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1 Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
2 Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
3 Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
Correspondence
Biswarup Mukhopadhyay
biswarup{at}vt.edu
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ABSTRACT |
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10 and 95 °C, respectively. At pH 7 and 83 °C, apparent Km values for NADH and O2 were 3 µM and 1.9 mM, respectively, and the specific activity at 1.4 mM O2 was 60 µmol min–1 mg–1; 62 % of NADH-derived reducing equivalents were recovered as H2O2 and the rest probably generated H2O. rMjNox had poor NADPH oxidase, NADH peroxidase and superoxide formation activities. It reduced ferricyanide, plumbagin and 5,5'-dithiobis(2-nitrobenzoic acid), but not disulfide coenzyme A and disulfide coenzyme M. Due to a high Km, O2 is not a physiologically relevant substrate for MJ0649; its true substrate remains unknown.
Present address: Yale University School of Medicine, Section of Microbial Pathogenesis, 295 Congress Avenue, BCMM 345, New Haven, CT 06536, USA.
A supplementary table describing the purification of rMjNox is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Jones et al., 1983). This strictly autotrophic anaerobe was isolated from a deep-sea hydrothermal vent (Jones et al., 1983). The genome of M. jannaschii carries an ORF (MJ0649) that has been annotated as an NADH oxidase (Nox) homologue (Bult et al., 1996) (Fig. 1), and such ORFs are present in most methanogens (Bult et al., 1996; Hendrickson et al., 2004; Smith et al., 1997). Methanogens are strict anaerobes (Boone et al., 1993). All anaerobic non-methanogenic archaea examined thus far also carry Nox homologues (Klenk et al., 1997; Maeder et al., 1999; Robb et al., 2001). Since the Noxs of bacteria and eukaryotes catalyse the reduction of molecular oxygen (O2) by either a two-electron transfer to produce hydrogen peroxide (H2O2) or a four-electron transfer to produce H2O (Claiborne et al., 1993; Toomey & Mayhew, 1998), it has been a puzzle why strictly anaerobic archaea carry Nox homologues. For this reason the Nox homologues of several non-methanogenic archaea have been investigated (Kengen et al., 2003; Reed et al., 2001; Ward et al., 2001). These investigations considered the fact that some of the Nox proteins transfer electrons to peroxides, disulfides and a variety of membrane-associated acceptors (Kengen et al., 2003; Reed et al., 2001; Ward et al., 2001). Some of these non-methanogenic archaea might use Nox homologues to counter the toxic effect of the molecular oxygen that they could be exposed to in their natural habitats (Kengen et al., 2003; Ward et al., 2001). Also, in Pyrococcus horikoshii and Pyrococcus furiosus, a Nox homologue has been demonstrated to act as a coenzyme A disulfide reductase (CoADR) (Harris et al., 2005) as well as sulfur reductase (Schut et al., 2007). None of the methanogen Nox homologues have been investigated for their catalytic or in vivo functions. A related enzyme, NADPH-dependent disulfide coenzyme M reductase (CoMDR), has been isolated from Methanothermobacter thermautotrophicus (Smith & Rouviere, 1990); however, the gene for this enzyme is unknown. As a result it is unclear whether the nox homologue of M. jannaschii (mj0649) represents a disulfide reductase. Also, in contrast to non-methanogenic anaerobic archaea, which carry three to nine nox homologues (Klenk et al., 1997; Maeder et al., 1999; Robb et al., 2001), M. jannaschii carries only one such ORF (Pagala et al., 2002). Methanothermobacter thermautotrophicus, Methanococcus maripaludis, Methanosarcina acetivorans and Methanosarcina barkeri, which are also methanogens, carry one, one, two, and one Nox homologues, respectively (Galagan et al., 2002; Hendrickson et al., 2004; http://genome.ornl.gov/microbial/mbar/; Smith et al., 1997). Therefore, we are interested in elucidating the role of mj0649, the nox homologue of M. jannaschii. To begin in this direction, we have expressed this ORF in Escherichia coli and determined the molecular and kinetic characteristics of the recombinant protein.
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METHODS |
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), and E. coli C41(DE3) (Miroux & Walker, 1996) carrying pRIL (Novagen) was used for the expression of recombinant Nox. These strains were grown in Luria–Bertani (LB) medium. During the cloning experiments, E. coli transformants were selected on plates or grown in liquid medium containing 100 µg ampicillin ml–1. For the expression of recombinant Nox, E. coli transformants were grown in liquid medium containing 100 µg ampicillin ml–1 and 25 µg chloramphenicol ml–1.
Construction of an overexpression plasmid.
Generally, all DNA manipulations were performed according to standard methods (Sambrook et al., 1989). mj0649 coding sequence was amplified from M. jannaschii genomic DNA with DeepVentR DNA polymerase (New England Biolabs) using the oligonucleotide primers MJ0649xp/1F (5'-GGGGATCCAATGAGAGCAATAA TAATAGGAAGTGGAGCTG-3') and MJ0649xp/2R (5'-CATAAACTGCAGCAATTATTTA TTACTCAACTTTTTCAAAGC-3'); the underlined sequences indicate engineered BamHI and PstI restriction sites, respectively. The resulting PCR product was cloned into the EcoRV site of pBluescript II SK(+) (Stratagene,) to obtain the plasmid pMJ0649-1, and the cloned mj0649 gene was determined to be free of mutation through sequencing of both of its strands. The mj0649 insert of pMJ0649-1 was excised by restriction digestion with BamHI and PstI and ligated to the similarly digested pETDuet1 vector (Novagen) to obtain pMJ0649-2. In pMJ0649-2, the mj0649 coding sequence was fused to an NH2-terminal His6 sequence and placed under the control of the T7 promoter. E. coli C41(DE3)(pRIL) was transformed with pMJ0649-2 for the expression of recombinant Nox (rMjNox).
Expression and purification of rMjNox.
E. coli C41(DE3)(pRIL)(pMJ0649-2) was grown to an optical density of 0.6–0.8 (as measured using a model Lambda 25 UV-visible spectrophotometer, PerkinElmer instruments), followed by induction with 1 mM IPTG. After 3 h of induction, cells were harvested by centrifugation at 7000 g for 10 min at 4 °C. The resulting 3 g cell pellet was resuspended in 3 ml 100 mM potassium phosphate buffer, pH 7.0. The cell suspension was passed three times through a French pressure cell at a pressure of 1.28x108 Pa. The resulting cell lysate was centrifuged at 18 000 g for 1 h at 4 °C to remove cell debris. The supernatant from this step was heated to 80 °C for 15 min and then centrifuged at 12 000 g for 10 min to remove the denatured proteins. The resulting 3.5 ml of supernatant was diluted to 7.0 ml with a solution containing 600 mM NaCl and 20 mM imidazole, pH 7.0, to give the following composition: 50 mM potassium phosphate buffer, pH 7.0, 300 mM NaCl, 10 mM imidazole, and cell extract. This solution was loaded onto a 3 ml column bed (13 mm diameter) of nickel-nitrilotriacetic acid (Ni-NTA) Superflow agarose (Qiagen) that was pre-equilibrated with a 15 ml solution containing 50 mM potassium phosphate buffer, pH 7.0, 300 mM NaCl and 10 mM imidazole (buffer A). The column was washed with 15 ml buffer A, and then, to remove weakly bound contaminants, the column was further washed with 3 ml 0.5 M NaCl, 3 ml 1 M NaCl and 6 ml 2 M NaCl, sequentially; each wash contained 50 mM potassium phosphate buffer, pH 7.0, and 10 mM imidazole. To lower the salt concentration in the column, a final wash with 3 ml buffer A was performed. rMjNox was eluted from the column with a 60 ml linear gradient of 10–300 mM imidazole containing 50 mM potassium phosphate buffer, pH 7.0, and 300 mM NaCl. Fractions at about 250 mM imidazole contained most of the Nox activity and were pooled and concentrated on a YM-30 membrane (Amicon). The protein retained on the membrane was washed three times, each time with 5 ml 25 mM potassium phosphate buffer, pH 7.0, and recovered in 1 ml of the same buffer. The concentrated and desalted protein was loaded onto a 4 ml column bed (13 mm diameter) of Cibacron Blue 3GA agarose (Sigma-Aldrich) pre-equilibrated with 20 ml 25 mM potassium phosphate buffer, pH 7.0. The column was washed with 20 ml 25 mM potassium phosphate buffer, pH 7.0, and 50 mM NaCl, and rMjNox was eluted by sequential addition of 4 ml each of 100, 200, 300, 400, 500, 750 and 1000 mM NaCl containing 25 mM potassium phosphate buffer, pH 7.0. rMjNox activity was found in fractions containing 750 and 1000 mM NaCl. These fractions were pooled, concentrated, desalted and placed in 1 ml 25 mM potassium phosphate buffer, pH 7.0 as described above. This concentrate contained 1 mg homogeneous rMjNox.
Expression and purification of recombinant form of P. furiosus rubredoxin (rPfRd).
E. coli NCM533 carrying pPfRd1 (Jenney & Adams, 2001), a plasmid allowing the expression of P. furiosus rubredoxin (PfRd) under the control of a T7 promoter, was obtained from Drs Francis E. Jenney, Jr, and Michael W. W. Adams, University of Georgia. The strain was transformed with pRIL. The cultivation of E. coli NCM533 (pPfRd1)(pRIL), the induction of the expression of PfRd, and the harvesting of the cells were carried out as described in the preceding section. E. coli cells carrying overexpressed PfRd from a 1 l culture were resuspended in 4 ml of a solution containing 150 mM NaCl and 25 mM potassium phosphate buffer, pH 7 (Solution B). The cells in the suspension were lysed and the resulting extract was heat-treated as described above for the purification of rMjNox. The heat-treated cell extract supernatant was then loaded onto a 50 ml 1.5x27 cm column bed of Sephadex G-200 (Pharmacia), which was pre-equilibrated with solution B. The column was then developed with solution B. The elution of rPfRd was followed by its characteristic deep burgundy colour. SDS-PAGE analysis showed that a pool of the coloured fractions was a homogeneous preparation of a protein with denatured molecular mass of 6 kDa, which corresponded well to PfRd (Jenney & Adams, 2001).
SDS-PAGE, size-exclusion chromatography, and protein assay.
SDS-PAGE was performed according to Laemmli (1970) and protein assays were performed according to Bradford (1976) using reagents from Bio-Rad Laboratories. Size-exclusion chromatography was performed as described previously (Patel et al., 2004) using molecular mass and size standards from Bio-Rad Laboratories with the following characteristics (standard, mass, Stokes radius; Mukhopadhyay & Purwantini, 2000): bovine thyroglobulin, 670 kDa, 85 Å (8.5 nm); bovine gamma globulin, 158 kDa, 52.5 Å (5.25 nm); chicken ovalbumin, 44 kDa, 30.5 Å (3.05 nm); horse myoglobin, 7 kDa, 19 Å (1.9 nm); vitamin B12, 1.357 kDa.
Preparation of disulfide coenzyme M and coenzyme A.
Disulfide coenzyme M (CoM-S-S-CoM) was prepared by dissolving 1 g 2-mercaptoethanesulfonate (HS-CoM) in 10 ml aqueous 30 % NH4OH and bubbling air through this solution for 72 h (Smith & Rouviere, 1990). The resulting disulfide coenzyme M solution was evaporated at 70 °C to dryness under vacuum in a rotary evaporator (model R-205, Büchi Labortechnik). This product was determined to be free of coenzyme M via an assay for free thiols (Ellman, 1958; Smith & Rouviere, 1990). Disulfide coenzyme A (CoA-S-S-CoA) was purchased from Sigma-Aldrich.
Nox assays.
Assays were conducted in round glass cuvettes sealed with a cut-off butyl rubber stopper (Daniels & Wessels, 1984). The gas atmospheres in the tubes were manipulated via one of the following three methods. (I) For a moderate level (0.5–0.72 mM) of dissolved oxygen, a tube containing assay mixture was sealed under air and then pressurized with 100 % O2 to a desired total pressure. (II) For a low level (0.066–0.33 mM) of dissolved O2, a sealed tube was made anaerobic via evacuation and pressurization with N2 as described previously (Daniels & Wessels, 1984; Mukhopadhyay & Daniels, 1989). The final pressure was 1.2x102 kPa. Then, O2 was added to this tube to the desired pressure. (III) To provide a high level of dissolved O2 (0.96–1.4 mM), the assay tubes were evacuated and pressurized with O2 to the desired partial pressure. The concentration of dissolved O2 was calculated from Henry's relationship: 
, where pO2 is the partial pressure of O2 in atm, 
is the mole fraction of dissolved O2, and 
is Henry's constant. From the available data in the 0–100 °C range (Liley et al., 1984), the value of at 83 °C was calculated to be 69 000 atm.
In most cases, prior to assays, the enzyme was incubated for 30 min at room temperature with FAD. The composition of this incubation mixture was 0.1 mg enzyme ml–1, 25 mM potassium phosphate buffer, pH 7.0, and 0.1 mM FAD. Standard Nox assays were performed at 83 °C with oxygen as the electron acceptor in a 1 ml reaction mixture containing 50 mM HEPES-NaOH buffer, pH 7.0, 0.15 mM NADH, 1.4 mM dissolved O2 [supplied by method (III), as described above] and 1 µM FAD (carried over from the enzyme stock). For pH studies the HEPES–NaOH buffer was replaced with constant ionic strength buffers containing 100 mM Tris, 50 mM glacial acetic acid and 50 mM MES (Mukhopadhyay et al., 2000). The progress of the NADH oxidase reaction was followed spectrophotometrically at 340 nm. The initial velocity rates were calculated using a value of 6.22 mM–1 cm–1 for the absorption coefficient of NADH at 340 nm and were corrected for the chemical oxidation of NADH as measured in the absence of enzyme.
Other electron acceptors.
For experiments involving electron acceptors other than oxygen, all assay tubes and assay components were made anaerobic by evacuating and pressurizing with nitrogen (1.4x102 kPa) three times. Additions of substrates were made from aqueous anaerobic stocks using an anaerobic syringe fitted with a needle (Daniels & Wessels, 1984; Mukhopadhyay & Daniels, 1989). For some of the electron acceptors, the progress of the reaction was followed at wavelengths other than 340 nm, and the initial rates were calculated by using the following absorption coefficient values (compound, assay wavelength, absorption coefficient): plumbagin, 419 nm, 3.95 mM–1 cm–1 (Rothery et al., 1998); ferricyanide, 420 nm, 1.00 mM–1 cm–1 (Kengen et al., 2003); 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 412 nm, 13.6 mM–1 cm–1 (Kengen et al., 2003); recombinant P. furiosus rubredoxin (rPfRd), 494 nm, 9.22 mM–1 cm–1 (Ma & Adams, 2001).
Determination of H2O2.
Hydrogen peroxide produced in an NADH oxidase assay was assayed as outlined in Ward et al. (2001) but with some modifications as detailed here. An NADH oxidase assay mixture containing 110 µM NADH and 1.4 mM dissolved O2 was incubated at 83 °C until the A340 of the solution was <0.01, indicating that almost all of the NADH had been oxidized. Two-hundred microlitres of this solution was combined with 800 µl 125 mM potassium phosphate buffer, pH 7. The mixture was allowed to cool to room temperature and was then combined with 150 µl of a solution containing 2-2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Sigma-Aldrich; 22 mg ml–1) and 100 units horseradish peroxidase ml–1 (Sigma-Aldrich). After incubation at room temperature for 30 min, the A725 was measured. From this absorbance value and a standard curve, the amount of H2O2 produced in the assay was determined. The standard curve was generated by assaying 0–30 µM H2O2 solutions that were prepared by diluting a 3 % (v/v) stock (Cumberland Swan Holdings) in 100 mM potassium phosphate buffer, pH 7. A control NADH oxidase assay mixture containing added H2O2 to 5.4 µM, but lacking NADH, was processed through heating, cooling and mixing steps similar to those described above. This mixture showed a 4 % loss in H2O2 during processing and this loss was considered in calculating the amount of H2O2 produced in the NADH oxidase assay.
Assay of superoxide production.
Superoxide formation was measured by the reduction of nitro blue tetrazolium (NBT) to formazan (Hillar & Loewen, 1995). The assay was conducted at 83 °C and various pHs (6–10) in a 0.8 ml reaction mixture with 1.4 mM O2, 200 µM NBT, 150 µM NADH, and the constant ionic strength buffer described above in Methods, Nox assays. The reaction was initiated by the addition of enzyme. The reduction of NBT was monitored at 560 nm. An absorption coefficient of 15 mM–1 cm–1 for the formazan product at 560 nm (Hillar & Loewen, 1995) was used to calculate the initial velocity.
Analysis of kinetic data.
All initial rate data were analysed according to Cleland (1979) using KinDist, a PC graphics program obtained from Professor Bryce V. Plapp, University of Iowa. The data were fitted to the Henri–Michaelis–Menten relationship: v=VmaxxS/(Km+S).
Determination of the identity and content of flavin.
The identity of the flavin bound to purified rMjNox was determined according to Mayhew & Massey (1969), with some modifications. During the following steps, exposure of the sample to light was avoided as much as possible. To extract flavin from the enzyme, 0.05 ml of a solution of rMjNox (1 mg ml–1) was mixed with 5.5 µl 50 % TCA. The mixture was held on ice for 5 min and then centrifuged at 10 000 g for 10 min to pellet the precipitated protein. The resulting supernatant of 50 µl was neutralized with 20 µl 1 M K2HPO4. This product was analysed via TLC on silica gel 60 F254 (Merck) using 1-butanol : acetic acid : water (10 : 5 : 5) as the resolving solvent. The resolved bands were viewed under a hand-held short-wavelength UV light. Solutions (0.1 mM) of FAD and FMN in 5 % TCA and 5 % K2HPO4 were used as standards.
The quantity of FAD bound per mole of rMjNox was determined as follows. For this work the following two homogeneous enzyme preparations were analysed: as isolated and FAD-incubated. The latter preparation was generated by incubating 300 µg purified protein with excess FAD (0.2 mM) at 4 °C for 5 h. Free flavin was removed by filtration and three 1 ml washes with 50 mM potassium phosphate buffer, pH 7.0, on the membrane of a YM-10 concentrator (Amicon). The product was recovered in 50 mM potassium phosphate buffer, pH 7.0, and assayed for protein content. From each of these two preparations, bound flavin was extracted as described in the preceding paragraph, except that the pellet obtained after the first centrifugation step was resuspended in 50 µl 5 % aqueous TCA and recentrifuged to recover an additional supernatant that was combined with that from the first centrifugation. Flavin in this combined supernatant was quantified by measuring the A450. The calculation was based on an absorption coefficient value of 10.6 mM–1 cm–1 for FAD in 5 % aqueous TCA as determined from the reported value of 11.3 mM–1 cm–1 at pH 7 (Dawson et al., 2002).
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RESULTS |
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, lane 2). When a cell lysate of E. coli C41(DE3)(pRIL)(pMJ0649-2) containing overexpressed rMjNox was centrifuged at 18 000 g for 1 h at 4 °C, the recombinant protein was almost exclusively found in the supernatant. This conclusion was based on an SDS-PAGE analysis (data not shown). Therefore, rMjNox was expressed in E. coli as a soluble protein. The 18 000 g supernatant exhibited NADH oxidase activity. However, a supernatant similarly obtained from E. coli C41(DE3)(pRIL) also had NADH oxidase activity. When the two cell extract preparations were heat-treated at 80 °C the resulting supernatant of E. coli C41(DE3)(pRIL)(pMJ0649-2), but not that of E. coli C41(DE3)(pRIL), contained the NADH oxidase activity. These results established that overexpressed MJ0649 carrying a His6-tag had NADH oxidase activity. From a heat-treated cell extract supernatant, rMjNox was purified to homogeneity through Ni-NTA and Cibacron Blue agarose chromatography steps (Fig. 2b). Typically, from a wet cell paste corresponding to a dry cell weight of 0.3 g, up to 2 mg of rMjNox (Supplementary Table S1) with specific NADH oxidase activity of 36–60 units mg–1 was obtained. The kinetic data reported in this paper were generated with a preparation exhibiting a specific activity of 60 units mg–1. At a later stage of this work it was found that chilling of the heat-treated extract on ice for 2 h prior to its centrifugal clarification led to the generation of homogeneous enzyme at the Ni-NTA chromatography step, and therefore removed the need for the Cibacron Blue agarose chromatography step.
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) showed that rMjNox had a subunit molecular mass of 
50 kDa. This value is consistent with the nucleic acid sequence-derived mass of 50.5 kDa for the recombinant protein. In size-exclusion chromatography on a HiPrep 16/60 Sephacryl S-300 HR column (Amersham Pharmacia Biotech), the homogeneous enzyme exhibited a single peak. From the retention time in this column, the apparent molecular mass of the native protein was determined to be 86±2 kDa. This value could indicate that the protein was a compact dimer or a monomer with a non-globular structure. With the latter possibility, the hydrodynamic radius of the protein would be larger than would be expected for a globular protein of equivalent mass. These possibilities will be investigated in the future through a more accurate method such as analytical ultracentrifugation (Cantor & Schimmel, 1980).
Flavin content of rMjNox
The UV-visible spectra of the enzyme as isolated from Ni-NTA chromatography as well as after incubation with FAD were characteristic of a flavoprotein (Fig. 3a, b). The absorbance at >500 nm exhibited by rMjNox has been seen with other group 3 flavin-dependent disulfide reductase (FDR) Nox enzymes, where it is indicative of a weak charge-transfer interaction between the active Cys thiolate and FAD (Ahmed & Claiborne, 1989a, b; Mallett & Claiborne, 1998). The absorbance peaks at 368 and 443 nm indicated that the enzyme contained bound flavin. TLC analysis of a TCA extract of the protein as isolated identified the bound flavin to be FAD. Further analysis showed that rMjNox as isolated contained 0.55±0.02 mole FAD per mole of the dimeric protein. After incubation with excess FAD, the flavin content value of rMjNox increased to 1.9±0.2 moles per dimer.
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The response of the NADH oxidase activity of rMjNox to changes in pH is shown in Fig. 4(a). In the pH range 6.0–7.5, the enzyme showed an activity peak at pH 7.0 (Fig. 4a). However, a rise in pH above 8.0 increased activity steadily and to substantially higher values. The activity at pH 10 was 178 µmol min–1 mg–1, which was nearly threefold higher than that at pH 7.0. Activities at pH higher than 10.0 were not determined. rMjNox showed significant activity over a broad temperature range (Fig. 4b). The optimum temperature was >95 °C (Fig. 4b); activities at higher temperatures were not determined due to technical limitations. From the straight-line section of the Arrhenius plot corresponding to the range 25–65 °C, the activation energy of the rMjNox-catalysed NADH oxidase reaction was determined to be 46 kJ mol–1.
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). However, the enzyme was extremely sensitive to NADH concentration (Fig. 5b). When dissolved O2 was fixed at 1.4 mM (subsaturating), rMjNox showed an apparent Km of 3 µM for NADH. The apparent Vmax value for these assays was 66 units mg–1. The enzyme also oxidized NADPH with oxygen. However, at dissolved O2 and NADPH concentrations of 1.4 and 0.15 mM, respectively, the activity of rMjNox was only 3 units mg–1, a value 95 % lower than that obtained with 0.15 mM NADH. Since the chemical breakdown of NADPH at 83 °C was faster than the enzymic reaction, a detailed kinetic analysis for the NADPH oxidation activity was not pursued.
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) showed that rMjNox produced a minor amount of superoxide while oxidizing NADH with oxygen. For the reason given in the Discussion, this activity was measured in the pH range 6–10. As shown in Fig. 4(a), superoxide formation activity reached a plateau at pH 7–8.5, and at higher pH values it increased about 2.5-fold above this plateau value. Although the nature of the pH profile for the superoxide formation activity was not exactly similar to that for the NADH oxidation activity, the fold increases between pH 7 and 10 were similar for both.
Alternate electron acceptors of rMjNox
The ability of rMjNox to use electron acceptors other than oxygen was tested. These assays were performed in the absence of oxygen and with NADH as the electron donor and the results are shown in Table 1. rMjNox catalysed the peroxidase reaction, although the value of this activity was much lower than that of the NADH oxidase activity. It exhibited high activity with ferricyanide and plumbagin, which have been used as artificial electron acceptors for enzyme assays. rMjNox also reduced DTNB, a disulfide compound and an artificial electron acceptor. However, no activity was seen with disulfide coenzyme A, disulfide coenzyme M or P. furiosus rubredoxin as electron acceptor.
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; Claiborne et al., 1999). We discuss this finding below. In the literature, a Nox enzyme from P. furiosus has been called Nox1 (Ward et al., 2001) and NoxA-3 (Maeder et al., 1999; Robb et al., 2001); we refer to this enzyme as NoxA-3.
Group 3 of the FDR family includes NADH oxidases (Nox), NADH peroxidases (Npx) and CoADRs (Argyrou & Blanchard, 2004; Claiborne et al., 1999) (Fig. 1). These enzymes use a reactive cysteine that lies within a conserved SFXXC element and form a Cys-sulfenic acid or Cys-SOH in the catalytic cycle (Claiborne et al., 1999). They also possess FAD and NADH binding sites (Fig. 1) (Argyrou & Blanchard, 2004; Claiborne et al., 1999). MJ0649 and its archaeal homologues possessed all these attributes and additionally certain distinct features (Fig. 1). In these proteins, the reactive Cys-bearing sequence element of MJ0649 (39YSPC42AIPY46) was more strictly conserved than that seen in their bacterial homologues (Fig. 1). Also, MJ0649 had CoADR-type sequence features. In the CoADR of Staphylococcus aureus the Tyr361 and Tyr419 residues of the 361YYPG364 and 419YAPP422 elements help to stabilize Cys43-thiolate, and during the CoA-disulfide reduction, Tyr361 is the primary player in the protonation of the CoASH thiolate (Mallett et al., 2006). These Tyr residues and the corresponding sequence elements were almost fully conserved in archaeal Nox homologues, but are absent in the Enterococcus faecalis Npx and Nox (Fig. 1). Also, the Arg395 and Asp428 of S. aureus CoADR, which further differentiate this enzyme from Ent. faecalis Npx and Nox (Mallett et al., 2006), were almost fully conserved in the archaeal proteins and corresponded to Arg399 and Glu432 of MJ0649 (Fig. 1). However, rMjNox did not reduce the disulfide of coenzyme A or disulfide of coenzyme M, but oxidized NADH with molecular oxygen (Table 1). These mixed properties make MJ0649 an unusual member of the FDR family. Interestingly, when tested with DTNB, a disulfide compound of no physiological relevance, rMjNox showed significant disulfide reductase activity. MJ0649 exhibits 62 % identities and 16 % strong similarities to a putative NAD(P)H : rubredoxin oxidoreductase of Methanococcus maripaludis (MMP1259) (Fig. 1). Since a clone of P. furiosus rubredoxin (PfRd; ORF PAF1282) is available (Jenney & Adams, 2001), the ability of rMjNox to reduce PfRd was tested. PfRd shows high homologies to putative M. jannaschii rubredoxins (49 % identities and 19 % strong similarities to MJ0740, and 33 % identities and 11 % strong similarities to MJ0735). However, rMjNox did not reduce recombinant PfRd (Table 1).
In its subunit size, flavin content (1 mole per subunit), and requirement for FAD for activity as established through an enhancement of activity via incubation with this coenzyme, rMjNox was similar to most group 3 FDR enzymes (Table 2). The described group 3 FDR enzymes contain one FAD per subunit (Argyrou & Blanchard, 2004; Claiborne et al., 1999) and transfer electrons from nicotinamides via flavin for the purpose of oxygen, peroxide or disulfide coenzyme A reduction (Argyrou & Blanchard, 2004; Claiborne et al., 1999). The observed temperature optimum for the activity of rMjNox (95 °C) was consistent with the growth temperature range (48–94 °C) and optimal growth temperature (85 oC) for the host organism (Jones et al., 1983). The pH versus activity data for rMjNox showed two activity peaks (at pH 7.0 and pH >10.0) and the NADH oxidase activity dramatically increased as pH was increased above 7.0 (Fig. 4a). It is possible that in the pH ranges of 
7.0 and >7.0, two different sets of amino acid residues of the protein play critical roles in catalysis. Another explanation is that at pH >7.0, rMjNox produces superoxide; certain Fe-containing Nox enzymes, which do not belong to the FDR family, exhibit such a behaviour (Singh et al., 2004). rMjNox did produce superoxide and the activity was elevated at pH >8.5. However, this action accounted for only 3.0 % of the total NADH oxidation activity.
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) clearly indicated that NADH is a physiologically relevant electron donor for MJ0649. rMjNox also oxidized NADPH with O2, but the rate of this reaction was about 5 % of that found with NADH. This property is consistent with the presence of a characteristic NADH binding motif (GXGXXG) (Bellamacina, 1996) in MJ0649 (GAGAIG, Fig. 1). In an NADP-utilizing enzyme the C-terminal Gly of this motif is replaced with Ala, Pro or Ser, perhaps to accommodate the pyrophosphate group of NADP (Bellamacina, 1996). It should be noted that CoADRs from Bacillus anthracis and P. horikoshii carry the GXGXXG motif, yet oxidize both NADH and NADPH (Harris et al., 2005; Wallen et al., 2008).
The apparent Km for O2 of rMjNox (1.9 mM) was much higher than that for Nox enzymes for which a role in oxygen detoxification has been proposed (Table 2). It is possible that in rMjNox, bound FAD is not easily accessible to the oxygen. However, the Vmax value of this enzyme (60 µmol min–1 mg–1, at 1.4 mM dissolved O2) was much higher than that of many NADH oxidases (Table 2) and rMjNox produced H2O2. Also only 62 % of the reducing equivalents provided by NADH were recovered as H2O2 and the rest likely yielded H2O; P. furiosus NoxA-3 behaves similarly (Ward et al., 2001). As is true for the NADH oxidases of the FDR family (Mallett & Claiborne, 1998), the oxidation of NADH with O2 by MjNox probably involved intermediary formation of enzyme-bound C(4a)-peroxyflavin, which was attacked by the thiolate of the reactive Cys residue (Cys42), generating H2O and a Cys-SOH species (Mallett & Claiborne, 1998); reduction of Cys-SOH with NADH generated H2O and the overall reaction produced 2H2O. Perhaps in MjNox and P. furiosus NoxA-3, the attack by the Cys thiolate is slow or very inefficient, which causes peroxyflavin to release H2O2. This hypothesis is supported by the observation that the C42S variant of Ent. faecalis Nox produces exclusively H2O2 from the NADH-dependent reduction of oxygen (Claiborne et al., 1999; Mallett & Claiborne, 1998); Cys42-SOH/SH (sulfenic acid/thiol) acts as the peroxidatic centre for the wild-type enzyme. Release of H2O2 from the peroxyflavin intermediate has been proposed previously for P. furiosus NoxA-3 (Ward et al., 2001). However, it is not clear how the observed low-level peroxidase activity (Table 2) and superoxide formation activity (Fig. 4a) of MjNox fit the mechanism described above or whether the enzyme utilizes yet unknown ways of accomplishing these tasks.
It is tempting to speculate about an oxygen detoxification role for MjNox or MJ0649. Geological data (Corliss et al., 1979; Jannasch & Mottl, 1985; Jones et al., 1983) suggest that M. jannaschii could experience minor oxygen exposure within the submarine hydrothermal vents (Johnson & Mukhopadhyay, 2005) or during transport through cold oxygenic water (Huber et al., 1990; Stetter et al., 1993). Also, during low-temperature storage, hyperthermophilic anaerobes are relatively resistant to O2 exposure (Huber et al., 1990; Jannasch et al., 1992). However, a high Km value (1.9 mM) of the MjNox enzyme for O2 makes an oxygen-detoxification role of MJ0649 less likely. At a typical seawater dissolved O2 concentration of 8 µM and temperature of 2 °C (McCollom & Shock, 1997), the activity of the enzyme would be only 0.006 µmol min–1 mg–1; this calculation is based on the values of Km for oxygen (1.9 mM) and activation energy (46 kJ mol–1) for rMjNox. The abilities of rMjNox to catalyse the reduction of plumbagin, ferricyanide and DTNB with NADH suggest that MJ0649 might reduce an as yet unidentified substrate.
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ACKNOWLEDGEMENTS |
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Edited by: J. van der Oost
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REFERENCES |
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Received 27 April 2008;
revised 14 September 2008;
accepted 17 September 2008.
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) and superoxide formation (
) activities of rMjNox. (b) Temperature study: assays were performed at various temperatures under otherwise standard conditions; inset, a replot of temperature data according to the Arrhenius relationship. The activation energy value was calculated from the slope of the linear segment (at 25–65 °C) of the replot; slope = –Ea/R, where Ea is the activation energy in kJ mol–1 and R is the universal gas constant (8.314 J K–1 mol–1).