INTRODUCTION

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The sulfur oxide sulfate is the most favored electron acceptor in anoxic environments where sulfate predominates. Dissimilatory sulfate reducers use sulfate as an electron sink in the generation of energy. The evolved product of sulfate reduction, H₂S, can be used as a substrate for growth by other organisms or is released into the environment, where it may be involved in the detoxification or precipitation and removal of compounds such as iron in a sulfide complex (FeS).

Sulfate reducers play an integral part in the complex redox cycle for sulfur. Archaeoglobus fulgidus is a hyperthermophilic anaerobe that can use D- or L-lactate, pyruvate, or hydrogen as an energy source (34). Members of this genus are the only known sulfate reducers in the domain Archaea. As a dissimilatory sulfate reducer, A. fulgidus transfers electrons through intermediary carriers to ATP-activated sulfate to obtain energy. The enzymes from sulfate-reducing organisms involved in the later stages of sulfate respiration (ATP sulfurylase, adenylylsulfate reductase, and sulfite reductase) have been characterized (7, 32). However the mechanisms by which respiratory enzymes such as lactate dehydrogenases and NADH dehydrogenase interact with electron carriers such as cytochromes and quinones during sulfate reduction are less well known (4, 10).

To better understand the physiology involved in lactate catabolism and the transfer of electrons during anaerobic sulfate respiration in the archaeon Archaeoglobus, we previously characterized the D-lactate dehydrogenase (Dld) from A. fulgidus (28). During purification of the Dld, an enzyme with NADH oxidase (Nox) activity remained closely associated with the fractions having Dld activity. The role for NADH oxidase in A. fulgidus, a strict anaerobe, is enigmatic because these enzymes catalyze the oxidation of NADH, with subsequent electron transfer to oxygen (oxidase activity). NADH oxidases carry out the bivalent reduction of oxygen to peroxide or the tetravalent reduction of oxygen to water, and they may also transfer electrons univalently to oxygen to form superoxides (20). A subset of NADH oxidases have diaphorase activity and can donate electrons to an acceptor like cytochrome c.

NADH oxidase (EC 1.6.99.3) can be classified as an oxidoreductase, dehydrogenase, diaphorase, or cytochrome c reductase. This type of Nox acts on NADH and transfers electrons to an acceptor. In addition, NAD(P)H dehydrogenase (quinone) (EC 1.6.99.2), mitochondrial NADH oxidase (ubiquinone) (EC 1.6.5.3), and NADH peroxidase (EC 1.11.1.1) are sometimes referred to as NADH oxidases. NADH oxidase (Nox) has been characterized from the eukaryotic mitochondria and eubacteria and more recently in archaea (23). Nox enzymes purified thus far come in a wide range of molecular weights (M_r 29,000 to 215,000) as monomeric, dimeric, or hexameric forms and are often associated with flavin cofactors and iron-sulfur complexes.

NADH oxidase (Nox) activity in anaerobes may contribute to antioxidant activities. For example, during aeration the catalytic activity of Nox in Lactobacillus results in a significant accumulation of H₂O₂, yet Nox has a very minimal energetic role in the cell (22). This suggests that the role of Nox in this organism is to remove oxygen (22). In Streptococcus mutans growing anaerobically, expression of Nox is induced with increased oxygen levels, probably in response to oxygen toxicity (12).

Evidence exists for NADH oxidoreductase involvement in the electron transfer reactions of respiration. A 47-kDa Nox from Escherichia coli has a noncovalently bound flavin adenine dinucleotide (FAD) that catalyzes the reduction of a quinone, probably ubiquinone-8, in vivo (27). Additionally, a defect in Nox in E. coli can be complemented by the respiratory D-lactate dehydrogenase, hinting at a common role for these enzymes in respiration (37). The sequence of the hmc operon in the sulfate reducer Desulfovibrio includes a cytochrome c reductase gene, believed to be part of a protein complex involved in hydrogen oxidation and sulfate reduction (30). Desulfovibrio's flavin mononucleotide (FMN)-containing Nox can transfer electrons from NADH directly to the adenylyl phosphosulfate (APS) reductase, suggesting a role for Nox in sulfate reduction (5). Although some data indicate that the NADH oxidase in sulfate-reducing bacteria can reduce menaquinones, NADH is not generally used as the carrier of the reducing equivalents from the substrate to the electron transport chain of sulfate reducers (9, 10).

To begin to understand the type of NADH oxidase (dehydrogenase) produced by A. fulgidus, the sequence of the N-terminal end of Nox that copurifies with D-lactate dehydrogenase was obtained. Sequence showed Nox to be coded for by a gene adjacent to the gene that codes for the Dld. Together these data suggested that Dld and NoxA2 might have related roles in electron transfer reactions or that Nox acts as a shield to protect components involved in lactate metabolism from O₂. Here we describe the expression of open reading frame (ORF) AF0395 (noxA2) in E. coli and biochemical characterization of NoxA2 enzyme.

	MATERIALS AND METHODS

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Strains, vectors, and reagents. Archaeoglobus fulgidus VC-16 (DSM4303) was obtained from Karl Stetter (Lehrstuhl für Mikrobiologie, Universität Regensburg). E. coli strains BL21(DE3) and JM107 were used for cloning DNA and expression of recombinant protein (26, 36). E. coli strains carrying plasmids were grown in Luria-Bertani (LB) medium supplemented with kanamycin sulfate (40 µg/ml). All antibiotics and chemicals were obtained from Aldrich, Fisher, and Sigma. Restriction enzymes obtained from New England Biolabs (NEB; Beverly, Mass.) were used as recommended by the manufacturer. Vector pET24b DNA was obtained from Novagen (Madison, Wis.). PCR product was purified using the Gibco PCR purification resins (Rockville, Md.). Plasmid DNA was purified over Qiagen columns (Valencia, Calif.). Talon affinity resin for recombinant protein purification was obtained from Clonetech (Palo Alto, Calif.). Fast protein liquid chromatography (FPLC) columns and column resins were purchased from Amersham-Pharmacia (Piscataway, N.J.). Ultrafiltration cartridges and membranes were obtained from Millipore-Amicon (Beverly, Mass.).

Growth of Archaeoglobus. To obtain protein and DNA from A. fulgidus, cells were grown anaerobically at 83°C in glass carboys. Modified sulfate-thiosulfate-lactate (STL) medium 3, growth (1), and harvest conditions are described elsewhere (11, 28). The STL buffer was modified by using 20 mM Tris and 20 mM maleic acid, adjusted to a pH of 6.1 with NaOH. The medium contained 22 mM DL-sodium lactate, 0.5 g of yeast extract per liter, basal salts, and trace minerals. The medium was reduced with 1 mM Na₂S and 1 mM Na₂S₂O₃, and 0.2% resazurin was added as a reduction indicator. A 40-liter carboy was inoculated with 500 ml of logarithmic-phase A. fulgidus cells. Growth was monitored with a Milton-Roy Spectronic 21D spectrophotometer. Following harvest of cells by ultrafiltration with an S3Y100 spiral cartridge and centrifugation, cell mass was stored under O₂-free N₂ gas at 70°C.

Purification of NADH oxidase. To purify the NADH oxidase, NoxA2, from A. fulgidus, frozen cell paste was thawed and cells were washed in salt solution (50 mM Tris [pH 7.8], 5 mM KCl, 300 mM NaCl, 15 mM MgCl₂, 6 mM Na₂S₂O₃, 1 mM DL-dithiothreitol [DTT]). Cells were centrifuged under anoxic conditions at 21,000 × g for 10 min at 4°C and suspended in one-third volume of suspension buffer (5 mM DTT, 50 mM Tris, pH 7.8). Cells were lysed by passage through a cold French pressure cell at 20,000 lb/in² three times and collected under a stream of N₂ gas. Microscopic analysis showed that lysis of A. fulgidus cells was complete after passage through the French pressure cell. After centrifugation at 14,000 × g for 30 min to remove insoluble salts and debris, the supernatant was collected for subsequent protein purification.

Gel analysis. To analyze protein purity, samples were heated at 95°C for 5 min in sample buffer (18), then separated in 10% sodium dodecyl sulfate (SDS)-Tricine-polyacrylamide gels (31) and stained with Coomassie GelCode blue staining reagent according to the manufacturer's specifications (Pierce, Rockford, Ill.). Protein concentration was determined by the Bradford method (3). Bovine serum albumin (BSA) and -globulin were used as standards.

N-terminal sequence. To sequence partially purified protein from A. fulgidus exhibiting Nox activity, samples were heated to 50°C for 10 min in SDS sample buffer and separated by 10% Tricine-SDS-PAGE at 4°C. The gel was allowed to polymerize for 36 h prior to electrophoresis to eliminate compounds that might artificially modify the N terminus of proteins and inhibit sequencing. The gel was rinsed in transfer buffer (10 mM 3-[cyclohexylamino]-1-propanesulfonic acid [CAPS, pH 11.0], 10% methanol) for 20 min, and proteins were transferred by tank blotting to polyvinylidene difluoride (PVDF) nylon membrane (Immobilon; p^sq) in transfer buffer. The membrane was rinsed with water for 5 min, stained with 0.2% Ponceau S dye in 3% trichloroacetic acid, and destained with water for 5 min. Protein bands in the membrane were excised, and the N terminus of the protein was sequenced with an Applied Biosystems 475A protein sequencer.

A. fulgidus DNA purification and cloning of ORF AF0395. Chromosomal DNA was prepared by suspending harvested A. fulgidus cells in 25% sucrose and 10 mM Tris (pH 7.5) and then treating the cells with one-half volume of lysis solution (5% SDS, 0.125 M EDTA, 0.5 M Tris, pH 9.4) for 1 h at 50°C (28). Lysis was complete following an overnight incubation at 37°C with pronase E (2 mg/ml). Following the addition of 0.5 M potassium acetate for 10 min at 37°C and then 1 h at 4°C, protein and cell debris were precipitated by a 15-min centrifugation (10,000 × g). DNA was spooled from the supernatant with a glass rod following the addition of 2 volumes of absolute ethanol. DNA was washed (70% ethanol, 10 mM Tris, 10 mM MgCl₂, 1 mM EDTA, pH 8.0), dried, and dissolved in TE (10 mM Tris, 1 mM EDTA, pH 8.0).

Induction and expression of NoxA2 protein in E. coli. Plasmid pDR8 was transformed into E. coli strain BL21, a DE3 lysogen containing a copy of the inducible gene for T7 RNA polymerase, to express the recombinant form of NoxA2. To determine if the NoxA2 C-terminal His6-tagged protein was produced in E. coli, strain BL21 containing pDR8 was induced with isopropyl--D-thiogalactopyranoside (IPTG), and cell extracts were analyzed by SDS-PAGE for the appearance of a 48-kDa protein.

Purification of recombinant NoxA2. The 48-kDa recombinant NoxA2-His6 protein (rNoxA2) was purified from E. coli using the Talon-immobilized metal affinity chromatography resin. Frozen cell suspensions in Tris buffer were quickly thawed, an equal volume of cold lysis buffer (100 mM NaCl, 20 mM Tris, pH 8.0) was added, and cells were lysed by three passages through a French pressure cell. Cell debris was removed by ultracentrifugation at 300,000 × g for 15 min at 4°C, and resin in lysis buffer (12.5 volumes) was added to the supernatant. The sample was gently agitated on a rocker for 20 min at room temperature and centrifuged at 700 × g for 2 min. Twelve bed volumes of lysis buffer were added to the rNoxA2 protein-bound resin, and the sample was agitated for 10 min at 25°C and centrifuged at 700 × g for 5 min. This step was repeated. The resin was suspended in 8 bed volumes of lysis buffer, transferred to a gravity flow column, and washed two times with 6 bed volumes of lysis buffer, and the recombinant protein was eluted with 100 mM NaCl, 50 mM imidazole, and 20 mM Tris (pH 8.0). The eluate was concentrated (to 1.5 to 2 mg/ml) by ultrafiltration with a Microcon YM10 membrane and washed in 25 mM NaPO₄ (pH 6.0) with 50 mM NaCl, and the rNoxA2 histidine-tagged protein was stored in the same buffer at 4°C or in 25% glycerol at 70°C.

Spectrophotometric enzyme assays. Enzyme activity was assayed in quartz cuvettes (Starna) on a dual-beam Perkin Elmer Lambda 12 UV/VIS spectrophotometer equipped with a PTP-6 temperature block and supported by a Dell Optiplex XMT590 work station with UV-Winlab software. Absorption spectra were taken from 200 to 900 nm.

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Cofactor analysis. Purified rNoxA2 was examined spectrophotometrically to identify potential cofactors. Cofactor was released from the protein and identified by ascending paper chromatography analysis (14). Cofactor was extracted from rNoxA2 (7 to 60 µg) using 10% trichloroacetic acid (TCA) (17), heat, or hot methanol (33). Extracts containing the cofactor and controls (200 µM FAD, 200 µM FMN, and 300 µM riboflavin) were spotted on Whatman no. 1 paper or Silica Gel 60 plates and allowed to dry. Samples were chromatographed in the dark with 5% Na₂HPO₄, n-butanol-acetic acid-H₂O (4:5:1) or n-butanol-acetic acid-H₂O (12:3:5), and analyzed with long-UV (365 nm).

NADH oxidase, H₂O₂, and cytochrome c assays. To determine if oxygen is a terminal electron acceptor for the oxidation of NADH by NoxA2, rNoxA2 (190 µg) was incubated with FAD and then dialyzed to remove excess FAD (see above). The sample was flushed with a stream of O₂-free N₂ gas for 1 h to remove oxygen from the sample, the volume was adjusted for loss of water due to evaporation, and the absorbance from 250 to 550 nm was measured in an anaerobic cuvette at 25°C. Anoxic NADH (400 µM) was added using a gas-tight syringe, and the temperature was increased to 55°C. After monitoring the absorbance for 30 min, the stopper was removed and the sample was stirred to introduce O₂. After 10 min, spectral readings were taken.

Yeast two-hybrid analysis. S. cerevisiae strain PJ69-4A (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4 gal80 LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ) is Ura, Met, and Lys⁺ but His⁺, Ade⁺, and LacZ⁺ in the presence of functional GAL4 (15). Plasmids pDR6 and pDR11 were engineered to express D-lactate dehydrogenase as a fusion to the GAL4 binding domain (BD) and NoxA2 as a fusion to the GAL4 activation domain (AD), respectively. To confirm the protein-protein interaction, reciprocal two-hybrid tests were performed. The dld and noxA2 fragments in the pDR6 and pDR11 vectors were removed after digestion with BglII and EcoRI purified from an agarose gel, and subcloned into pGAD and pGBD vectors, respectively, digested with the same enzymes to produce pDR9 (AD-Dld fusion) and pDR10 (BD-Nox fusion).

	RESULTS

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Archaeoglobus species, in the domain Archaea, are the only hyperthermophiles that can use sulfate as the terminal electron acceptor to generate energy. To better understand the processes involved in the initial stages of sulfate respiration, enzymes involved in electron capture have been studied. The D-lactate dehydrogenase (Dld) coded for by AF0394 (dld) is the enzyme responsible for D-lactate catabolism leading to the eventual transfer of electrons to sulfate. Analysis of the A. fulgidus genome indicates that dld is a third gene of five that likely comprise an operon. AF0395, the gene adjacent to the gene encoding Dld, is predicted to encode a 47-kDa NADH oxidase (NoxA2).

To determine if A. fulgidus encodes an NADH oxidase, cell extracts were separated by nondenaturing gel electrophoresis and assayed for activity with NADH as the electron donor and NBT as the artificial electron acceptor. Multiple proteins, ranging in size from about 45 to 90 kDa, had NADH oxidase activity (Fig. 1, lane 1). This is consistent with the fact that the A. fulgidus genome includes at least eight genes that code for proteins having identity with NADH oxidases. A least four nox genes (noxA-1, noxA-2, noxA-4, and noxA-5) code for proteins of about 48 kDa, noxA-3 codes for a 60-kDa protein, and noxB-1 and noxB-2 code for 68-kDa proteins. The noxC gene codes for a 19-kDa protein, which was not detected in our gel assay. Because dld and noxA2 are transcribed together (V. Pagala and P. Hartzell, unpublished results), NoxA2 and Dld might function in the same energy-yielding pathway. Indeed, a 47-kDa protein with Nox activity was present in samples of partially purified Dld from A. fulgidus after salt precipitation and two chromatographic steps (Fig. 1, lane 2).

View larger version (40K): [in this window] [in a new window]   FIG. 1.   Purified NoxA2 activity. Samples were separated by nondenaturing polyacrylamide and assayed for NADH oxidase (diaphorase) activity with Tris, menadione, NBT, and NADH. Activity was detected by the appearance of dark formazan precipitate. Lanes: 1, French-pressed cells (20 µg); 2, phenyl-Sepharose-purified fraction (0.16 µg). Sizes are shown in kilodaltons.

N-terminal sequence of NADH oxidase. Column fractions containing Nox were identified after gel electrophoresis and treatment with NBT or MTT to assay for activity. Polyacrylamide gel strips stained by Coomassie dye or with enzyme assay reagents were treated for the same time period to preserve the position of each protein. Activities for Dld and Nox correlated precisely with two distinct protein bands in the Coomassie-stained gel lane (Fig. 2). The 47-kDa band with Nox activity was targeted for sequencing.

View larger version (77K): [in this window] [in a new window]   FIG. 2.   Purification and comigration of Dld and NoxA2 enzyme activities. Lane 1, partially purified sample from A. fulgidus obtained after phenyl-Sepharose fractionation (3.3 µg) was separated by SDS-10% PAGE and stained with Coomassie dye. Lane 2, the same phenyl-Sepharose fraction was incubated in Tris (pH 7.5) and then assayed with Tris, MgSO4, PMS, MTT, and D-lactate to detect Dld activity and with NaPi, menadione, NBT, and NADH to detect NoxA2 activity.

Expression of AF0395 and purification of NoxA2 from E. coli. To confirm that AF0395 codes for a protein with Nox activity, the gene was expressed in E. coli. The 1,300-bp AF0395 gene was amplified by PCR using chromosomal DNA from A. fulgidus as the template and cloned into pET24b to generate plasmid pDR8. This construct is expected to generate a 48-kDa protein (NoxA2-His6) that includes six His residues at the carboxyl end of the protein.

NoxA2 requires FAD for activity. Some NADH oxidases are flavin-containing enzymes, including the NoxA2-related Enterococcus faecalis 10C1 FAD- and Desulfovibrio vulgaris FMN-containing NADH oxidases (5, 20). Purified rNoxA2 has absorption maxima at 373 and 451 nm with a shoulder at 480 nm, spectral features which are characteristic of proteins with bound flavin cofactors.

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View larger version (19K): [in this window] [in a new window]   FIG. 3.   Absorption spectra of rNADH oxidase A2. Purified rNoxA2 (190 µg) was incubated with 25 mM NaPi (pH 6.0) and 50 mM NaCl with 320 µM FAD and then dialyzed in the same buffer to remove unbound FAD. The spectrum shows peaks characteristic of FAD at 373 and 451 nm and a shoulder at 480 nm. The ratio of FAD to rNoxA2 was 1.1:1. (Insets) Redox peaks of FAD (lower) at 451 nm and NADH (upper) at 340 nm. Dotted line, absorption spectrum of enzyme (190 µg) following flushing with nitrogen gas; solid line, spectrum of rNoxA2 following addition of NADH (400 µM); dashed line, spectrum of rNoxA2 following the mixing of oxygen with the enzyme.

View larger version (78K): [in this window] [in a new window]   FIG. 4.   Reconstitution of active NADH oxidase after extraction of cofactor. (A) rNoxA2 (40 µg) and (B) NoxA2 (13 µg, total protein) were electrophoresed under denaturing conditions, renatured, and then assayed in the presence of cofactors for Nox activity. Activity was observed by the formation of a blue formazan precipitate from MTT. Lanes: 1, benchmark prestained protein standards; 2, no cofactor; 3, FAD; 4, F420; 5, FMN; 6, riboflavin.

rNoxA2 is an FAD-dependent NADH oxidase. To show that rNoxA2 is an NADH oxidase using oxygen as a terminal electron acceptor, an O₂-free NoxA2 sample was assayed at 55°C for FAD reduction at 450 nm and NADH oxidation at 340 nm before and after addition of saturating NADH. As indicated in Fig. 3 (insets), the addition of NADH resulted in rapid reduction of the FAD cofactor, which shows that electrons are transferred from NADH to FAD in the absence of oxygen. Following the introduction of O₂ to the sample and regeneration of FAD, the absorbance at 340 nm decreased due to the oxidation of NADH, which was present in excess. These results show that the FAD moiety of NoxA2 can accept electrons from NADH and transfer them to O₂.

H₂O₂ is the product of NADH oxidation. To identify the end product of NADH oxidation, NoxA2 and NADH and control reactions without enzyme or NADH were incubated at 55°C in the presence of O₂ and then assayed with horseradish peroxidase. If NADH-dependent reduction of O₂ by NoxA2 yields H₂O₂, the chromogenic substrate TMBZ will be oxidized upon the addition of horseradish peroxidase at 25°C. The change in absorbance of TMBZ at 650 nm showed that H₂O₂ is an end product of rNoxA2 under aerobic conditions. An assay of rNoxA2 using TMBZ as the sole electron acceptor failed to show a color change, showing that rNoxA2 did not transfer electrons directly to TMBZ.

rNoxA2 has diaphorase activity, including affinity to cytochrome c. Like the native NoxA2 from A. fulgidus, the recombinant NoxA2 has diaphorase activity. Under anaerobic conditions, NBT, MTT, DCIP, potassium ferricyanide [K₃Fe(CN)₆], menadione, and cytochrome c each served as a terminal electron acceptor during oxidation of NADH. MTT and K₃Fe(CN)₆ served as electron acceptors even in the presence of oxygen, suggesting that some factors may serve as better electron acceptors than oxygen. The addition of H₂O₂ (50 µM) to the diaphorase reaction did not affect the rate of reduction for MTT and K₃Fe(CN)₆, implying that these acceptors receive electrons directly from the enzyme cofactor.

Kinetic analysis of rNoxA2. NADH (NADPH) oxidation by rNoxA2 at 55°C was monitored at 340 nm. Using oxygen as an electron acceptor, -NADH but not -NADPH served as an electron donor in A. fulgidus. The maximum catalytic activity of the enzyme was observed in NaPi buffer at pH 7.6 (Fig. 5). Increasing the ionic strength of phosphate from 10 to 100 mM increased rNoxA2 activity only slightly. The anionic detergent SDS (0.5%) completely inhibited the reaction, whereas sodium deoxycholate (0.5%) slightly decreased activity. The nonionic detergents Tween 20 (1.25%), Triton X-100 (0.1 to 0.5%), and n-dodecyl--D-maltoside (0.5%) and the zwitterionic detergent CHAPS (0.2 to 0.5%) each increased activity about twofold.

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Effect of pH and buffers on rNoxA2 activity. rNoxA2 (1 µg) was assayed spectrophotometrically at 340 nm with 50 mM buffer, 0.5% CHAPS, 5 µM FAD, and 100 µM NADH at 55°C. The pH of each buffer was measured at 55°C. Buffers tested included sodium acetate (), Tris maleate (), NaPi (), and Tris-HCl ().

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View larger version (20K): [in this window] [in a new window]   FIG. 6.   Hanes-Woolf plot of rNoxA2-catalyzed oxidation of NADH shows Michaelis-Menten kinetics. rNoxA2 (0.2 µg) was assayed spectrophotometrically at 340 nm with 50 mM NaPi (pH 7.6), 0.2% CHAPS, 5 µM FAD, and 0 to 500 µM NADH at 55°C. A Km of 3 µM for NADH was determined from the plot at 98% confidence. NADH approached saturation for rNoxA2 near 10 µM. (Inset) Expanded plot near the y intercept. x intercept = Km (3.1 µM); slope = Vmax1 (1.3 µmol mg1 min1).

View larger version (26K): [in this window] [in a new window]   FIG. 7.   Thermal stability of the recombinant form of NoxA2. rNoxA2 was incubated at 83°C in 50 mM NaPi (pH 7.6, as determined at 55°C) (open circles) or 50 mM Tris (pH 7.8, as determined at 55°C) (solid circles). Aliquots of rNoxA2 (0.5 µg) were removed and monitored at 340 nm for NADH oxidation in 50 mM NaPi (pH 7.6), 0.2% CHAPS, 20 µM FAD, and 100 µM NADH at 55°C. Half-life of rNox following incubation in buffer was estimated to be 40 min in NaPi and 12 min in Tris. (Inset) NoxA2 (phenyl-Sepharose fraction) from A. fulgidus was incubated at 83°C in NaPi prior to assaying as explained above. One unit equals 1 µg of total protein; the half-life of NoxA2 averaged 35 h.

View larger version (21K): [in this window] [in a new window]   FIG. 8.   Effect of temperature on rNoxA2 activity. The activity of rNoxA2 (1 µg) was monitored at 340 nm for NADH oxidation at the temperatures indicated. The enzyme was assayed in 50 mM NaPi (pH 7.6, as determined at 55°C), 0.2% CHAPS, 20 µM FAD, and saturating 200 µM NADH. (Inset) An average of maximum velocities determined by the method of Arrhenius.

Interaction between NADH oxidase and D-lactate dehydrogenase. The association of NoxA2 with Dld during the purification of Dld hints that these proteins interact in vivo. To test this idea independently, the yeast two-hybrid system was used to check for protein-protein interactions. The yeast strain PJ-694A, constructed by James et al. (15), has been designed to eliminate the risk of false-positive tests by placing the HIS, ADE, and lacZ genes under control of a GAL4-dependent promoter. Transformants that are able to grow in medium lacking His and Ade likely carry fusion proteins that interact to produce active GAL4 protein and can be tested further for -galactosidase activity. When pDR6 and pDR11 were introduced into PJ-694A, the transformants were able to grow on medium lacking His and Ade. Similarly, pDR10 and pDR9 also passed this initial screen. Controls, pDR6 plus pGAD and pDR11 plus pGBD, failed to grow on medium without His and Ade. Yeast carrying pDR6 and pDR11 produced pale blue colonies on yeast medium with X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside). The positive controls, pGAD-Fos and pGBD-Jun, produced strong blue colonies on X-Gal-containing medium. These results show that Dld and NoxA2 interact, albeit weakly. Yeast carrying pDR6 (GBD-Dld) and pDR9 (GAD-Dld) also showed a weak interaction, which suggests that Dld interacts with itself. This is consistent with earlier observations that some Dld migrates as a dimer on a Superose gel filtration column. Attempts to show an interaction between NoxA2 and the A. fulgidus c-type cytochrome (the product of ORF AF503) in the yeast two-hybrid system were unsuccessful.

	DISCUSSION

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During dissimilatory sulfate respiration, electrons are removed from reduced substrates such as D- and L-lactate and eventually are passed to oxidized sulfur compounds, such as sulfate, to generate a proton motive force. The D-lactate dehydrogenase (Dld) transfers electrons from D-lactate to its FAD cofactor to other intermediate carriers. To begin to identify proteins that might interact with Dld during electron transfer, we examined a protein that remains associated with Dld during purification. N-terminal sequence of this protein matches two proteins, both putative NADH oxidases, encoded by AF395 and AF400 on the A. fulgidus genome. The protein that copurifies with Dld is about 48 kDa. AF0395 codes for the expected size (47 kDa) of the protein, whereas AF0400 codes for a much larger protein (60 kDa).

AF0395 (noxA2) is the gene immediately upstream of dld within a cluster of five genes that appear to form an operon (V. Pagala and P. Hartzell, unpublished results). NoxA2 has homology with NADH oxidases from organisms such as E. (Streptococcus) faecalis and Deinococcus radiodurans. Like other NADH oxidases, the predicted product of noxA2 has an N-terminal binding site for the ADP moiety of FAD, a binding site for the flavin moiety of FAD, a putative internal NAD⁺ binding site, and a conserved cysteine (Cys42) that has been shown to be critical for redox chemistry of Nox from E. faecalis (Fig. 9). Consistent with this, we found that both partially purified and recombinant NoxA2 proteins contain a tightly bound FAD cofactor and can oxidize NADH in the presence of various electron acceptors.

View larger version (52K): [in this window] [in a new window]   FIG. 9.   Primary sequence of noxA2 derived from A. fulgidus AF0395. Conserved ADP binding residues are in bold; conserved redox-active cysteine (Cys42) is starred; the flavin ribityl binding site is underlined (conserved amino acids are italicized).

By the classic definition, NADH oxidases donate electrons to molecular oxygen and regenerate critical reserves of NAD⁺. NoxA2 has oxidase activity because in the presence of NADH, O₂ can serve as an electron acceptor. In the presence of oxygen, NoxA2 catabolism of NADH results in the production of H₂O₂. NoxA2 also has diaphorase activity, because it can donate electrons to acceptors such as NBT, MTT, DCIP, potassium ferricyanide, menadione, and cytochrome c. These activities are similar to the NADH oxidase activities of thermophilic prokaryotes that transfer electrons to O₂ and artificial electron carriers (25, 35).

The FAD cofactor is required for oxidase and diaphorase activities of both recombinant and native NoxA2. NoxA2 has a K_m of about 3 µM for NADH, similar to the K_m determined for NADH oxidase from Thermus thermophilus (25) and a V_max near 1.3 µmol mg¹ min¹ was similar to the V_max of 0.21 µmol mg¹ min¹ reported for Desulfovibrio vulgaris (5). Like the partially purified enzyme from A. fulgidus, rNoxA2 was stable in the presence of O₂ and was active over a range of temperatures from 25 to 85°C. rNoxA2 oxidase and diaphorase activities were optimal at temperatures near 80°C, indicating that AF0395 codes for an enzyme that folds properly when expressed at 37°C under aerobic conditions in E. coli. Although the half-life of the recombinant enzyme ranged from 12 to 40 min at 83°C, preparations of the partially purified form from A. fulgidus had a half-life of about 35 h. Hence, the native NoxA2 may be protected from denaturation by additional proteins or a heat-stable conformation only attainable in A. fulgidus.

The role of Nox in vivo and the need for so many forms of Nox, which appear to be produced constitutively under anaerobic conditions, is mysterious. Because A. fulgidus is a strict anaerobe, it is unlikely that Nox acts in the classic sense to transfer electrons to O₂ for the purpose of regenerating NAD⁺. Nox enzymes may instead play a protective role and/or interact with other electron transfer components in novel ways. In E. faecalis and Streptococcus mutans, the NADH oxidase (peroxidase) is involved in defense against oxygen toxicity by direct reduction of O₂ to water (6, 12). Typically these enzymes are found in organisms that are unable to synthesize heme, which is needed to produce catalase. The NADH oxidase in A. fulgidus may similarly be involved in protecting the cell from oxidative stress. In this case NoxA2 may reduce oxygen toxicity by the formation of H₂O₂. Although H₂O₂ can potentially cause cellular damage, A. fulgidus has a gene, AF2233, which is predicted to encode a peroxidase to detoxify H₂O₂. Indeed, although A. fulgidus can only grow under anaerobic conditions, cells are not killed upon exposure to oxygen, but rather form a protective biofilm (19). Cells remain viable for up to 24 h in medium contaminated with oxygen, particularly if the temperature is below 80°C.

The finding that NoxA2 specifically copurifies and interacts with the D-lactate dehydrogenase hints that Nox may play a role in energy-yielding reactions. NoxA2 may behave like the respiratory NADH dehydrogenase in E. coli that has oxidase activity and is involved in electron transfer reactions (27). The bacterial sulfate reducer D. vulgaris produces an H₂O₂-forming NADH oxidase that transfers electrons to APS reductase (4). Because this NADH oxidase is functionally related to NoxA2, it is exciting to speculate that NoxA2 might interact with APS reductase in A. fulgidus. In D. vulgaris, NADH oxidase and cytochrome c reductase are postulated to form part of a membrane complex that, together with ubiquinol, catalyzes the reduction of sulfate (30). Likewise, the A. fulgidus Nox may interact directly with cytochrome c or indirectly with an intermediate, such as the A. fulgidus 7-menaquinone, to catalyze sulfate reduction. This is consistent with the finding that rNoxA2 can donate electrons to the quinoline-like compound menadione.

The ability of NoxA2 to reduce yeast and horse heart cytochrome c is of interest because cytochrome c is a potential electron carrier in vivo. Cytochrome c in A. fulgidus is located in the "periplasm" (between the membrane and the proteinaceous S-layer), where it associates with the membrane. If NoxA2 interacts with a c-type cytochrome in vivo, then NoxA2 must be located in the periplasm. Indeed, we found that NoxA2 is located almost exclusively in the periplasmic space in A. fulgidus cells (V. Pagala and P. Hartzell, unpublished results).

Although the topology of other membrane-bound NADH oxidases (2, 13, 16) is unclear, they may have periplasmic domains that facilitate interaction with periplasmic electron carriers. If cytochrome c is an electron acceptor of NADH oxidation by NoxA2 in A. fulgidus, then NoxA2 may have a respiratory role under anaerobic conditions. We have been unable to detect a change in absorbance of cytochrome using partially purified NoxA2 and crude A. fulgidus membrane preparations. This assay may not be sensitive enough if the level of cytochrome is low or if other factors interfere in a crude preparation. Alternatively, the c-type cytochrome may not interact with NoxA2 under these or any other conditions.