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Enzymatic Properties and Effect of Ionic Strength on Periplasmic Nitrate Reductase (NAP) fromDesulfovibrio desulfuricansATCC 27774
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
239, 816–822 (1997)
RC977560
Enzymatic Properties and Effect of Ionic Strength on Periplasmic Nitrate Reductase (NAP) from Desulfovibrio desulfuricans ATCC 27774 S. A. Bursakov,*,† C. Carneiro,* M. J. Almendra,* R. O. Duarte,* J. Caldeira,* I. Moura,* and J. J. G. Moura* *Departamento de QuıB mica (and Centro de QuıB mica Fina e Biotecnologia), Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, 2825 Monte de Caparica, Portugal; and †A.N.Bach Institute of Biochemistry RAS, Leninsky str.33, 117071 Moscow, Russia
Received September 5, 1997
Some sulfate reducing bacteria can induce nitrate reductase when grown on nitrate containing media being involved in dissimilatory reduction of nitrate, an important step of the nitrogen cycle. Previously, it was reported the purification of the first soluble nitrate reductase from a sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774 (S.A.Bursakov, M.-Y. Liu, W.J. Payne, J. LeGall, I. Moura, and J.J.G. Moura (1995) Anaerobe 1, 55–60). The present work provides further information about this monomeric periplasmic nitrate reductase (Dd NAP). It has a molecular mass of 74 kDa, 18.6 U specific activity, KM (nitrate) Å32 mM and a pHopt in the range 8-9.5. Dd NAP has peculiar properties relatively to ionic strength and cation/anion activity responses. It is shown that monovalent cations (potassium and sodium) stimulate NAP activity and divalent (magnesium and calcium) inhibited it. Sulfate anion also acts as an activator in KPB buffer. NAP native form is protected by phosphate anion from cyanide inactivation. In the presence of phosphate, cyanide even stimulates NAP activity (up to 15 mM). This effect was used in the purification procedure to differentiate between nitrate and nitrite reductase activities, since the later is effectively blocked by cyanide. Ferricyanide has an inhibitory effect at concentrations higher than 1 mM. The N-terminal amino acid sequence has a cysteine motive C-X2-C-X3C that is most probably involved in the coordination of the [4Fe-4S] center detected by EPR spectroscopy. The active site of the enzyme consists in a molybdopterin, which is capable for the activation of apo-nit-1 nitrate reductase of Neurospora crassa. The oxidized product of the pterin cofactor obtained by acidic hidrolysis of native NAP with sulfuric acid was identified by HPLC chromatography and characterized as a molybdopterin guanine dinucleotide (MGD). q 1997 Academic Press
Key Words: Desulfovibrio desulfuricans ATCC 27774; 0006-291X/97 $25.00
nitrate reductase; molybdenum enzymes; molybdopterin guanine dinucleotide; molybdenum; iron-sulfur cluster.
Sulfate reducing bacteria are spread in different extreme ecosystems with important impact in the environment and have broad metabolic capacities (1,2). Among them, the ability to reduce nitrate is quite rare but has been found in the genera Desulfovibrio (3,4,5), Desulfobulbus (6) and Desulfomonas (7). The first soluble nitrate reductase of this type has been isolated and purified to homogeneity from Desulfovibrio desulfuricans ATCC 27774 (8). It has been shown that this periplasmic enzyme is monomeric, with a molecular weight of 74 kDa and has some unique chemical environment on the Mo(V) site. D. desulfuricans ATCC 27774 is a slightly halophilic bacteria and has some specific behaviour in the presence of salts in vitro conditions. It can grow in a salinity range 0.5-6% of NaCl (1,2). However, not enough information is available about the specific adaptation of proteins to these conditions. In this paper, we present further characterization of the molybdenum site of this novel nitrate reductase and some unique enzymatic properties of NAP related to salt dependence, which are unusual when compared to other nitrate reductases. We also take advantage of cyanide inhibition reactions, and the insensitivity of some Mo-containing enzymes in the as prepared form (9-10), as a key step for nitrite determination in the presence of nitrite reductase (NIR) that has a higher turnover for converting nitrite than NAP for conversion of nitrate. MATERIALS AND METHODS Growth conditions. D. desulfuricans ATCC 27774 was grown in anaerobic conditions in the medium described by Liu and Peck (15).
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Nitrate, rather than sulfate was used as terminal electron acceptor to induce the production of nitrate reductase. Harvested cells were suspended in 10 mM Tris-HCl in a 1/1 ratio (weight/volume), pH 7.6, and passed through a Manton-Gaulin press at 9,000 psi. The extract was centrifuged at 10,000xg for 1 h and then at 30,000xg for 45 min to eliminate the membrane fraction. NAP purification. Purification procedure was adapted and modified from the one published earlier (8). All purification procedures were performed at 4 7C and all buffers were pH 7 or otherwise special mentioned. The crude extract (600ml) was loaded in to DEAE-Biogel, pre-equilibrated with 10 mM Tris-HCl. NAP fraction was eluted between 75-100 mM with a linear gradient from 10-500 mM of the same buffer. The active fractions were joined, concentrated and dyalised in a Amicon ultrafiltration cell with a YM-10 membrane. After dialysis it was loaded in a DEAE-Waters column using the same buffers as previously. NAP was eluted at 100 mM, concentrated and loaded in a Superdex-75 using 300 mM Tris-HCl. The resultant active fraction was applied in a Q-Resourse (6 ml) and after this step NAP was pure. The purity of the fraction was confirmed by polyacrylamide gel electrophoresis. The activity was determined along the purification process at each step and the ratio A400/A280 Å 0.15 was determined in the last step of purification. Enzyme and protein assay. The NAP enzyme was routinely assayed at 37 7C in 0.5 M KPB buffer, pH 8.0, or AMPSO 0.5 M pH 9, by measuring the nitrite produced (12). The reaction mixture contains: 1.4 mM methyl viologen, 20 mM NaNO3 , 50 mM NaCl with 20 mM KCN in a final volume of 0.5 ml. The reaction was started by adition of 30 ml of sodium dithionite (8 mg/ml) in 8 % NaHCO3 . After incubation 3-5 min the reaction was stopped by vigorously shaking. The nitrite formed was measured with sulfanilamide and N(naphthyl)ethylene diamine hydrochloride (13) and the absorbance measured at 540 nm. The enzymatic unit is defined by mmol of nitrate reduced to nitrite per minute. The specific activity (U/mg) is the activity per mg of protein The NIR activity was determined by the same methodology except nitrate was replaced by nitrite and incubation was 1-2 min. Protein concentration. Was estimated using the Bradford (14) and Lowry (15) method with bovine serum albumine as a standard. Inactivation experiments. Except otherwise stated, in all experiments the enzyme was introduced in the reaction mixture containing all reagents for reaction with methyl viologen. In some other assays (ferricyanide effect) the enzyme was incubated 5 min at 20 7C with the reagents and small aliquots withdrawn at intervals and transferred to the assay mixture. Molybdenum-cofactor assay. Assays of the molybdenum-cofactor content of enzyme samples were carried out by using the apo-(nit1)-nitrate reductase assay of Hawkes & Bray (16). After liberating NAP cofactor anaerobically by heat shock (17) reconstituted NADPHdependent nitrate reductase activity was measured under aerobic conditions. To establish standard conditions for Mo-cofactor determination the same conditions were used in the extraction of Mo-co from purified xanthine oxidase and assayed using the reconstitution assay. Acid hydrolysis of the MPT dinucleotide. NAP (0.145 mg of protein, i.e., 1.96 nmol) has been made in 0.05 ml of 50 mM ammonium acetate buffer, pH 6.0 and treated with sulfuric acid as described previously (18, 19). Denaturated protein was removed by centrifugation at 10,000xg for 10 min. Nucleotides were analyzed in the supernatant using a HPLC column (Merck Supersher 100 RP-18, 250 1 4 mm). Isocratic elution was performed using ammonium acetate buffer, 50 mM pH 6.0, as a mobile phase with a flow rate of 1 ml min01. Organic compounds were identified with a UV- detector (model L-7400, LaChrom HPLC Systems). N-terminal amino acid sequence. N-terminal sequence was determined by automated Edman degradation in a Applied Biosystem
FIG. 1. Effect of cyanide on D. desulfuricans ATCC 27774 nitrate and nitrite reductases activities. Nitrite reductase (l), nitrate reductase (j).
Protein Sequencer Model 477 A coupled to an Applied Biosystem 120 Analyser following the manufacter’s instrutions (675 pmol of NAP were used). Chemicals and chromatographic materials. Low molecular mass protein standards were purchased from Bio-Rad and DEAE-52 from Watman. All other chemicals were of reagent grade or of highest available purity.
RESULTS In a earlier report periplasmic nitrate reductase (NAP) from a sulfate reducer D.desulfuricans ATCC 27774 was purified to homogeneity as a monomer (circa 70 kDa) with a specific activity of 5.4 U (8). The purification procedure performed this time involved only four chromatographic steps and it produced NAP with a highest specific activity 18.6 U. To follow NAP activity during purification a special reaction mixture is used, as described in materials and methods. It includes KCN, a well known inhibitor, due to the fact that nitrite reductase, also present in the first steps of purification has a very high activity and does not allow the determination of the nitrite produced by NAP. In the reaction mixture KCN concentration is such that NIR is inactive but it does not inhibit NAP. NAP is not inhibited by 15 mM KCN, but is even stimulated, where pure NIR can be inhibited by a concentration of KCN of 0.11 mM (Fig. 1). Nitrite formation is proportional with the incubation time in the first 15-20 min with and without NaCl. The purified enzyme is active with viologen dyes such as methyl and benzyl viologen and bromophenolblue, with specific activity 18.6, 5.0 and 0.3 U respectively. No activity is observed with reduced nucleotides: flavine (FADH2) and pyridines (NAD(P)H). Formate is not effective as an electron donor with pure NAP, but only with free living cells. Nitrate formation is greatly influenced by pH in the range of 4.2-12.8. NAP activity was studied in different buffer systems. The pH shape dependency is found to
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In the first system a sligth inactivation is observed, while in the second activation occurs. KCN effect in AMPSO, with or without NaCl, reveal the usual inhibition behavior (data not shown); meanwhile in phosphate buffer it is observed an increase of activity followed by inactivation that is dependent on the ionic strengh of the reaction mixture, suggesting a protective effect of phosphate ions. The KM for the substrate was determined and it was obtained a KM (nitrate) Å 32 mM in the absence of NaCl. The KM in the presence of NaCl is similar KM (nitrate) Å 12 mM. The determined KM for the most effective electron donor - methylviologen is 81 mM, with a 20 mM nitrate concentration. Several storage conditions were tested using low protein concentration (11 mg/ml) in various buffer systems. The mixture of phosphate buffer (10mM), glycerol (50%), EDTA (0.4mM) (without and with DTT (1.4 mM)) are effective, and preserve NAP from rapid inactivation (70% residual activity after 15 days). If the storage conditions are made in AMPSO (132 mM) or
FIG. 2. pH dependence of D. desulfuricans ATCC 27774 nitrate reductase activity. (Panel A) Ionic buffers: Citric acid (,), Phosphate (j), Tris-HCl (l), Glicine/NaOH (h), Bicarbonate/NaOH(m). (Panel B) Unionic buffers: MES (.), MOPS (h), HEPES (s), Tricine (j), Bicine (n), AMPSO (l), CAPS (,).
be sharp and very similar in both nonionic and ionic buffers, with an optimum pH between 8.0-9.5 as shown in the curve (Fig. 2). The effect of different salts in MVH-NAP activity was studied in AMPSO and phosphate buffers (Fig. 3, Panel A). No difference was found when the two buffer systems were compared. Monovalent cations K/ and Na/ stimulate this activity, but divalent cations Ca2/ and Mg2/, known as protein destabilizers (20), produce strong inhibition. Ammonium cation practically does not affect the enzymatic activity. In particular, the NAP activity followed in the presence of sodium and potassium sulfate, in buffers such as AMPSO and phosphate, show a different behavior.
FIG. 3. Salt dependence of D. desulfuricans ATCC 27774 nitrate reductase activity. (Panel A) KCl (n), NaCl (j),NH4Cl (l), MgCl2 (h), CaCl2 (m) (AMPSO and KPB). (Panel B) K2SO4/ AMPSO (j), K2SO4/KPB (s).
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FIG. 4. Ferricyanide effect on D. desulfuricans ATCC 27774 nitrate reductase activity. 0 mM (h), 0.1 mM (m), 1 mM (l), 10 mM (n).
in phosphate buffer (400 mM) with NaCl (400 mM and 960 mM) this leads to 22 % and 8 % of the initial activity respectively. D. desulfuricans NAP has a high similarity with some proteins from extremly halophilic bacteria which are stabilized at low salt concentrations (21). The purified NAP is stable if frozen at 020 7C with no lost of specific activity, even after several months, but cycles of frozen-defrozen causes partial precipitation, indicating protein denaturation with consequent lost of activity. The effect of prolonged ferricyanide treatment, until 6 hrs on NAP, as prepared, was investigated within a range of 0 to 10 mM (Fig. 4). Zero concentration is taken as a standard; for 0.1 mM the specific activity remains practically the same, but with 1-10 mM strong and rapid (in the second case) inhibition is observed till zero value of specific activity. This effect was reproducible with different loading reagents order (AMPSO, MV and nitrate) and the reaction started by dithionite addition. With exception of nitrogenase, all molybdenum-containing enzymes have a molybdenum cofactor in which molybdenum is bound with a 6-substituted pterin. We examined the presence of active molybdenum cofactor in NAP, by using the apo-(nit-1) nitrate reductase assay. The experimental conditions used, provide a quantitative basis for the assay of the Mo cofactor (16). Measurements were carried out with the addition of Na2MoO4 (10 mM) to the complementation media. The extraction procedure and complementation was also checked for xanthine oxidase (XO) under the same conditions. Increasing amounts of Mo cofactor, extracted from NAP and XO, were incubated with nit-1 mutant crude extract and finally assayed for NADPH-dependent NAR activity. The slopes of the plots, of the liberated nitrate against the correspondent added enzyme, were found to be linear. The highest nitrate reductase activity, after complementation with nit-1, was 8.68
mmol of nitrite per mg of protein for XO and 10.5 mmol of NO02 per mg for NAP, equivalent to 7% and 9% of recovered activity respectively. This cofactor extraction and transfer efficiency, for both enzymes, is in good agreement with heat treatment type of extraction procedure (22). It states that NAP contains an Mo cofactor able for the activation of apo-(nit-1) nitrate reductase. Upon acid hydrolysis 5*-GMP identification was possible, indicating the presence of a MGD type molybdenum cofactor. The MGD quantity determined is 1.3 mol per 1 mol of NAP suggesting that there are two MGD moieties per protein. The N-terminal amino acid sequence (Fig. 5) reveal a cysteine rich motif, which may be involved in the coordination of the iron-sulfur center (23); homologies with other molybdenum cofactor-containing enzymes is shown: nitrate reductase from E. coli (24), periplasmic nitrate reductase from Alcaligenes eutrophus H16 (25), formate dehydrogenase from Methanobacterium formicicum (26), assimilatory nitrate reductase of Klebsiella pneumonia M5 (27), polysulfide reductase chain A from Wolinella succinogenes (28) and a nitrate-inducible formate dehydrogenase from E. coli (29). The homology with NAP A from E. coli is 54 % and with NAP from A. eutrophus is 56 %. DISCUSSION D. desulfuricans is a slightly halophilic bacterium, and NAP properties were compared with other enzymes from different halophilic bacteria. Two classes were proposed (30), one resembling non-halophilic proteins which are either active or stable in the absence of salt (21, 32-34), and the other, hallophilic, showing unique properties that may be related to salt dependence (31). The isoelectric point of most of the proteins from extremely halophilic bacteria are between 4.0 and 4.65 which is in agreement with the generally high content of glutamic and aspartic acid in proteins from halobacteria (30). It was found that NAP has similar properties, concerning salts, with the nitrate reductase from Halobacterium marismortui (35), suggesting that the excess of acidic over basic amino acids can explain this behavior. More, both have high specific activity in the presence of salts, but are more stable if low salt concentration are used in the storage conditions. Also, a stimulatory effect on yeast Candida nitratophila assimilatory nitrate-reducing activity was observed upon increasing ionic strength (36). Salts can affect in widely different manner the properties of enzymes like their stability, solubility and activity (20). At low concentrations, salts can stabilize through nonspecific electrostatic interactions with the enzyme, depending only on the ionic strength of the medium (37). For high concentrations, however, salts have specific effects dependent on the nature of salt and its concentration, resulting in either stabilization
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FIG. 5. Comparison N-terminal sequence of the periplasmic nitrate reductase from D. desulfuricans ATCC 27774 with sequences of the following molybdenum cofactor-containing enzymes: periplasmic nitrate reductases of E.coli (NAPA E.coli) (25), Alcaligenes eutrophus H16 (NAPA A.eutr.) (26), formate dehydrogenase of Methanobacterium formicicum (FDHA M. form.) (27), assimilatory nitrate reductase of Klebsiella pneumonia M5 (NASA K.pneu.) (28), polysulfide reductase chain A Wolinella succinogenes (PSRA W.succ.) (29), nitrate-inducible formate dehydrogenase of E.coli (FDNG)(30).
or denaturation. NAP activity in different salts conditions reveal that potassium and sodium chlorides, both in ionic and nonionic buffer, in high concentrations, have an activation effect. Sulfate anion on the contrary has a different behavior in the two buffer systems, activation only occurs in phosphate buffer, pointing out a dependence with the ionic strength. These facts suggest that the enzyme may suffer changes in the global charge of the surface modifying the molecule configuration and making the molybdenum site more accessible, justifying in this way the higher activities for high salt concentration. Calcium and magnesium turn the system thermodynamically less stable, so NAP activity decreases. This phenomenon may involve changes in the nature and polarity of NAP surface, hydration and charged state (38). In this case the molecule configuration changes in a way that the catalytic site is not easily accessible, decreasing the activity. The experiments performed in different buffer systems demonstrate that salts have an activation effect during the catalytic act, but are not so effective as a protector against NAP inactivation during long time storage. However, phosphate anion always has a positive effect on NAP activity and storage, which is very similar with what is observed with ferredoxin-nitrate reductase from Plectonema boryanum (39). D. desulfuricans ATCC 27774 pure NAP, as prepared, is not activated by short or prolonged incubation with ferricyanide oppositly to membrane bound nitrate reductase from E. coli (40) and assimilatory nitrate reductase from Chlorella vulgaris (10). Moreover, strong inhibition occurs with 1 to10 mM ferricyanide. Sequence comparisons between NAP with other molybdenum cofactor-containing enzymes such as nitrate reductases, polysulfide reductase and formate dehydrogenases from various sources revealed a conserved segment. Three cysteine residues coordinates an iron-sulphur center, as proposed for similar cysteine clusters found in formate dehydrogenase of E. coli (28) and NAD-reducing hydrogenase of A. eutrophus (41). They are located at the amino-terminal part of the polypeptide chain and form a structure C-X2-C-X3-C which is
very similar with the C-X2-C-X3-C-X27-C cluster, resembling the [4Fe-4S] centers found in bacterial ferredoxins (42). The characterization of the Mo site in NAP as well as the details on enzymatic properties described here, help to place this novel enzyme in the large family of monomeric molyddenum enzymes, further support by recent EXAFS studies (col. G.N.George) that indicated the presence of two pterins, one cysteinyl residue and one hydroxyl group in the Mo coordination sphere. D.desulfuricans ATCC 27774 has been a source of a variety of molybdopterin containing enzymes, namely aldehyde oxido-reductase (43), formate dehydrogenase (44) and nitrate reductase (45). These molybdenum proteins belong to sub-groups classified on the basis of whether the molydenum center of the active site possesses a MoO2 or MoOS unit (11) (see also footnote), defined as: (1) The xanthine oxidase family [(MCD - molybdopterin cytosine dinucleotide or MPT - molybdopterin) MoVI ÅO, ÅS, 0H2O] that includes enzymes such as xanthine oxidase/dehydrogenase, aldehyde oxidase, CO dehydrogenase and the aldehyde oxido-reductases isolataded in sulfate reducers (43, 46). Members of this family possess a MoVIOS center and one molybdopterin, and no covalent bonding between the cofactor and the polypeptide chain. In the coordination sphere is an additional water molecule or hydroxo group. A Mo(O)2 unit is generated after cyanide reaction. The inactivation mechanism involves cyanide abstraction of sulfur from the molybdenum center, in the form of thiocyanate (9, 47), which is essential for enzyme activity. (2) The DMSO reductase family [(MGD - molybdopterin guanine dinucleotide)2 MoVI ÅO, (-OSer, -SCys or -SeCys)] includes enzymes such as biotin-S-oxide reductase, dimethylsulfoxide reductase, dissimilatory nitrate reductase and formate dehydrogenase. Two molybdopterins (MGD) are found in the coordination sphere of Mo and an aminoacid side chain coordinates the metal: cysteine in the periplasmic nitrate redutase (8), selenum-cysteine in formate dehydrogenase (48) or serine in DMSO reductase (49, 50).
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A third subgroup includes the sulfite oxidase family [(MPT) MoVI ÅO, 0H2O, (-SCys)] that also includes assimilatory nitrate reductase. Enzymes within this family typically have one molybdopterin cofactor and a MoVIO2 nucleus and the polypeptide chain coordinates directly to the molybdenum site by a cysteinyl residue (51). Three crystal structures of molybdopterin-containing enzymes were reported for members of the first two families (aldehyde oxido-reductase from Desulfovibrio gigas (46, 52), DMSO reductases from Rhodobacter sphaeroides and capsulatus (50, 53) and formate dehydrogenase from E. coli (48)). Also, there is a preliminary report for the X-ray structure of chicken liver sulfite oxidase (51). Attempts to solve the 3D structure of D.desulfuricans ATCC 2774 are in progress (our work in collaboration with M.J.Roma˜o).1 ACKNOWLEDGMENTS This work was supported PRAXIS 2/2.1/BIO/05/94. CC thanks PhD Grants JNICT BD/2192/92-IF and PRAXIS BD/5402/95. SAB is PRAXIS Visiting Professor in Portugal. We wish to thank Rene´ Toci for the growth of cells and Manuela Regala for the amino acid Nterminal sequence and C.Costa for NIR preparation from D. desulfuricans ATCC 27774.
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Moura, J. J. G. (1996), 3rd Eur. Conf. Bioinorg. Chem., Noorwijkerhout, Netherlands, Aug, 4–10. Costa, C., Teixeira, M., LeGall, J., Moura, J. J. G., and Moura, I. (1997) J. Biol. Inorg. Chem. 2, 198–208. Hille, R. (1996) Chem. Rev. 96, 2757–2816. Roma˜o, M. J., Archer, M., Moura, I., Moura, J. J. G., LeGall, J., Engh, R., Schneider, M., Hof, P., and Huber, R. (1995) Science 167, 1170–1176. Massey, V., and Edmondson, D. (1970) J. Biol. Chem. 245, 6595– 6598. Boyington, J. C., Gladyshev, V. N., Khangulov, S. V., Stadtman, T. C., Sun, P. D. (1997) Science 275, 1305–1308.
49. Kna¨blein, J., Mann, K., Ehlert, S., Fonstein, M., Huber, R., and Schneider, F. (1996) J. Mol. Biol. 263, 40–52. 50. Schindelin, H., Kisker, C., Hilton, J., Rajagopalan, K. V., and Rees, D. C. (1996) Science 272, 1615–1621. 51. Kisker, C., Schindelin, H., and Rees, D. C., (1997) Molybdenum Enzymes Meeting, Univ. Sussex, April 1997. 52. Huber, R., Hof, P., Duarte, R. O., Moura, J. J. G., Moura, I., Liu. M-Y., LeGall, J., Hille, R., Archer, M., and Roma˜o, M. J. (1996) PNAS 93, 8846–8861. 53. Schneider, F., Lo¨we, J., Huber, R., Schindelin, H., Kisker, C., and Kna¨blein, J. (1996) J. Mol. Biol. 263, 53–69.
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Enzymatic Properties and Effect of Ionic Strength on Periplasmic Nitrate Reductase (NAP) from Desulfovibrio desulfuricans ATCC 27774 S. A. Bursakov,*,† C. Carneiro,* M. J. Almendra,* R. O. Duarte,* J. Caldeira,* I. Moura,* and J. J. G. Moura* *Departamento de QuıB mica (and Centro de QuıB mica Fina e Biotecnologia), Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, 2825 Monte de Caparica, Portugal; and †A.N.Bach Institute of Biochemistry RAS, Leninsky str.33, 117071 Moscow, Russia
Received September 5, 1997
Some sulfate reducing bacteria can induce nitrate reductase when grown on nitrate containing media being involved in dissimilatory reduction of nitrate, an important step of the nitrogen cycle. Previously, it was reported the purification of the first soluble nitrate reductase from a sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774 (S.A.Bursakov, M.-Y. Liu, W.J. Payne, J. LeGall, I. Moura, and J.J.G. Moura (1995) Anaerobe 1, 55–60). The present work provides further information about this monomeric periplasmic nitrate reductase (Dd NAP). It has a molecular mass of 74 kDa, 18.6 U specific activity, KM (nitrate) Å32 mM and a pHopt in the range 8-9.5. Dd NAP has peculiar properties relatively to ionic strength and cation/anion activity responses. It is shown that monovalent cations (potassium and sodium) stimulate NAP activity and divalent (magnesium and calcium) inhibited it. Sulfate anion also acts as an activator in KPB buffer. NAP native form is protected by phosphate anion from cyanide inactivation. In the presence of phosphate, cyanide even stimulates NAP activity (up to 15 mM). This effect was used in the purification procedure to differentiate between nitrate and nitrite reductase activities, since the later is effectively blocked by cyanide. Ferricyanide has an inhibitory effect at concentrations higher than 1 mM. The N-terminal amino acid sequence has a cysteine motive C-X2-C-X3C that is most probably involved in the coordination of the [4Fe-4S] center detected by EPR spectroscopy. The active site of the enzyme consists in a molybdopterin, which is capable for the activation of apo-nit-1 nitrate reductase of Neurospora crassa. The oxidized product of the pterin cofactor obtained by acidic hidrolysis of native NAP with sulfuric acid was identified by HPLC chromatography and characterized as a molybdopterin guanine dinucleotide (MGD). q 1997 Academic Press
Key Words: Desulfovibrio desulfuricans ATCC 27774; 0006-291X/97 $25.00
nitrate reductase; molybdenum enzymes; molybdopterin guanine dinucleotide; molybdenum; iron-sulfur cluster.
Sulfate reducing bacteria are spread in different extreme ecosystems with important impact in the environment and have broad metabolic capacities (1,2). Among them, the ability to reduce nitrate is quite rare but has been found in the genera Desulfovibrio (3,4,5), Desulfobulbus (6) and Desulfomonas (7). The first soluble nitrate reductase of this type has been isolated and purified to homogeneity from Desulfovibrio desulfuricans ATCC 27774 (8). It has been shown that this periplasmic enzyme is monomeric, with a molecular weight of 74 kDa and has some unique chemical environment on the Mo(V) site. D. desulfuricans ATCC 27774 is a slightly halophilic bacteria and has some specific behaviour in the presence of salts in vitro conditions. It can grow in a salinity range 0.5-6% of NaCl (1,2). However, not enough information is available about the specific adaptation of proteins to these conditions. In this paper, we present further characterization of the molybdenum site of this novel nitrate reductase and some unique enzymatic properties of NAP related to salt dependence, which are unusual when compared to other nitrate reductases. We also take advantage of cyanide inhibition reactions, and the insensitivity of some Mo-containing enzymes in the as prepared form (9-10), as a key step for nitrite determination in the presence of nitrite reductase (NIR) that has a higher turnover for converting nitrite than NAP for conversion of nitrate. MATERIALS AND METHODS Growth conditions. D. desulfuricans ATCC 27774 was grown in anaerobic conditions in the medium described by Liu and Peck (15).
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Nitrate, rather than sulfate was used as terminal electron acceptor to induce the production of nitrate reductase. Harvested cells were suspended in 10 mM Tris-HCl in a 1/1 ratio (weight/volume), pH 7.6, and passed through a Manton-Gaulin press at 9,000 psi. The extract was centrifuged at 10,000xg for 1 h and then at 30,000xg for 45 min to eliminate the membrane fraction. NAP purification. Purification procedure was adapted and modified from the one published earlier (8). All purification procedures were performed at 4 7C and all buffers were pH 7 or otherwise special mentioned. The crude extract (600ml) was loaded in to DEAE-Biogel, pre-equilibrated with 10 mM Tris-HCl. NAP fraction was eluted between 75-100 mM with a linear gradient from 10-500 mM of the same buffer. The active fractions were joined, concentrated and dyalised in a Amicon ultrafiltration cell with a YM-10 membrane. After dialysis it was loaded in a DEAE-Waters column using the same buffers as previously. NAP was eluted at 100 mM, concentrated and loaded in a Superdex-75 using 300 mM Tris-HCl. The resultant active fraction was applied in a Q-Resourse (6 ml) and after this step NAP was pure. The purity of the fraction was confirmed by polyacrylamide gel electrophoresis. The activity was determined along the purification process at each step and the ratio A400/A280 Å 0.15 was determined in the last step of purification. Enzyme and protein assay. The NAP enzyme was routinely assayed at 37 7C in 0.5 M KPB buffer, pH 8.0, or AMPSO 0.5 M pH 9, by measuring the nitrite produced (12). The reaction mixture contains: 1.4 mM methyl viologen, 20 mM NaNO3 , 50 mM NaCl with 20 mM KCN in a final volume of 0.5 ml. The reaction was started by adition of 30 ml of sodium dithionite (8 mg/ml) in 8 % NaHCO3 . After incubation 3-5 min the reaction was stopped by vigorously shaking. The nitrite formed was measured with sulfanilamide and N(naphthyl)ethylene diamine hydrochloride (13) and the absorbance measured at 540 nm. The enzymatic unit is defined by mmol of nitrate reduced to nitrite per minute. The specific activity (U/mg) is the activity per mg of protein The NIR activity was determined by the same methodology except nitrate was replaced by nitrite and incubation was 1-2 min. Protein concentration. Was estimated using the Bradford (14) and Lowry (15) method with bovine serum albumine as a standard. Inactivation experiments. Except otherwise stated, in all experiments the enzyme was introduced in the reaction mixture containing all reagents for reaction with methyl viologen. In some other assays (ferricyanide effect) the enzyme was incubated 5 min at 20 7C with the reagents and small aliquots withdrawn at intervals and transferred to the assay mixture. Molybdenum-cofactor assay. Assays of the molybdenum-cofactor content of enzyme samples were carried out by using the apo-(nit1)-nitrate reductase assay of Hawkes & Bray (16). After liberating NAP cofactor anaerobically by heat shock (17) reconstituted NADPHdependent nitrate reductase activity was measured under aerobic conditions. To establish standard conditions for Mo-cofactor determination the same conditions were used in the extraction of Mo-co from purified xanthine oxidase and assayed using the reconstitution assay. Acid hydrolysis of the MPT dinucleotide. NAP (0.145 mg of protein, i.e., 1.96 nmol) has been made in 0.05 ml of 50 mM ammonium acetate buffer, pH 6.0 and treated with sulfuric acid as described previously (18, 19). Denaturated protein was removed by centrifugation at 10,000xg for 10 min. Nucleotides were analyzed in the supernatant using a HPLC column (Merck Supersher 100 RP-18, 250 1 4 mm). Isocratic elution was performed using ammonium acetate buffer, 50 mM pH 6.0, as a mobile phase with a flow rate of 1 ml min01. Organic compounds were identified with a UV- detector (model L-7400, LaChrom HPLC Systems). N-terminal amino acid sequence. N-terminal sequence was determined by automated Edman degradation in a Applied Biosystem
FIG. 1. Effect of cyanide on D. desulfuricans ATCC 27774 nitrate and nitrite reductases activities. Nitrite reductase (l), nitrate reductase (j).
Protein Sequencer Model 477 A coupled to an Applied Biosystem 120 Analyser following the manufacter’s instrutions (675 pmol of NAP were used). Chemicals and chromatographic materials. Low molecular mass protein standards were purchased from Bio-Rad and DEAE-52 from Watman. All other chemicals were of reagent grade or of highest available purity.
RESULTS In a earlier report periplasmic nitrate reductase (NAP) from a sulfate reducer D.desulfuricans ATCC 27774 was purified to homogeneity as a monomer (circa 70 kDa) with a specific activity of 5.4 U (8). The purification procedure performed this time involved only four chromatographic steps and it produced NAP with a highest specific activity 18.6 U. To follow NAP activity during purification a special reaction mixture is used, as described in materials and methods. It includes KCN, a well known inhibitor, due to the fact that nitrite reductase, also present in the first steps of purification has a very high activity and does not allow the determination of the nitrite produced by NAP. In the reaction mixture KCN concentration is such that NIR is inactive but it does not inhibit NAP. NAP is not inhibited by 15 mM KCN, but is even stimulated, where pure NIR can be inhibited by a concentration of KCN of 0.11 mM (Fig. 1). Nitrite formation is proportional with the incubation time in the first 15-20 min with and without NaCl. The purified enzyme is active with viologen dyes such as methyl and benzyl viologen and bromophenolblue, with specific activity 18.6, 5.0 and 0.3 U respectively. No activity is observed with reduced nucleotides: flavine (FADH2) and pyridines (NAD(P)H). Formate is not effective as an electron donor with pure NAP, but only with free living cells. Nitrate formation is greatly influenced by pH in the range of 4.2-12.8. NAP activity was studied in different buffer systems. The pH shape dependency is found to
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In the first system a sligth inactivation is observed, while in the second activation occurs. KCN effect in AMPSO, with or without NaCl, reveal the usual inhibition behavior (data not shown); meanwhile in phosphate buffer it is observed an increase of activity followed by inactivation that is dependent on the ionic strengh of the reaction mixture, suggesting a protective effect of phosphate ions. The KM for the substrate was determined and it was obtained a KM (nitrate) Å 32 mM in the absence of NaCl. The KM in the presence of NaCl is similar KM (nitrate) Å 12 mM. The determined KM for the most effective electron donor - methylviologen is 81 mM, with a 20 mM nitrate concentration. Several storage conditions were tested using low protein concentration (11 mg/ml) in various buffer systems. The mixture of phosphate buffer (10mM), glycerol (50%), EDTA (0.4mM) (without and with DTT (1.4 mM)) are effective, and preserve NAP from rapid inactivation (70% residual activity after 15 days). If the storage conditions are made in AMPSO (132 mM) or
FIG. 2. pH dependence of D. desulfuricans ATCC 27774 nitrate reductase activity. (Panel A) Ionic buffers: Citric acid (,), Phosphate (j), Tris-HCl (l), Glicine/NaOH (h), Bicarbonate/NaOH(m). (Panel B) Unionic buffers: MES (.), MOPS (h), HEPES (s), Tricine (j), Bicine (n), AMPSO (l), CAPS (,).
be sharp and very similar in both nonionic and ionic buffers, with an optimum pH between 8.0-9.5 as shown in the curve (Fig. 2). The effect of different salts in MVH-NAP activity was studied in AMPSO and phosphate buffers (Fig. 3, Panel A). No difference was found when the two buffer systems were compared. Monovalent cations K/ and Na/ stimulate this activity, but divalent cations Ca2/ and Mg2/, known as protein destabilizers (20), produce strong inhibition. Ammonium cation practically does not affect the enzymatic activity. In particular, the NAP activity followed in the presence of sodium and potassium sulfate, in buffers such as AMPSO and phosphate, show a different behavior.
FIG. 3. Salt dependence of D. desulfuricans ATCC 27774 nitrate reductase activity. (Panel A) KCl (n), NaCl (j),NH4Cl (l), MgCl2 (h), CaCl2 (m) (AMPSO and KPB). (Panel B) K2SO4/ AMPSO (j), K2SO4/KPB (s).
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FIG. 4. Ferricyanide effect on D. desulfuricans ATCC 27774 nitrate reductase activity. 0 mM (h), 0.1 mM (m), 1 mM (l), 10 mM (n).
in phosphate buffer (400 mM) with NaCl (400 mM and 960 mM) this leads to 22 % and 8 % of the initial activity respectively. D. desulfuricans NAP has a high similarity with some proteins from extremly halophilic bacteria which are stabilized at low salt concentrations (21). The purified NAP is stable if frozen at 020 7C with no lost of specific activity, even after several months, but cycles of frozen-defrozen causes partial precipitation, indicating protein denaturation with consequent lost of activity. The effect of prolonged ferricyanide treatment, until 6 hrs on NAP, as prepared, was investigated within a range of 0 to 10 mM (Fig. 4). Zero concentration is taken as a standard; for 0.1 mM the specific activity remains practically the same, but with 1-10 mM strong and rapid (in the second case) inhibition is observed till zero value of specific activity. This effect was reproducible with different loading reagents order (AMPSO, MV and nitrate) and the reaction started by dithionite addition. With exception of nitrogenase, all molybdenum-containing enzymes have a molybdenum cofactor in which molybdenum is bound with a 6-substituted pterin. We examined the presence of active molybdenum cofactor in NAP, by using the apo-(nit-1) nitrate reductase assay. The experimental conditions used, provide a quantitative basis for the assay of the Mo cofactor (16). Measurements were carried out with the addition of Na2MoO4 (10 mM) to the complementation media. The extraction procedure and complementation was also checked for xanthine oxidase (XO) under the same conditions. Increasing amounts of Mo cofactor, extracted from NAP and XO, were incubated with nit-1 mutant crude extract and finally assayed for NADPH-dependent NAR activity. The slopes of the plots, of the liberated nitrate against the correspondent added enzyme, were found to be linear. The highest nitrate reductase activity, after complementation with nit-1, was 8.68
mmol of nitrite per mg of protein for XO and 10.5 mmol of NO02 per mg for NAP, equivalent to 7% and 9% of recovered activity respectively. This cofactor extraction and transfer efficiency, for both enzymes, is in good agreement with heat treatment type of extraction procedure (22). It states that NAP contains an Mo cofactor able for the activation of apo-(nit-1) nitrate reductase. Upon acid hydrolysis 5*-GMP identification was possible, indicating the presence of a MGD type molybdenum cofactor. The MGD quantity determined is 1.3 mol per 1 mol of NAP suggesting that there are two MGD moieties per protein. The N-terminal amino acid sequence (Fig. 5) reveal a cysteine rich motif, which may be involved in the coordination of the iron-sulfur center (23); homologies with other molybdenum cofactor-containing enzymes is shown: nitrate reductase from E. coli (24), periplasmic nitrate reductase from Alcaligenes eutrophus H16 (25), formate dehydrogenase from Methanobacterium formicicum (26), assimilatory nitrate reductase of Klebsiella pneumonia M5 (27), polysulfide reductase chain A from Wolinella succinogenes (28) and a nitrate-inducible formate dehydrogenase from E. coli (29). The homology with NAP A from E. coli is 54 % and with NAP from A. eutrophus is 56 %. DISCUSSION D. desulfuricans is a slightly halophilic bacterium, and NAP properties were compared with other enzymes from different halophilic bacteria. Two classes were proposed (30), one resembling non-halophilic proteins which are either active or stable in the absence of salt (21, 32-34), and the other, hallophilic, showing unique properties that may be related to salt dependence (31). The isoelectric point of most of the proteins from extremely halophilic bacteria are between 4.0 and 4.65 which is in agreement with the generally high content of glutamic and aspartic acid in proteins from halobacteria (30). It was found that NAP has similar properties, concerning salts, with the nitrate reductase from Halobacterium marismortui (35), suggesting that the excess of acidic over basic amino acids can explain this behavior. More, both have high specific activity in the presence of salts, but are more stable if low salt concentration are used in the storage conditions. Also, a stimulatory effect on yeast Candida nitratophila assimilatory nitrate-reducing activity was observed upon increasing ionic strength (36). Salts can affect in widely different manner the properties of enzymes like their stability, solubility and activity (20). At low concentrations, salts can stabilize through nonspecific electrostatic interactions with the enzyme, depending only on the ionic strength of the medium (37). For high concentrations, however, salts have specific effects dependent on the nature of salt and its concentration, resulting in either stabilization
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FIG. 5. Comparison N-terminal sequence of the periplasmic nitrate reductase from D. desulfuricans ATCC 27774 with sequences of the following molybdenum cofactor-containing enzymes: periplasmic nitrate reductases of E.coli (NAPA E.coli) (25), Alcaligenes eutrophus H16 (NAPA A.eutr.) (26), formate dehydrogenase of Methanobacterium formicicum (FDHA M. form.) (27), assimilatory nitrate reductase of Klebsiella pneumonia M5 (NASA K.pneu.) (28), polysulfide reductase chain A Wolinella succinogenes (PSRA W.succ.) (29), nitrate-inducible formate dehydrogenase of E.coli (FDNG)(30).
or denaturation. NAP activity in different salts conditions reveal that potassium and sodium chlorides, both in ionic and nonionic buffer, in high concentrations, have an activation effect. Sulfate anion on the contrary has a different behavior in the two buffer systems, activation only occurs in phosphate buffer, pointing out a dependence with the ionic strength. These facts suggest that the enzyme may suffer changes in the global charge of the surface modifying the molecule configuration and making the molybdenum site more accessible, justifying in this way the higher activities for high salt concentration. Calcium and magnesium turn the system thermodynamically less stable, so NAP activity decreases. This phenomenon may involve changes in the nature and polarity of NAP surface, hydration and charged state (38). In this case the molecule configuration changes in a way that the catalytic site is not easily accessible, decreasing the activity. The experiments performed in different buffer systems demonstrate that salts have an activation effect during the catalytic act, but are not so effective as a protector against NAP inactivation during long time storage. However, phosphate anion always has a positive effect on NAP activity and storage, which is very similar with what is observed with ferredoxin-nitrate reductase from Plectonema boryanum (39). D. desulfuricans ATCC 27774 pure NAP, as prepared, is not activated by short or prolonged incubation with ferricyanide oppositly to membrane bound nitrate reductase from E. coli (40) and assimilatory nitrate reductase from Chlorella vulgaris (10). Moreover, strong inhibition occurs with 1 to10 mM ferricyanide. Sequence comparisons between NAP with other molybdenum cofactor-containing enzymes such as nitrate reductases, polysulfide reductase and formate dehydrogenases from various sources revealed a conserved segment. Three cysteine residues coordinates an iron-sulphur center, as proposed for similar cysteine clusters found in formate dehydrogenase of E. coli (28) and NAD-reducing hydrogenase of A. eutrophus (41). They are located at the amino-terminal part of the polypeptide chain and form a structure C-X2-C-X3-C which is
very similar with the C-X2-C-X3-C-X27-C cluster, resembling the [4Fe-4S] centers found in bacterial ferredoxins (42). The characterization of the Mo site in NAP as well as the details on enzymatic properties described here, help to place this novel enzyme in the large family of monomeric molyddenum enzymes, further support by recent EXAFS studies (col. G.N.George) that indicated the presence of two pterins, one cysteinyl residue and one hydroxyl group in the Mo coordination sphere. D.desulfuricans ATCC 27774 has been a source of a variety of molybdopterin containing enzymes, namely aldehyde oxido-reductase (43), formate dehydrogenase (44) and nitrate reductase (45). These molybdenum proteins belong to sub-groups classified on the basis of whether the molydenum center of the active site possesses a MoO2 or MoOS unit (11) (see also footnote), defined as: (1) The xanthine oxidase family [(MCD - molybdopterin cytosine dinucleotide or MPT - molybdopterin) MoVI ÅO, ÅS, 0H2O] that includes enzymes such as xanthine oxidase/dehydrogenase, aldehyde oxidase, CO dehydrogenase and the aldehyde oxido-reductases isolataded in sulfate reducers (43, 46). Members of this family possess a MoVIOS center and one molybdopterin, and no covalent bonding between the cofactor and the polypeptide chain. In the coordination sphere is an additional water molecule or hydroxo group. A Mo(O)2 unit is generated after cyanide reaction. The inactivation mechanism involves cyanide abstraction of sulfur from the molybdenum center, in the form of thiocyanate (9, 47), which is essential for enzyme activity. (2) The DMSO reductase family [(MGD - molybdopterin guanine dinucleotide)2 MoVI ÅO, (-OSer, -SCys or -SeCys)] includes enzymes such as biotin-S-oxide reductase, dimethylsulfoxide reductase, dissimilatory nitrate reductase and formate dehydrogenase. Two molybdopterins (MGD) are found in the coordination sphere of Mo and an aminoacid side chain coordinates the metal: cysteine in the periplasmic nitrate redutase (8), selenum-cysteine in formate dehydrogenase (48) or serine in DMSO reductase (49, 50).
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A third subgroup includes the sulfite oxidase family [(MPT) MoVI ÅO, 0H2O, (-SCys)] that also includes assimilatory nitrate reductase. Enzymes within this family typically have one molybdopterin cofactor and a MoVIO2 nucleus and the polypeptide chain coordinates directly to the molybdenum site by a cysteinyl residue (51). Three crystal structures of molybdopterin-containing enzymes were reported for members of the first two families (aldehyde oxido-reductase from Desulfovibrio gigas (46, 52), DMSO reductases from Rhodobacter sphaeroides and capsulatus (50, 53) and formate dehydrogenase from E. coli (48)). Also, there is a preliminary report for the X-ray structure of chicken liver sulfite oxidase (51). Attempts to solve the 3D structure of D.desulfuricans ATCC 2774 are in progress (our work in collaboration with M.J.Roma˜o).1 ACKNOWLEDGMENTS This work was supported PRAXIS 2/2.1/BIO/05/94. CC thanks PhD Grants JNICT BD/2192/92-IF and PRAXIS BD/5402/95. SAB is PRAXIS Visiting Professor in Portugal. We wish to thank Rene´ Toci for the growth of cells and Manuela Regala for the amino acid Nterminal sequence and C.Costa for NIR preparation from D. desulfuricans ATCC 27774.
REFERENCES 1. Postgate, J. R. (1984) The Sulfate-Reducing Bacteria, pp. 42– 43, Cambridge University Press. 2. Fauque, G. D. (1995) in Sulfate Reducing Bacteria, (Barton, L. L., Ed.), Chap. 8, pp. 217–241, Plenum Press, New York. 3. Senez, J. C., and Pichinoty, F. (1958) Bull. Soc. Chim. Biol. Paris 40, 2099–2117. 4. Liu, M. C., and Peck, H. D., Jr. (1981) J. Biol. Chem. 256, 13159– 13164. 5. McCready, R. G. L., Gould, W. D., and Cook, F. D. (1983) Arch. Microbiol. 135, 182–185. 6. Widdel, F., and Pfenning, N. (1982) Arch. Microbiol. 131, 360– 365. 7. Widdel, F., and Pfenning, N. (1984) in Bergey’s Manual of Systematic Bacteriology (Krieg, N. R., and Holt, J. G., Eds.), Vol. 1, pp. 663–679, Williams and Wilkins Co., Baltimore. 8. Bursakov, S. A., Liu, M-Y., Payne, W. J., LeGall, J., Moura, I., and Moura, J. J. G. (1995) Anaerobe 1, 55–60. 9. Coughlan, M. P., Johnson, J. L., and Rajagopalan, K. V. (1980) J. Biol. Chem. 255, 2694–2699. 10. Solomonson, L. P. (1974) Biochem. Biophys. Acta 334, 297–308. 11. Liu, M. C., and Peck, H. D., Jr. (1981) J. Biol. Chem. 256, 13159– 13164. 12. Lowe, R. H., and Evans, H. J. (1964) Biochim. Biophys. Acta. 85, 377–389.
1 This classification is based on the structure of the molybdenum center. With the exception of DMSO reductases, other additional redox-active sites are present (iron-sulfur centers, hemes and/or flavins) that participate in electron transfer from the molybdenum center to an external electron acceptor.
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