TY - JOUR
AU - Butler, Clive, S.
AB - Abstract Paracoccus pantotrophus grown anaerobically under denitrifying conditions expressed similar levels of the periplasmic nitrate reductase (NAP) when cultured in molybdate- or tungstate-containing media. A native PAGE gel stained for nitrate reductase activity revealed that only NapA from molybdate-grown cells displayed readily detectable nitrate reductase activity. Further kinetic analysis showed that the periplasmic fraction from cells grown on molybdate (3 µM) reduced nitrate at a rate of Vmax=3.41±0.16 µmol [NO3−] min−1 mg−1 with an affinity for nitrate of Km=0.24±0.05 mM and was heat-stable up to 50°C. In contrast, the periplasmic fraction obtained from cells cultured in media supplemented with tungstate (100 µM) reduced nitrate at a much slower rate, with much lower affinity (Vmax=0.05±0.002 µmol [NO3−] min−1 mg−1 and Km=3.91±0.45 mM) and was labile during prolonged incubation at >20°C. Nitrate-dependent growth of Escherichia coli strains expressing only nitrate reductase A was inhibited by sub-mM concentrations of tungstate in the medium. In contrast, a strain expressing only NAP was only partially inhibited by 10 mM tungstate. However, none of the above experimental approaches revealed evidence that tungsten could replace molybdenum at the active site of E. coli NapA. The combined data show that tungsten can function at the active site of some, but not all, molybdoenzymes from mesophilic bacteria. Nitrate reductase, Molybdenum, Tungsten, Enzyme activity 1 Introduction Nitrate (NO3−) is an important component of the biological nitrogen cycle. It serves as the substrate for denitrification, whereby nitrate is reduced via nitrite (NO2−), nitric oxide (NO) and nitrous oxide (N2O) to dinitrogen (N2), but can also be reduced via nitrite to ammonium as part of nitrogen assimilation and respiration [1]. Many bacteria can express multiple, biochemically distinct, nitrate reductases. For example, Paracoccus pantotrophus expresses three nitrate reductases [2,3]. Two of these, NAR and NAP, are linked to respiratory electron transport systems, and are located in the membrane and periplasm, respectively. The third enzyme, NAS, is located in the cytoplasmic compartment, is ammonium-repressible, and participates in nitrogen assimilation [1]. Although Escherichia coli also expresses three nitrate reductases, two of them are membrane-bound respiratory orthologues of nitrate reductase A (NAR). The third, NAP, is encoded by a seven-gene operon, napFDAGHBC[4]. Bacterial nitrate reductases are members of the sub-group of molybdoenzymes that bind the molybdopterin guanine dinucleotide (MGD) form of the molybdenum cofactor. The periplasmic nitrate reductases (NAP) from P. pantotrophus and E. coli contain a soluble 16-kDa di-haem c-type cytochrome subunit (NapB) and an 80–90-kDa catalytic subunit (NapA) that binds an N-terminal [4Fe–4S] cluster and the MGD cofactor [4–6]. A third component, NapC, is a membrane-anchored tetra-haem c-type cytochrome that mediates electron flow between membrane quinols/quinones and the NapAB complex [7]. The sequence of NapA is highly similar to the MGD-binding polypeptides of bacterial assimilatory nitrate reductases (NAS) and formate dehydrogenases, both of which also bind an N-terminal iron–sulfur cluster and MGD. Analysis of the emerging genome sequences suggests that novel thermostable nitrate reductases may also be utilised in the anaerobic respiration of the hyperthermophilic archaea. Interestingly, the growth of the hyperthermophilic archaea appears to be obligately dependent upon tungsten and they are incapable of utilising molybdenum: consequently, they express a number of novel tungsto-enzymes [8]. This raises two questions. Are the novel thermostable nitrate reductases tungsto-enzymes? And, can tungsten catalyse nitrate reduction? Given that the atomic and ionic radii and the chemical properties (oxidation states IV, V and VI) of tungsten are very similar to those of molybdenum, there has been much interest to see if tungsten can substitute for molybdenum in a range of molybdoenzymes and retain their activity [9]. Many publications have reported that the replacement of molybdenum by tungsten results in inactive proteins, including nitrogenase from Azotobacter vinelandii[10], nitrate reductase from plants [11], hepatic sulfite oxidase [12] and formate dehydrogenase from Methanobacterium formicicum[13]. It has been suggested that molybdenum is more suitable for catalysing reactions with relatively high redox potentials and the reason why tungsten-substituted enzymes are inactive is because of the lower reduction potential of the W(VI)/(V) and W(V)/(IV) couples. However, two bacterial periplasmic molybdo-enzymes, the dimethyl sulfoxide reductase (DMSOR) from Rhodobacter capsulatus[14], and the periplasmic trimethylamine N-oxide reductase (TMAOR) from E. coli[15], can function with tungsten at the active site, resulting in an enzyme with increased catalytic efficiency, increased thermal stability and broader substrate selectivity [15]. Clearly, in some enzyme systems both molybdenum and tungsten can not only catalyse the same chemical reaction, but also catalyse reactions at relatively high redox potential (TMAO/TMA, Em=+130 mV; DMSO/DMS, Em=+160 mV). We now report studies of the different effects of tungstate on NAP-catalysed nitrate reduction in both P. pantotrophus and E. coli. 2 Materials and methods 2.1 Growth of bacteria and the production of periplasmic extracts P. pantotrophus M6, a membrane-bound nitrate reductase mutant (narH::Tn5) [16], was grown anaerobically in minimal salts medium, supplemented with Vishniac and Santer trace element solution [17] and containing succinate as the sole carbon and energy source, and nitrate as the sole electron acceptor. E. coli strain LCB2048 lacks the two membrane-bound nitrate reductases, A and Z [18], but is a prolific source of NAP activity [19]. Strains JCB4011, expressing only nitrate reductase A, and JCB4024, expressing only NAP, were described previously [20]. Bacteria were grown without aeration either in 2-l conical flasks filled with medium, or in a 2-l Mini-bioreactor fermenter [20]. E. coli strains were grown in a minimal medium (designated GM) that contained 10.5 g K2HPO4, 5.44 g KH2PO4, 2 g (NH4)2SO4, 16.26 ml of 50% (v/v) glycerol, 0.5 g of casein hydrolysate, 19 mg of thiamine, 1 µM selenate and 1 ml of E. coli sulfur-free salts per litre of ultra-pure distilled water. For growth in tungstate-supplemented medium the trace element solution was modified by replacing ammonium molybdate ((NH4)6Mo7O24·4H2O) with sodium tungstate (Na2WO4) at the required concentration. Control experiments replacing (NH4)6Mo7O24·4H2O with Na2MoO4 had no obvious effect on cell growth. To ensure that tungstate media were molybdate-free all glassware was acid-washed and only ultra-pure H2O was used during medium preparation. Periplasmic fractions were produced as described previously [21] and stored on ice. Protein concentration was determined using the Bio-Rad protein assay following the manufacturer's instructions. 2.2 Raising antibodies to NapA from P. pantotrophus The periplasmic nitrate reductase NapAB complex from P. pantotrophus was purified as described by Berks et al. [21]. The protein components were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) [22] and the ~90-kDa band corresponding to NapA was extracted using a gel electro-elution technique (Bio-Rad) following the manufacturer's instructions. Antibodies to NapA were raised in a New Zealand White rabbit [23]. 2.3 Polyacrylamide gel electrophoresis, Western blotting and activity staining Periplasmic proteins were separated by SDS–PAGE [22] and blotted onto nitrocellulose membranes [24]. Blots were probed with a primary antibody to NapA (diluted 1:5000) and developed using a secondary antibody (goat anti-rabbit) linked to a colorimetric horseradish peroxidase assay. For activity staining, periplasmic fractions were separated using native PAGE and gels were stained with reduced methyl viologen (10 mM) under anaerobic conditions. Protein bands with nitrate reductase activity were determined by the addition of 10 mM nitrate for 5 min, and observed as clear bands due to the nitrate-dependent re-oxidation of reduced methyl viologen, changing from dark blue to colourless. The relative intensities of the bands were determined by densitometry and analysed using the computer program Quantity One. To determine rates of transcription from the E. coli napF promoter, strain JCB4024 was transformed with the napF::lacZ reporter plasmid, pDF102 [25], and assayed for β-galactosidase activity after anaerobic growth to the middle of the exponential phase in the presence of glycerol, nitrate and either molybdate or tungstate. 2.4 Enzyme activity assays For studies of NAP from P. pantotrophus, Mo-NAP and W-NAP activities were measured at 20°C by following the oxidation of reduced methyl viologen (MV+) spectrophotometrically at 600 nm, coupled to the reduction of nitrate as described in earlier work [26]. For heat stability assays, periplasmic fractions were incubated at the selected temperature for 30 min prior to the activity assay measurement. Methyl viologen is a poor electron donor for NAP in the periplasmic fraction from E. coli. NAP activity was therefore assayed quantitatively using permeabilised bacteria [27]. Various concentrations of Na+ phosphate buffer, pH 7.4, NaCl, KCl or sucrose were added, as noted in the text. Rates of nitrate reduction by physiological substrates were determined using the nitrate electrode assay [28]. 3 Results 3.1 The effect of Mo and W on growth of P. pantotrophus M6 and E. coli JCB 4024 The effect of Mo and W on growth of P. pantotrophus M6 and E. coli JCB4024 was investigated. Both P. pantotrophus and E. coli can express membrane-bound (NAR) and periplasmic (NAP) nitrate reductases. These strains were selected because they only express NAP and in both cases this can support anaerobic growth. The denitrifying P. pantotrophus was grown with succinate as the carbon source whilst E. coli was grown with glycerol. In both cases nitrate was initially present as the sole electron acceptor. Typical growth curves for P. pantotrophus in the presence of increasing concentrations of tungstate (3 µM–100 mM Na2WO4) are shown in Fig. 1. No growth was observed at the highest concentration of tungstate used (100 mM), suggesting that tungstate at this concentration is highly toxic to P. pantotrophus. Some growth was observed in media containing tungstate at concentrations 10–30 mM, with cultures reaching a maximum optical density at 650 nm after 70 h of only ~0.15 units. At the lower tungstate concentrations used (3–100 µM), cell growth was similar to that observed in ‘normal’ growth medium containing 3 µM molybdate, with the cultures reaching a maximum optical density (OD600nm) of ~1 unit in 40–50 h. Cell growth was also observed in cultures containing tungstate at 1 mM, but the growth rate was slower reaching only an OD600nm of ~0.7 units in 70 h. When both molybdate and tungstate were omitted from the culture medium, the cell density reached only ~0.4 units in 70 h. 1 Open in new tabDownload slide Growth curves for P. pantotrophus grown anaerobically using nitrate as the sole electron acceptor in minimal media supplemented with various concentrations of Na2WO4. The inset key provides actual [Na2WO4] added. The data presented are typical of the results obtained from two independent experiments. Control cells were grown under identical conditions in minimal medium supplemented with 3 µM sodium molybdate. 1 Open in new tabDownload slide Growth curves for P. pantotrophus grown anaerobically using nitrate as the sole electron acceptor in minimal media supplemented with various concentrations of Na2WO4. The inset key provides actual [Na2WO4] added. The data presented are typical of the results obtained from two independent experiments. Control cells were grown under identical conditions in minimal medium supplemented with 3 µM sodium molybdate. Similar growth analysis for E. coli JCB4024 showed that the growth rate (~0.7 h−1) and final optical density (~0.6) were unaffected by the absence of supplementary molybdate. These results indicated that, despite the use of ultra-pure water, acid-washed glassware and a defined medium, E. coli was able to scavenge residual traces of molybdate extremely effectively. Growth of the NAP+ strain was only slightly inhibited by 15 mM tungstate, but was much more strongly inhibited at higher concentrations. Glycerol- or formate-dependent rates of nitrate reduction by bacteria from cultures supplemented with tungstate, 70 units (nmol [NO3−] min−1 mg−1), were typically ~50% of those of the molybdate-supplemented culture. However, studies with a nap-lacZ promoter fusion also confirmed that transcription from the napF promoter was not inhibited under these growth conditions and was approximately 11 µmol of o-nitrophenol β-d-galactoside hydrolysed min−1 (mg dry mass)−1 in the presence of either molybdate or tungstate. 3.2 Synthesis of P. pantotrophus and E. coli NapA during nitrate-dependent anaerobic growth in tungsten media Synthesis of the periplasmic nitrate reductase in cells of P. pantotrophus and E. coli grown anaerobically in the presence of tungstate was determined by a combination of SDS–PAGE analysis and immunoblotting. Fig. 2A shows duplicate samples of periplasmic proteins prepared from P. pantotrophus grown in the presence of 3 µM Mo, 100 µM W plus 6 nM Mo and 100 µM W alone. The profile of proteins observed appeared to be the same for all three growth conditions, however, a small protein at ~25 kDa does appear to be up-regulated in the presence of tungstate (or absence of molybdate). We have not identified this protein but speculate that it might be the periplasmic molybdate-binding protein (ModA) which migrates on SDS–PAGE at ~22.5 kDa [29]. In the absence of molybdate the ModA regulator protein (ModE) is repressed, thus up-regulating ModA to transport more Mo. The level of expression of NapA appears to be similar under all three different growth conditions (Fig. 2). Western blot analysis using an antibody raised to purified NapA confirmed that the ~90-kDa band detected by SDS–PAGE was NapA, and that a band of similar intensity is observed in all samples (Fig. 2B). Furthermore, there appears to be no obvious difference in the level of expression of NapA in cells supplemented with 6 nM Mo, demonstrating unequivocally that NapA is expressed under tungstate-enriched conditions. Additional control experiments have shown this band migrates to the same position on a gel as purified NapA and that it is absent in the napA::Ω mutant HJS10 [30] demonstrating that the band can only arise from NapA (not shown). 2 Open in new tabDownload slide NAP expression in P. pantotrophus and E. coli grown in tungstate-supplemented medium. A: SDS–PAGE-resolved periplasmic fractions (10 µg total protein in duplicate) from P. pantotrophus cells grown in the presence of 3 µM Mo, 100 µM W plus 6 nM Mo and 100 µM W alone. Molecular mass marker sizes are indicated. Gel stained with Coomassie blue stain. B: Western blot of gel shown in A probed with antibodies to NapA. C: Native PAGE-resolved periplasmic fractions (10 µg total protein in duplicate) from P. pantotrophus cells grown in the presence of 3 µM Mo, 100 µM W plus 6 nM Mo and 100 µM W alone as above. Gel stained with reduced methyl viologen (10 mM) and developed by the addition of nitrate (10 mM) for 5 min under anaerobic conditions. NapA and the NapAB complex are indicated. D: NAP expression in E. coli grown in molybdate- or tungstate-supplemented medium. a: Coomassie blue-stained SDS–PAGE-resolved periplasmic fractions (20 µg total protein) from cells grown in the presence of 1 µM Mo or 15 mM W. No NapA is detected in the NapA− strain JCB4011. b: Native PAGE-resolved periplasmic fractions (10 µg total protein) from cells grown in the presence of 1 µM Mo or 15 mM W. Activity staining was as described for C. 2 Open in new tabDownload slide NAP expression in P. pantotrophus and E. coli grown in tungstate-supplemented medium. A: SDS–PAGE-resolved periplasmic fractions (10 µg total protein in duplicate) from P. pantotrophus cells grown in the presence of 3 µM Mo, 100 µM W plus 6 nM Mo and 100 µM W alone. Molecular mass marker sizes are indicated. Gel stained with Coomassie blue stain. B: Western blot of gel shown in A probed with antibodies to NapA. C: Native PAGE-resolved periplasmic fractions (10 µg total protein in duplicate) from P. pantotrophus cells grown in the presence of 3 µM Mo, 100 µM W plus 6 nM Mo and 100 µM W alone as above. Gel stained with reduced methyl viologen (10 mM) and developed by the addition of nitrate (10 mM) for 5 min under anaerobic conditions. NapA and the NapAB complex are indicated. D: NAP expression in E. coli grown in molybdate- or tungstate-supplemented medium. a: Coomassie blue-stained SDS–PAGE-resolved periplasmic fractions (20 µg total protein) from cells grown in the presence of 1 µM Mo or 15 mM W. No NapA is detected in the NapA− strain JCB4011. b: Native PAGE-resolved periplasmic fractions (10 µg total protein) from cells grown in the presence of 1 µM Mo or 15 mM W. Activity staining was as described for C. The antibody raised against P. pantotrophus NAP did not recognise the E. coli NAP, which prevented immunochemical analysis of cell extracts. However, a 90-kDa band could be resolved in SDS–PAGE gels of E. coli periplasmic extracts which was absent from NapA mutants and thus corresponded to the NapA polypeptide (Fig. 2D). Densitometry indicated that this polypeptide, similarly to P. pantotrophus NapA, was present at equivalent amounts in all of the cell extracts, regardless of the tungstate concentrations tested (up to 15 mM; data not shown). The assembly of a functional NapA from the tungstate-grown cells was probed using native PAGE gels stained with dithionite-reduced methyl viologen. In P. pantotrophus a clear doublet band at a position corresponding to NapA (or the NapAB complex) was obtained only from cells grown in media supplemented with molybdate (3 µM) (Fig. 2C). It is expected that the functional periplasmic Nap is a NapAB dimer under these non-denaturing conditions and that the NapAB complex corresponds to the larger, more intense band on the activity-stained gel. The slightly lower, less intense band probably arises from a small fraction of monomeric NapA that is still functional using methyl viologen as the electron donor. No rapid nitrate-dependent re-oxidation of the methyl viologen was seen in the lanes resolving proteins from the periplasms of cells grown in tungstate- or tungstate/molybdate-supplemented media. Control experiments confirmed that purified NapAB showed a single activity-staining band that migrated at the same position as the activity-staining band in molybdenum-grown P. pantotrophus M6. This band was again absent in the napA::Ω mutant HJS10 (data not shown). The observed lack of a fully functional NapA in periplasms from cells grown in the presence of 100 µM W supplemented with 6 nM Mo also suggests that NapA cannot preferentially scavenge Mo, when the tungsten:molybdenum ratio is >104. Despite not detecting any obvious rapid nitrate reductase activity by native PAGE, prolonged incubation (up to 30 min) of the native gel with nitrate resolved faint clear bands in the periplasms isolated from the cells grown on both 100 µM W plus 6 nM Mo and 100 µM W alone, corresponding to W-NAP with very low activity or a very low percentage of Mo-NAP with full activity (data not shown). The intensities of these would represent at most 1% of those observed in periplasmic fractions prepared from cells grown in the absence of W. In E. coli, a ~90-kDa activity-staining band was also seen in periplasmic fractions grown with 1 µM molybdate (Fig. 2D). This band was absent in NapA mutants [28]. In periplasmic fractions prepared from cells grown in the presence of 15 mM tungstate an activity-staining band could also be observed that was around 10% of the intensity of that observed in equal loadings of periplasmic fractions prepared for Mo-grown cells (Fig. 2D). In some gels, an additional ~50-kDa activity-staining band was apparent, due to the cytochrome c nitrite reductase (NrfA) which will react with nitrite, the product of the NAP enzyme activity. 3.3 The effect of tungsten on P. pantotrophus and E. coli NAP enzyme activity In order to probe further this apparent low level of activity in P. pantotrophus and determine whether it represents a functional W-NAP, nitrate reductase activity of periplasmic fractions from cells grown in the presence of either Mo (3 µM) or W (100 µM) was measured spectrophotometrically (OD600nm) in an anaerobic cuvette using reduced methyl viologen as the electron donor, at a range of nitrate concentrations (0.1–20 mM). From the dependence of the observed rate of methyl viologen oxidation on nitrate concentration (Fig. 3A,B), a Km of 0.24±0.05 mM and a Vmax=3.41±0.16 µmol [NO3−] min−1 mg−1 was calculated for the periplasmic nitrate reductase from molybdate-grown cells, consistent with those published previously [21]. In contrast, a Km of 3.91±0.45 mM and a Vmax=0.05±0.002 µmol [NO3−] min−1 mg−1 was calculated for the periplasmic nitrate reductase from tungstate grown cells. No activity towards the substrate analogues selenate and DMSO was detected in the periplasms from cells grown in tungstate-supplemented media. No MV+-dependent nitrate reductase activity could be detected in solution assays from periplasms of HJS10 (napA::Ω) confirming that the activities measured come from NapA alone. 3 Open in new tabDownload slide The effect of tungstate on P. pantotrophus NAP kinetics. Graphs show the dependence of the rate of nitrate reduction on nitrate concentration for periplasmic fractions from cells grown in the presence of (A) 3 µM Mo and (B) 100 µM W. Each point represents the average±S.E.M., where n=3. The line is the best fit, and values for Km and Vmax cited in the text were calculated using non-linear regression analysis done using Grafit v3.0 (Erithacus software). 3 Open in new tabDownload slide The effect of tungstate on P. pantotrophus NAP kinetics. Graphs show the dependence of the rate of nitrate reduction on nitrate concentration for periplasmic fractions from cells grown in the presence of (A) 3 µM Mo and (B) 100 µM W. Each point represents the average±S.E.M., where n=3. The line is the best fit, and values for Km and Vmax cited in the text were calculated using non-linear regression analysis done using Grafit v3.0 (Erithacus software). In E. coli, the maximal methyl viologen-dependent NAP activity in Mo-grown cells and W-grown cells was 0.07 and 0.01 µmol [NO3−] min−1 mg−1, respectively, consistent with the observations reported previously [31]. The latter value was too low for accurate kinetic analysis comparable to that performed with periplasmic extracts of P. pantotrophus. However, comparison of the activity at saturating substrate concentrations revealed that the pH optimum of NAP activity was 8.5 for periplasmic fractions from both Mo- and W-grown cultures and no differences were detected in sensitivity to inhibition by 2 M sucrose (~50% inhibition), 2 M NaCl (~25% inhibition) or 2 M sodium phosphate buffer, pH 7.4 (~50% inhibition). Furthermore, NAP activity from molybdate- and tungstate-supplemented cultures was strongly activated by 0.5 M KCl (~40% stimulation). The comparable catalytic properties of NapA within periplasmic fractions of Mo-grown and W-grown E. coli cells strongly suggest that, in contrast to P. pantotrophus, molybdo-NapA had not been replaced by an active tungsto-NapA during growth in tungstate-supplemented cultures. Rather, the lower specific nitrate reductase activities of the fractions prepared from the W-grown cells, despite comparable levels of NAP polypeptide and nap transcription, suggest that W prevents correct Mo incorporation, most likely through incorporation of the W itself leading to an inactive enzyme. 3.4 Thermostability of tungsten-substituted NAP from P. pantotrophus The thermal stability of NAP from P. pantotrophus grown in both molybdenum- and tungsten-supplemented media was assessed (Fig. 4A). Periplasmic fractions from cells grown on either molybdate (3 µM) or tungstate (100 µM) were incubated at a range of temperatures from 0 to 100°C for 30 min prior to assaying for nitrate reductase activity using reduced methyl viologen. Taking the activity of periplasms incubated on ice (0°C) for 30 min prior to assay to represent 100% active enzyme, the graph (Fig. 4A) clearly shows that increased activity (up to a maximum of 160% activity retained) is observed when periplasmic fractions from the cells grown on molybdate are incubated at 23°C (room temperature), 40°C and 50°C for 30 min prior to assay. Samples incubated at 60°C retained only ~10% of the activity. Above 70°C no activity was detected. For periplasmic fractions isolated from tungsten-grown cells the heat stability profile was markedly different. Again, taking the activity recorded after 30 min on ice (0°C) to represent 100% activity, a substantial decrease in activity was observed upon incubation of the periplasmic fraction at 23°C (room temperature) for 30 min, retaining only approximately 30% activity, demonstrating the poor thermal stability of the nitrate reductase produced in the presence of 100 µM tungstate. As the temperature was increased to 40–60°C, the percentage activity retained remained at ~20%. As with the periplasmic fraction from molybdenum-grown cells, the tungsten-substituted periplasmic fraction displayed no activity above 70°C. These different thermal stability profiles again strongly suggest that in the presence of a high concentration of tungstate, P. pantotrophus can assemble the periplasmic nitrate reductase with tungsten at the active site. However, in contrast to the tungsten-substituted TMAOR, tungsten-substituted NAP appears to have significantly decreased thermal stability. 4 Open in new tabDownload slide Thermal stability of nitrate reductase activity in periplasmic fractions isolated from cells of P. pantotrophus M6 and E. coli LCB2048 grown in the presence of either molybdate or tungstate. Enzyme assays monitoring the nitrate-dependent re-oxidation of methyl viologen were performed under anaerobic conditions at 20°C. A: Periplasmic fractions from P. pantotrophus cells grown in media containing 3 µM Mo (◯) or 100 µM W (●) were incubated at various temperatures for 30 min prior to activity assay. Each point represents the average±S.E.M., where n=3. B: E. coli cells grown in media containing 1 µM Mo (●) or 15 mM W (◯) were incubated at various temperatures for 15 min prior to activity assay. Each point represents the average of two determinations from separate experiments±S.D. 4 Open in new tabDownload slide Thermal stability of nitrate reductase activity in periplasmic fractions isolated from cells of P. pantotrophus M6 and E. coli LCB2048 grown in the presence of either molybdate or tungstate. Enzyme assays monitoring the nitrate-dependent re-oxidation of methyl viologen were performed under anaerobic conditions at 20°C. A: Periplasmic fractions from P. pantotrophus cells grown in media containing 3 µM Mo (◯) or 100 µM W (●) were incubated at various temperatures for 30 min prior to activity assay. Each point represents the average±S.E.M., where n=3. B: E. coli cells grown in media containing 1 µM Mo (●) or 15 mM W (◯) were incubated at various temperatures for 15 min prior to activity assay. Each point represents the average of two determinations from separate experiments±S.D. In E. coli, NAP activity from Mo-grown cells was also remarkably heat-stable with >25% of the activity still detectable after 15 min at 80°C (Fig. 4B). However, in contrast to P. pantotrophus (Fig. 4A) the NAP activity of W-grown E. coli cells showed comparable stability (Fig. 4B), again suggesting that NAP was incorporating Mo under both culture conditions. This was further supported by analysis of the half-lives for thermal inactivation, which were insignificantly different, 17 min for the molybdate- and 18 min for the tungstate-grown cultures. 4 Discussion We have examined the effect of tungstate on the periplasmic nitrate reductases from P. pantotrophus and E. coli. Our results show that at concentrations >15 mM, tungstate alone is highly toxic to both types of bacteria and toxicity might result from a highly elevated level of intracellular tungsten. In E. coli the modABCD operon, which encodes the molybdate transporter, is repressed by the ModE gene product. ModE is activated in the presence of molybdate, but is much less sensitive to tungstate [32]. However, the ModA transporter has an equal affinity for both molybdate and tungstate [33]. Consequently, in the absence of molybdate, tungstate may accumulate in the cell to a toxic level, which is significantly greater than the usual physiological level of intracellular molybdate. In media containing less tungstate (100 µM), in either the absence or presence of trace molybdate (6 nM), the periplasmic nitrate reductase in P. pantotrophus M6 can support anaerobic denitrification. Kinetic analysis of isolated periplasmic fractions demonstrated functional assembly of the periplasmic nitrate reductase, but with a ~70-fold decrease in catalytic activity and a 16-fold increase in the Km for nitrate. The observation that anaerobic growth can be supported while the catalytic activity of NAP has decreased ~70-fold demonstrates that nitrate reduction by W-Nap is not the rate-limiting step during nitrate respiration. It has been reported previously that NAP activity using the artificial electron donor MV+ is at least an order of magnitude faster than the physiological rate of nitrate respiration [34,35], again showing clearly that the rate of respiration is not limited by NAP turnover. Western blotting clearly demonstrated that the level of expression of NapA in the periplasm of cells grown in the presence of either molybdate or tungstate is the same. Given that NapA is a substrate for the twin arginine translocation apparatus [36], and that insertion of the MGD cofactor occurs in the cytoplasm, it is unlikely that the NapA apo-protein gets exported, as this would not be folded correctly. The high level of NapA detected in the periplasmic space of the tungstate-grown P. pantotrophus cells provides suggestive evidence for tungsto-pterin cofactor insertion. We therefore consider that the observed decrease in catalytic activity in the periplasmic fraction may result from only one of two possibilities: (1) poorly active enzyme with W at the catalytic site, or (2) the majority of enzyme is W-substituted and inactive, but a very small percentage of fully functional enzyme with Mo at the active site is also present. The apparent 16-fold increase in Km, indicating a significant decrease in the affinity for nitrate, provides strong evidence against the latter, and suggests the formation of a poorly functional NapA with W at the active site. However, the possibility of tungsten binding non-specifically to Mo-NAP, inhibiting nitrate reductase activity, cannot be excluded, but in the absence of any added molybdate to the growth medium, this is considered highly unlikely. Our model for nitrate reduction by NAP based upon spectroscopic studies [5] predicts that NO3− binds to Mo(IV) and is reduced to NO2− by the oxidation of Mo(IV) to Mo(VI). The midpoint redox potential, Em, for the NO3−/NO2− couple is +420 mV [37], suggesting that the Mo(IV)–NO3− complex in NAP can be stabilised at ~+400 mV. The reported midpoint redox potentials of the W(IV/V) and W(V/VI) couples of tungsten-substituted DMSOR from R. capsulatus are −203 mV and −105 mV, respectively [14]. Consequently, the low catalytic activity of W-NAP might result from the poor stability of the W(IV) at higher redox potentials. Previous attempts to produce a tungsto-derivative of the membrane-bound nitrate reductase from E. coli have resulted in inactive enzyme [38] and results presented herein demonstrate that there is no evidence for the incorporation of redox-active tungsten into the active site of NapA from E. coli. Thus the ability to generate a functional W-substituted NAP in P. pantotrophus, albeit with low catalytic activity, might reflect a distinct difference in the active sites of these iso-functional enzymes. Note that W substitution of the periplasmic DMSOR from R. capsulatus results in a functional enzyme [14], whereas W substitution of the membrane-bound DMSOR in E. coli does not [15]. Other differences between the P. pantotrophus and E. coli NapA include the failure of the anti-NapA antiserum used to recognise the E. coli NapA and the much greater tendency of NapA from P. pantotrophus to co-purify with NapB [21,31]. A number of differences in the amino acid composition of the active site pocket are also apparent which may underpin the present observation that these two enzymes also differ in their ability to function with tungsten rather than molybdenum at their active sites. The kinetic analysis shows that W-NAP catalyses the oxygen atom transfer from nitrate at a rate ~70-fold slower than Mo-NAP and is consistent with the enhanced bond strength of W complexes due to stronger π interactions. This enhanced bond strength has also been reported to account for the enhanced thermal stability of tungsto-enzymes [8], and Buc and co-workers [15] have demonstrated enhanced thermal stability of the tungsten-substituted TMAOR from E. coli. In contrast, the observed P. pantotrophus NAP activity reported in the present study is highly temperature-labile and activity is lost even after short incubation at room temperature. W complexes are often very oxygen-sensitive compared to their Mo counterparts [8] and the loss of nitrate reductase activity in the periplasmic fraction may also have resulted in part due to oxidative damage. Finally, are hyperthermostable nitrate reductases likely to be tungsto-enzymes? The low specific activity, low affinity for nitrate and poor thermal stability of tungsten-substituted NAP would suggest against this hypothesis. However, the fact that some activity is retained clearly highlights a distinct possibility that tungsten derivatives of nitrate reductase may exist. The only nitrate reductase from the hyperthermophilic archaea to be purified and characterised to date is that from Pyrobaculum aerophilum[39]. The enzyme is associated with the cytoplasmic membrane and is a complex of three subunits. Unlike the nitrate reductase from the mesophilic bacteria, the P. aerophilum nitrate reductase has very high specific activity (Vmax 1162 s−1 and Km 58 µM) and functions at an optimal temperature of >95°C [39]. The enzyme purified from cells grown in the presence of both molybdate and tungstate has been reported to contain molybdenum (0.8 mol mol complex−1) suggesting a ‘normal’ molybdo-enzyme. Acknowledgements This work was partially supported by BBSRC Research Grants 83/P13842 and 13/P17219 to D.J.R. and C.S.B., 6/P11528 to D.J.R. and J.A.C., and a Royal Society Research Grant to C.S.B. A.J.G. was in receipt of a Biochemical Society Vacation Studentship. We acknowledge Dr Ben Berks and Prof. Stuart Ferguson, University of Oxford, for useful discussions. We also thank Judith Nuttall and Dr Ben Berks for their help with raising the antibodies to NapA. References [1] Richardson D.J. Watmough N.J. ( 1999 ) Inorganic nitrogen metabolism in bacteria . Curr. Opin. Chem. Biol. 3 , 207 – 219 . Google Scholar Crossref Search ADS PubMed WorldCat [2] Berks B.C. Ferguson S.J. Moir J.W.B. Richardson D.J. ( 1995 ) Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions . Biochim. Biophys. Acta 1232 , 97 – 173 . Google Scholar Crossref Search ADS PubMed WorldCat [3] Sears H.J. Little P.J. Richardson D.J. Spiro S. Berks B.C. Ferguson S.J. ( 1997 ) Identification of an assimilatory nitrate reductase in mutants of Paracoccus denitrificans GB17 deficient on nitrate respiration . Arch. Microbiol. 167 , 61 – 66 . Google Scholar Crossref Search ADS PubMed WorldCat [4] Potter L. Angove H. Richardson D. Cole J.A. ( 2001 ) Nitrate reduction in the periplasm of Gram-negative bacteria . Adv. Microb. Physiol. 45 , 51 – 112 . Google Scholar Crossref Search ADS PubMed WorldCat [5] Butler C.S. Charnock J.M. Bennett B. Sears H.J. Reilly A.J. Ferguson S.J. Garner C.D. Lowe D.J. Thomson A.J. Berks B.C. Richardson D.J. ( 1999 ) Models for molybdenum co-ordination during the catalytic cycle of periplasmic nitrate reductase from Paracoccus denitrificans derived from EPR and EXAFS spectroscopy . Biochemistry 38 , 9000 – 9012 . Google Scholar Crossref Search ADS PubMed WorldCat [6] Butler C.S. Ferguson S.J. Berks B.C. Thomson A.J. Cheesman M.R. Richardson D.J. ( 2001 ) Assignment of haem ligands and detection of electronic absorption bands of molybdenum in the di-haem periplasmic nitrate reductase of Paracoccus pantotrophus . FEBS Lett. 500 , 71 – 74 . Google Scholar Crossref Search ADS PubMed WorldCat [7] Roldan M.D. Sears H.J. Cheesman M.R. Ferguson S.J. Thomson A.J. Berks B.C. Richardson D.J. ( 1998 ) Spectroscopic characterization of a novel multiheme c-type cytochrome widely implicated in bacterial electron transport . J. Biol. Chem. 273 , 28785 – 28790 . Google Scholar Crossref Search ADS PubMed WorldCat [8] Johnson M.K. Rees D.C. ( 1996 ) Tungstoenzymes . Chem. Rev. 96 , 2817 – 2839 . Google Scholar Crossref Search ADS PubMed WorldCat [9] Garner C.D. Stewart L.J. ( 2002 ) Tungsten-substituted molybdenum enzymes . Metal Ions Biol. Syst. 39 , 699 – 726 . OpenURL Placeholder Text WorldCat [10] Hales B.J. Case E.E. ( 1987 ) Nitrogen fixation by Azotobacter vinelandii in tungsten-containing medium . J. Biol. Chem. 262 , 16205 – 16211 . Google Scholar PubMed OpenURL Placeholder Text WorldCat [11] Deng M.D. Moureaux T. Caboche M. ( 1989 ) Tungstate, a molybdate analog inactivating nitrate reductase, deregulates the expression of the nitrate reductase structural gene . Plant Physiol. 91 , 304 – 309 . Google Scholar Crossref Search ADS PubMed WorldCat [12] Johnson J.L. Cohen H.J. Rajagopalan K.V. ( 1974 ) Molecular basis of the biological function of molybdenum. Molybdenum-free sulfite oxidase from livers of tungsten-treated rats . J. Biol. Chem. 249 , 5046 – 5055 . Google Scholar PubMed OpenURL Placeholder Text WorldCat [13] May H.D. Patel P.S. Ferry J.G. ( 1988 ) Effect of molybdenum and tungsten on synthesis and composition of formate dehydrogenase in Methanobacterium formicicum . J. Bacteriol. 170 , 3384 – 3389 . Google Scholar Crossref Search ADS PubMed WorldCat [14] Stewart L.J. Bailey S. Bennett B. Charnock J.M. Garner C.D. McAlpine A.S. ( 2000 ) Dimethylsulfoxide reductase: An enzyme capable of catalysis with either molybdenum or tungsten at the active site . J. Mol. Biol. 299 , 593 – 600 . Google Scholar Crossref Search ADS PubMed WorldCat [15] Buc J. Santini C.-L. Giordani R. Czjzek M. Wu L.-F. Giordano G. ( 1999 ) Enzymatic and physiological properties of the tungsten-substituted molybdenum TMAO reductase from Escherichia coli . Mol. Microbiol. 32 , 159 – 168 . Google Scholar Crossref Search ADS PubMed WorldCat [16] Bell L.C. Page M.D. Berks B.C. Richardson D.J. Ferguson S.J. ( 1993 ) Insertion of transposon Tn5 into a structural gene of the membrane-bound nitrate reductase of Thiosphaera pantotropha results in anaerobic overexpression of periplasmic nitrate reductase activity . J. Gen. Microbiol. 139 , 3205 – 3214 . Google Scholar Crossref Search ADS PubMed WorldCat [17] Robertson L.A. Kuenen J.G. ( 1983 ) Thiosphaera pantotropha gen. nov. sp. nov., a facultatively anaerobic, facultatively autotrophic sulphur bacterium . J. Gen. Miocrobiol. 129 , 2847 – 2855 . OpenURL Placeholder Text WorldCat [18] Blasco F. Nunzi F. Pommier J. Brasseur R. Chipaux M. Giordano G. ( 1992 ) Involvement of the narJ or narW gene production formation of active nitrate reductase in Escherichia coli . Mol. Microbiol. 6 , 209 – 219 . Google Scholar Crossref Search ADS PubMed WorldCat [19] Potter L.C. Millington P.D. Thomas G.H. Rothery R.A. Giordano G. Cole J.A. ( 2000 ) Novel growth characteristics and high rates of nitrate reduction of an Escherichia coli strain, LCB2048, that expresses only a periplasmic nitrate reductase . FEMS Microbiol. Lett. 185 , 51 – 57 . Google Scholar Crossref Search ADS PubMed WorldCat [20] Potter L. Millington P. Thomas G. Cole J. ( 1999 ) Competition between Escherichia coli strains expressing either a periplasmic or a membrane-bound nitrate reductase: does Nap confer a selective advantage during nitrate-limited growth? Biochem. J. 344 , 77 – 84 . Google Scholar PubMed OpenURL Placeholder Text WorldCat [21] Berks B.C. Richardson D.J. Robinson C. Reilly A. Aplin R.T. Ferguson S.J. ( 1994 ) Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha . Eur. J. Biochem. 220 , 117 – 124 . Google Scholar Crossref Search ADS PubMed WorldCat [22] Laemmli U.K. ( 1970 ) Cleavage of structural head proteins during assembly of the head bacteriophage T4 . Nature 227 , 280 – 285 . Google Scholar Crossref Search ADS WorldCat [23] Harboe N. Ingild A. ( 1973 ) Immunisation, Isolation of immunoglobulins and estimation of antibody titre . Scand. J. Immunol. 2 ( Suppl. 1 ), 161 – 164 . Google Scholar Crossref Search ADS WorldCat [24] Towbin H. Staehelin T. Gordon J. ( 1979 ) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets; procedure and some applications . Proc. Natl. Acad. Sci. USA 76 , 4350 – 4357 . Google Scholar Crossref Search ADS WorldCat [25] Brondijk T.H.C. Fiegen D. Richardson D.J. Cole J.A. ( 2002 ) Roles of NapF, NapG and NapH, subunits of the Escherichia coli periplasmic nitrate reductase, in ubiquinol oxidation . Mol. Microbiol. 44 , 245 – 255 . Google Scholar Crossref Search ADS PubMed WorldCat [26] Craske A.L. Ferguson S.J. ( 1986 ) The respiratory nitrate reductase from Paracoccus denitrificans. Molecular characterisation and kinetic properties . Eur. J. Biochem. 158 , 429 – 436 . Google Scholar Crossref Search ADS PubMed WorldCat [27] Showe M.K. DeMoss J.A. ( 1968 ) Localisation of synthesis of of nitrate reductase in Escherichia coli . J. Bacteriol. 95 , 1305 – 1313 . Google Scholar PubMed OpenURL Placeholder Text WorldCat [28] Potter L.C. Cole J.A. ( 1999 ) Essential roles for the products of the napABCD genes, but not napFGH, in periplasmic nitrate reduction by Escherichia coli K-12 . Biochem. J. 344 , 69 – 76 . Google Scholar PubMed OpenURL Placeholder Text WorldCat [29] Rech S. Wolin C. Gunsalus R.P. ( 1996 ) Properties of the periplasmic ModA molybdate-binding protein of Escherichia coli . J. Biol. Chem. 271 , 2557 – 2562 . Google Scholar Crossref Search ADS PubMed WorldCat [30] Sears H.J. Little P.J. Richardson D.J. Spiro S. Berks B.C. Ferguson S.J. ( 1997 ) Identification of an assimilatory nitrate reductase in mutants of Paracoccus denitrificans GB17 deficient on nitrate respiration . Arch. Microbiol. 167 , 61 – 66 . Google Scholar Crossref Search ADS PubMed WorldCat [31] Thomas G. Potter L. Cole J.A. ( 1999 ) The periplasmic nitrate reductase from Escherichia coli: a heterodimeric molybdoprotein with a double-arginine signal sequence and an unusual leader peptide cleavage site . FEMS Microbiol. Lett. 174 , 167 – 171 . Google Scholar Crossref Search ADS PubMed WorldCat [32] Grunden A.M. Self W.T. Villain M. Blalock J.E. Shanmugam K.T. ( 1999 ) An analysis of the binding repressor protein ModE to modABCD (molybdate transport) operator/promoter DNA of Escherichia coli . J. Biol. Chem. 274 , 24308 – 24315 . Google Scholar Crossref Search ADS PubMed WorldCat [33] Imperial J. Hadi M. Amy N.K. ( 1998 ) Molybdate binding by ModA, the periplasmic componente of the Escherichia coli mod molybdate transport system . Biochim. Biophys. Acta 1370 , 337 – 346 . Google Scholar Crossref Search ADS PubMed WorldCat [34] Richardson D.J. Ferguson S.J. ( 1992 ) The influence of carbon substrate on the activity of the periplasmic nitrate reductase in aerobically grown Thiosphaera pantotropha . Arch. Microbiol. 157 , 535 – 537 . OpenURL Placeholder Text WorldCat [35] Bell L.C. Page M.D. Berks B.C. Richardson D.J. Ferguson S.J. ( 1993 ) Insertion of transposon Tn5 into a structural gene of the membrane-bound nitrate reductase of Thiosphaera pantotropha results in anaerobic overexpression of periplasmic nitrate reductase activity . J. Gen. Microbiol. 139 , 3205 – 3214 . Google Scholar Crossref Search ADS PubMed WorldCat [36] Berks B.C. Sargent F. Palmer T. ( 2000 ) The Tat protein export pathway . Mol. Microbiol. 35 , 260 – 274 . Google Scholar Crossref Search ADS PubMed WorldCat [37] Nicholls D.G. Ferguson S.J. ( 2002 ) Bioenergetics 3 . Elsevier Science , London . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC [38] Enoch H.G. Lester R.L. ( 1972 ) Effects of molybdate, tungstate, and selenium compounds on formate dehydrogenase and other enzymes systems in Escherichia coli . J. Bacteriol. 110 , 1032 – 1040 . Google Scholar PubMed OpenURL Placeholder Text WorldCat [39] Afshar S. Johnson E. De Vries S. Schröder I. ( 2001 ) Properties of a thermostable nitrate reductase from the hyperthermophilic Archaeon Pyrobaculum aerophilium . J. Bacteriol. 183 , 5491 – 5495 . Google Scholar Crossref Search ADS PubMed WorldCat © 2003 Federation of European Microbiological Societies
TI - Properties of the periplasmic nitrate reductases from Paracoccus pantotrophus and Escherichia coli after growth in tungsten-supplemented media
JO - FEMS Microbiology Letters
DO - 10.1016/S0378-1097(03)00122-8
DA - 2003-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/properties-of-the-periplasmic-nitrate-reductases-from-paracoccus-K0cpuEfHT1
SP - 261
EP - 269
VL - 220
IS - 2
DP - DeepDyve
ER -