oxidase activity of the wild-type protein (Fig. 5C). These results are similar to those obtained with a NOX from E. faecalis, where a serine substitution of its active site residue Cys42 (C42S) resulted in approximately 3% of the activity of the wild-type under the same conditions [4,28]. Considering these results, Cys45 may provide the essential second redox center in addition to the fla- vin. We further examined the products of NOXtp and its mutants. NAD(P)H oxidation was allowed to go to completion, and the amount of H 2 O 2 formed in the reaction was quantified using the peroxi-DETECT kit. The NOXtpC122A mutant produced a similar amount of H 2 O as the wild-type under the same conditions and with the same substrates (Fig. 5D). Oxidation of NADH and NADPH by NOXtpC45A and NOX- tpC45A ⁄ C122A led to the formation of about one equivalent of H 2 O 2 (Fig. 5D), demonstrating that H 2 O 2 production by these two mutants is stoichiome- tric with NADH and NADPH oxidation. The activity and product assays using the wild-type and mutants clearly demonstrated that Cys45 participates in the direct four-electron reduction of O 2 to H 2 O, and the Cys45 mutation alters the reaction to produce H 2 O 2 instead of H 2 O. Discussion In this study, we have demonstrated that NOXtp has a hexameric ring-shaped structure. Gel filtration under nondenaturing conditions revealed that NOXtp is com- posed of six subunits. Moreover, upon electron micro- scopic analysis, NOXtp was found to predominantly exhibit a hexameric structure that contained a middle region of high density with heavy stain accumulation in its center. However, the crystal structure of NOX from L. sanfranciscensis revealed a dimeric form with an N-terminal oxidoreductase domain and a C-termi- nal dimerization domain [12]. NPX from Streptococ- cus faecalis, catalyzing the conversion of H 2 O 2 to H 2 O, was reported to be a homotetrameric structure [29]. These two mesophilic proteins show different types of subunit oligomerization and low sequence identity (Fig. S1), but each of their subunits shows high structural similarity and their folding patterns are similar to that of GR [12,29]. In contrast, NOX from Thermoanaerobium brockii was found to have a hexa- meric quaternary structure by gel filtration [15]. Elec- tron microscopic analysis has revealed that NOXtp has a hexameric ring-shaped structure composed of two 0 1 2 3 4 5 6 7 8 Specific activity (U·mg –1 ) 12 dcbadcba 100 nm 100 nm 100 nm100 nm a b c d 0 20 40 60 80 100 H 2 O 2 production (µmol) 12 dcbadcba 123 45 669 kDa 440 67 140 232 ABC D Fig. 5. Comparisons of activity, products and structures between NOXtp and the mutants. (A) Electron micrographs of NOXtp (a), NOX- tpC45A (b), NOXtpC122A (c) and NOXtpC45A ⁄ C122A (d). The bar represents 100 nm. (B) Native PAGE of wild-type NOXtp and its mutants. Lanes 1–4 correspond to (a), (b), (c) and (d) in (A), respectively; lane 5 is the molecular weight marker. The lower part shows the correspond- ing proteins determined by SDS ⁄ PAGE. (C) Specific activity of wild-type NOXtp (a), NOXtpC45A (b), NOXtpC122A (c) and NOX- tpC45A ⁄ C122A (d) with NADH (bar 1) and NADPH (bar 2) as substrates. (D) The amount of H 2 O 2 produced by NOXtp (a), NOXtpC45A (b), NOXtpC122A (c) and NOXtpC45A ⁄ C122A (d) when 100 l M NADH (bar 1) or 100 lM NADPH (bar 2) was oxidized. Table 2. Determination of the sulfhydryl contents of wild-type and mutant NOXtp using Ellman’s reagent. Data shown are means of triplicate determinations ± SD. Protein No. cysteines per protein NOXtp 1.84 ± 0.31 NOXtpC45A 0.85 ± 0.34 NOXtpC122A 0.84 ± 0.18 NOXtpC45A ⁄ C122A 0.14 ± 0.07 B. Jia et al. Hexameric thermophilic NADH oxidase-producing H 2 O FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS 5361 stacked rings of different diameters (19 and 15 nm respectively) that encompass a central opening; this is the first hexameric NOX determined by electron microscopy. Significantly, this structural feature of NOXtp is highly similar to that of valosine-containing protein-like ATPase from Th. acidophilum, an archaeal member of the AAA family (ATPases associated with a variety of cellular activities) [24]. In addition, the structure of the cysteine mutants, NOXtpC45A, NOX- tpC122A and NOXtpC45A ⁄ C122A, was the same as that of the wild-type. Thus, it appears that a disulfide bond does not participate in the oligomerization and quaternary structure of NOXtp. NADH oxidase catalyzes the transfer of electrons from reduced pyridine nucleotides to O 2 [2,4]. Here we have demonstrated that NOXtp can efficiently reduce O 2 to produce H 2 O using both NADH and NADPH as electron donors. In addition, the activity and prod- uct assays of the wild-type and mutants showed that Cys45 is the active site residue and that Cys122 does not function in the NADH and NADPH oxidase activity. These results indicate that Cys45 participates in the direct four-electron transfer reduction of O 2 to H 2 O, and that the Cys45 mutant alters the reduction to produce H 2 O 2 instead of H 2 O, similar to NOX in E. faecalis [28]. NOX in E. faecalis belongs to a group of enzymes that use a cysteine sulfenic acid as the non- flavin redox center. These enzymes are found in Enterococcus and Streptococcus, which are aerotolerant anaerobes, where they play an important role in O 2 tolerance [4]. For example, H 2 O-forming NOX-defi- cient mutants of Streptococcus pyogenes are unable to grow under high-O 2 conditions, revealing the impor- tance of NOX-scavenging activity against harmful O 2 [30]. We therefore propose that NOXtp may decom- pose O 2 in the anaerobe T. profundus. The predominant production of H 2 O by NOXtp is in contrast to the exclusive production of H 2 O 2 by most NOXtp homologs in thermophiles, such as NOX in A. fulgidus, Desulfovibrio gigas, Thermot. maritima and Thermoanaerobium brockii [10,11,15,31]. Previously, the production of H 2 O 2 was considered to be the dis- tinctive property of NOX proteins from thermophiles [10,11], with the exception of NOX from P. furiosus, which produces both H 2 O 2 (77%) and H 2 O (23%) [9]. To our knowledge, NOXtp is the first NOX to be purified from thermophilic microorganisms that can catalyze electron transfer from NADH and NADPH to O 2 and predominantly produce H 2 O. NOXtp is therefore better for removing O 2 than other reported O 2 -scavenging systems, which must employ intermedi- ates to reduce H 2 O 2 produced by NAD(P)H oxidases, such as in D. gigas, where rubredoxin and neelaredoxin act as intermediates [31]. As NOXtp and the mesophil- ic enzymes that decompose injurious O 2 belong to the same group (discussed above), and NOXtp reduces O 2 to H 2 O directly, we propose that NOXtp may play an important role in O 2 removal or aerobic tolerance in thermophilic anaerobes. Experimental procedures Purification of NOXtp from T. profundus Thermococcus profundus cells (8 L) were grown at 80 °Cas reported previously [32]. After harvesting, the cells were dis- solved in 20 mm potassium phosphate buffer (pH 6.5), con- taining 5 mm MgCl 2 , 0.5 mm EDTA, 1 mm dithiothreitol and 10% glycerol (PMEDG buffer), and disrupted by soni- cation. The homogenates were centrifuged at 10 000 g for 30 min. The supernatant was loaded on a phosphocellulose column that had been equilibrated with PMEDG buffer. After being washed completely, the proteins were eluted by 100, 200, 300, 400, 500 and 1000 mm NaCl in a stepwise gradient, and the eluates in 200 mm NaCl were dialyzed with 50 mm Tris buffer (pH 8.0) containing 400 mm NaCl. The sample was then loaded on an amino-benzimide col- umn equilibrated with the same buffer. Unabsorbed pro- teins on the resin were collected and dialyzed with PMEDG buffer, concentrated using a centricon (Millipore, Billerica, MA, USA), and stored at )80 °C. Eluates in all steps were checked by transmission electron microscopy. The protein concentration was determined by the Bradford method, and BSA was used as standard. SDS/PAGE and N-terminal sequencing The purified enzyme was subjected to SDS ⁄ PAGE and elec- troblotted onto poly(vinylidene difluoride) membranes. The visible band was excised and applied to a protein sequence analyzer (Korea Basic Science Institute, Daejeon, Korea). Cloning of NOXtp from T. profundus Polymerase chain reaction experiments with T. profundus genomic DNA as a template were performed using degener- ate oligonucleotides (sense primer, 5¢-GTA GTA ATA ATA GGA GGA GGA GCN GCN GGN ATG-3¢; anti- sense primer, 5¢-TAN ACT TTY TCN CAN SWN GTY TGC AT-3¢; N = A, G, C and T; Y = C and T; W = A and T). The sense primer was designed from the known N-terminal sequence, and the antisense primer was from the conserved C-terminal sequence of NOX. The experi- ment using the two oligonucleotides afforded an amplificate of  1.3 kb, which was ligated into the pTOPO vector (Invitrogen, Carlsbad, CA, USA), transformed, and confirmed by sequencing. The resulting sequence was used Hexameric thermophilic NADH oxidase-producing H 2 O B. Jia et al. 5362 FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS for subsequent cloning. Full gene cloning of NOXtp was per- formed using the Universal Genomewalker kit (ClonTech, Mountain View, CA, USA). Briefly, the genomic DNA was digested with EcoRV, DrabI, PvuII and SspI separately, and ligated to the adaptor provided by the kit. PCR was per- formed with the adaptor primers (provided by the kit) and tail-specific primers (N-terminal genomewalker primer, 5¢-TGG AGG TCT TTG CCG CGC TTT TTG AT-3¢; C-terminal genomewalker primer, 5¢-GGT GTG CAG GCT GTA AAT GCC GAG AT-3¢), which corresponded to the known sequence detected by the degenerate primers. The PCR products were ligated into the pTOPO vector, transformed, and sequenced. Expression and purification of NOXtp in Es. coli The new primers (sense primer, 5¢-CGCGCG CCA TGG AGAGGAAACGCGTTGTTAT-3¢; antisense primer, 5¢-CGCG AAGCTT TAAAACTTTAGAACCCTG-3¢)were designed on the basis of the sequence of the Genomewalker result (the underlined bases indicate the restriction enzyme site). The PCR product and pET28-(a) were digested by NcoI and HindIII and ligated. The ligation product was transformed into Es. coli BL21(DE3) by electroporation. Finally, the recombinant vector (pENOXtp) was confirmed by sequencing. Recombinant Es. coli cells (2 L) were cultured in LB broth to a D 600 nm of 1.0, and then induced with 1 mm iso- propyl-thio-b-d-galactoside for 4 h. After harvesting, the cells were resuspended in PMEDG buffer and disrupted by sonication. After centrifugation (3000 g, 30 min), the super- natants were heated at 65 °C for 30 min, and then the denatured proteins were removed by centrifugation (3000 g, 30 min). The supernatants were loaded onto a phosphocel- lulose column that had been equilibrated with the same buffer. After being washed completely, the proteins were eluted with 200 mm NaCl. The purified protein was checked by SDS ⁄ PAGE and dialyzed with PMEDG buffer, concentrated, and stored at )80 °C. Mutagenesis The primers used for the single cysteine to alanine mutants were as follows: C45A, forward primer, 5¢-ACG GAA TGG GTG AGC CAC GCT CCC GCC GGT ATC CCC TAC GTA GTT GAG GGT-3¢; C45A, reverse primer, 5¢-ACC CTC A AC TAC GTA GGG G AT ACC GGC GGG AGC GTG GCT CAC CCA TTC CGT-3¢; C122A, for- ward primer, 5¢-CCG CAG GTT CCG GCG ATA GAG GGC GCC CAC CTG GAA GGA GTA TTC ACA GCA- 3¢; and C122A reverse primer, 5¢-TGC TGT GAA TAC TCC TTC CAG GTG GGC GCC CTC TAT CGC CGG AAC CTG CGG-3¢. The PCR was performed using Pfu polymer- ase (Takara, Kyoto, Japan), and the cycling parameters were: 95 °C for 5 min (one cycle), 95 °C for 30 s, and 68 ° C for 12 min (12 cycles). After amplification, the PCR mixture was digested with DpnI and then transformed into Es. coli BL21(DE3) by electroporation. The mutants were confirmed by DNA sequencing. The double cysteine mutants were produced by the same method, except that pENOXtpC45A was used as the template and C122A primers were used for the amplification. The mutant proteins were purified using the same method as used for wild-type purification. Gel filtration chromatography The sample (1 mgÆmL )1 ) was loaded onto a Superdex-200 column (Amersham Biotech, Piscataway, NJ, USA). Stan- dard proteins included ferritin (440 kDa), catalase (232 kDa), albumin (67 kDa) and ovalbumin (43 kDa). Apo-NOX preparation The purified NOXtp from Es. coli is a holoenzyme with FAD. The protein was dialyzed with 100 mm phosphate buffer (pH 7.2) containing 2.4 m (NH 4 ) 2 SO 4 ,1mm dith- iothreitol and 0.5 mm EDTA, and then loaded on the hydrophobic interaction chromatography column equili- brated with the same buffer. FAD was eluted with equili- bration buffer saturated with NaBr (pH 3.5). The column was balanced again with the equilibration buffer, and the apoprotein was eluted with 100 mm phosphate buffer [26,27]. Eluates were dialyzed with the PMEDG buffer described above, and stored at )80 °C. Enzyme assays The NADH or NADPH oxidase activity of the recombi- nant protein was examined by time-dependent removal of NAD(P)H in aerobic conditions. The assays were per- formed in 50 mm sodium or potassium phosphate buffer (pH 7.2), 0.5 mm NAD(P)H and 100 mm NaCl at the indi- cated temperatures. The reaction was started by adding NOXtp in the amounts indicated. The rate of NAD(P)H consumption was measured by monitoring the decrease in A 340 nm . One unit of activity was defined as the amount of enzyme catalyzing the oxidation of 1 lmol NADH per min at 75 °Cin50mm potassium phosphate buffer (pH 7.2) and 0.5 mm NADH. To measure kinetic parame- ters, reaction rates were measured at a series of NAD(P)H concentrations, and the rates at various substrate concen- trations were finally fitted by Lineweaver–Burk plots. The parameters (with standard deviation) were determined by three separate experiments. Determination of the sulfhydryl content The sulfhydryl contents were determined using Ellman’s reagent in anaerobic conditions according to a published B. Jia et al. Hexameric thermophilic NADH oxidase-producing H 2 O FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS 5363 method [17,33]. After the proteins and 5,5¢-dithiobis- (2-nitrobenzoic acid) (Nbs 2 ) were incubated for 15 min at 25 °C, A 412 nm was monitored to estimate the number of cysteine residues present as protein ⁄ Nbs 2 mixed disulfide. The sulfhydryl concentrations in these proteins were deter- mined from a calibration curve created using known con- centrations of standard l-cysteine solutions. H 2 O 2 detection H 2 O 2 was detected using the PeroxiDetect Kit (Sigma). Briefly, the assay was performed in 50 mm sodium phos- phate buffer (pH 7.2), 100 lmol NAD(P)H, 1 mm EDTA, 100 mm NaCl and 0.2 nmol NOXtp. The reaction was allowed to go to completion. Reaction buffer (100 lL) was added to the kit. Peroxides convert Fe 2+ to Fe 3+ ions under acidic conditions. Fe 3+ ions will then form a colored adduct with xylenol orange, which is observed at 560 nm. NAD(P)H will interfere with the H 2 O 2 assay, so all of the NAD(P)H must be consumed completely. Electron microscopy and image processing Purified NOX was applied to glow-discharged carbon- coated copper grids. After the proteins had been allowed to absorb for 1–2 min, the grids were rinsed with droplets of de-ionized water, and stained with 2% (w ⁄ v) uranyl acetate. Electron micrographs were recorded with an FEI TECH- NAI 12 microscope at a magnification of ·51 600 (nominal magnification of ·52 000) and an acceleration voltage of 120 kV. Light-optical diffractograms were used to select the micrographs, to examine the defocus and to verify that no drift or astigmatism was present. Suitable areas were digi- tized as arrays of 1024 · 1024 pixels with leaf scan 45 at a pixel size of 20 lm, corresponding to 0.38 nm at the spec- imen level. For image processing, the semper [34] and em [35] software packages were used. From digitized micro- graphs, smaller frames of 64 · 64 pixels containing individ- ual particles were extracted interactively. These images were aligned translationally and rotationally, using standard cor- relation methods [36,37]. An arbitrarily chosen reference was used for the first cycle of alignment and averaging, and the resulting average was used as a reference in the second refinement cycle. For analysis of the rotational symmetry of top-on-view images, the individual images were aligned translationally but not rotationally [38]. These aligned images were subjected to multivariate statistical analysis [39]. The resulting eigenimages represent all-important structural features of the original dataset. If the images had different rotational symmetries in the original dataset, the eigenimages would reveal the different symmetry axes. Moreover, these images can be distinguished and subse- quently separated on the basis on eigenimages. The rota- tionally aligned images were classified on the basis of eigenvector–eigenvalue data analysis, and subsequent aver- aging was performed for each class separately. The average was finally symmetrized on the basis of angular correlation coefficients [40]. Acknowledgements B. Jia, S. Lee, B. P. Pham, R. Yu and T. L. Le were supported by scholarships from the Brain Korea21 project in 2008, Korea. This work was supported by a grant from the MOST ⁄ KOSEF to the Environmental Biotechnology National Core Research Center (grant no. R15-2003-012-01003-0), and the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (grant no. KRF-2007-521-C00241), to G. W. Cheong. References 1 Fukui T, Atomi H, Kanai T, Matsumi R, Fujiwara S & Imanaka T (2005) Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraen- sis KOD1 and comparison with Pyrococcus genomes. Genome Res 15, 352–363. 2 Jenney FE Jr, Verhagen MF, Cui X & Adams MW (1999) Anaerobic microbes: oxygen detoxification with- out superoxide dismutase. Science 286, 306–309. 3 Miyoshi A, Rochat T, Gratadoux JJ, Le Loir Y, Oliveira SC, Langella P & Azevedo V (2003) Oxida- tive stress in Lactococcus lactis. Genet Mol Res 2, 348–359. 4 Argyrou A & Blanchard JS (2004) Flavoprotein disul- fide reductases: advances in chemistry and function. 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W, Dahlmann B, Hegerl R, Kopp F, Luehn L & Pfeifer G (1988) Electron microscopy and image analysis of the multicatalytic proteinase FEBS Lett 241, 239–245 37 Kim KI, Cheong GW, Park SC, Ha JS, Woo KM, Choi SJ & Chung CH (2000) Heptameric ring structure of the heat-shock protein ClpB, a protein-activated ATPase in Escherichia coli J Mol Biol 303, 655–666 38 Marco S, Urena D, Carrascosa JL, Waldmann . was also measured. These assays revealed that NADH oxidase activity was slightly higher than that of NADPH oxidase, and FAD significantly restored the oxidase. Hexameric ring structure of a thermophilic archaeon NADH oxidase that produces predominantly H 2 O Baolei Jia 1, *, Seong-Cheol Park 1, * , , Sangmin