Biochimica et Biophysica Acta, 1040(1990) 237-244
237
Elsevier BBAPRO33728
Purification and some properties of the nitrite reductase from the cyanobacterium Phormidium laminosum Jesfis M. A r i z m e n d i a n d J u a n L. S e r r a Departamento de Bioqulmica y Biologla Molecular, Facultad de Ciencias, Universidaddel Pals Vasco, Bilbao (Spain)
(Received12 October 1989) (Revised manuscriptreceived24 April 1990)
Key words: Nitritereductase;Enzymepurification; ( P. laminosum) Assimilatory ferredoxin-nitrite reductase (EC 1.7.7.1, ammonia: ferredoxin oxidoreductase) has been purified 5300-fold with a specific activity of 625 units/mg protein from the filamentous non-heterocystous cyanobacterium Phormidium laminosum. The enzyme was soluble and consisted of a single polypeptidic chain of 54 kDa. It catalyzed the reduction of nitrite to ammonia using ferredoxin or flavodoxin as electron donor. Methyl and benzyl viologens were also effective as electron donors but neither flavins nor NAD(P)H were. The apparent Michaelis constants for nitrite, ferredoxin and methyl viologen were 40, 22 and 215 FM, respectively. Nitrite reductase activity was inhibited effectively by cyanide and thiol reagents. The enzyme exhibited absorption maxima at 281, 391 (Sorer), 570 (a) and 695 nm, with ¢~391 of 4.3.104 M -I cm -t, and an absorbance ratio A2s,/A39, of 1.95, suggesting the presence of siroheme as prosthetic group. These results show that this enzyme is similar to those of eukaryotic organisms.
Introduction The assimilatory nitrate reduction is catalyzed by the sequential action of nitrate reductase and nitrite reductase. Assimilatory nitrite reductase catalyzes the six-electron reduction of nitrite to ammonia. In photosynthetic organisms the physiological electron donor is ferredoxin, whereas in non-photosynthetic organisms the donor is NAD(P)H [1]. Ferredoxin-nitrite reductase (ammonia: ferredoxin oxidoreductase, EC 1.7.7.1) has been purified to electrophoretic homogeneity from several higher plants and eukaryotic algae. The enzyme is located in the chloroplast and composed of one polypeptide of 60-63 kDa [2-6], although it has been also reported as a heterodimer composed of two subunits of 61-64 kDa and 24-35 kDa [7-9]. The spinach nitrite reductase is a heme-protein with an iron-sulfur duster. The heme moiety has been identified as siroheme, an iron tetrahydroporphyrin of the isobacteriochlorin type with eight carboxylic acid-containing side chains, which is also present in both assimilatory and dissimilatory sulfite reductases [10]. The presence of one mol siroheme/mol enzyme was classi-
cally reported [6,11], although, recently, a ratio of 2 mol siroheme/mol enzyme has been calculated for the native heterodimer of the spinach enzyme [12]. In addition to siroheme, nitrite reductase also contains an [Fe4-S4] cluster as a prosthetic group [13]. The available information about nitrite reductase from photosynthetic prokaryotes is very scarce. In this group of organisms, cyanobacteria have a special interest because of their phylogenetic situation and their relationship with the origin of the chloroplast. The nitrite reductase from the cyanobacteria Anabaena cylindrica [14], Anacystis nidulans [15], Anabaena sp. 7119 [16,17] and Spirulina platensis [18] have been partially characterized, although only the enzyme from S. platensis has been highly purified. In the present work, we report the purification of nitrite reductase from the filamentous non-heterocystous cyanobacterium Phormidium laminosum, as well as some of its molecular, catalytic and spectral properties. Results show that this enzyme is similar to those of eukaryotic organisms. Materials and Methods Materials
Correspondence: J.M. Afizmendi, Departamento de Bioquimica y BiologlaMolecular,Facultadde Ciencias,Universidaddel Pals Vasco, Apartado 644, 48080-Bilbao, Spain.
Mono-Q, Mono-P, Phenyl-Superose and Superose 12 columns, Sephadex G-50, PhastGel 8-25, SDS and native buffer strips, Coomassie PhastGel Blue R, Poly-
0167-4838/90/$03.50 © 1990 ElsevierSciencePublishersB.V. (BiomedicalDivision)
238 buffer 74 and SDS-polyacrylamide gel electrophoresis markers were obtained from Pharmacia (Uppsala, Sweden). DEAE-cellulose (DE-52) was from Whatman (Maidstone, U.K.). Phenyl-Sepharose, acrylamide, N,N'-methylenebisacrylamide, SDS, methyl viologen, sodium dithionite, N-l-naphthylethylenediamine and native-electrophoresis markers were purchased from Sigma (St. Louis, U.S.A.). Immersible CX-10 ultrafiltration units were from Millipore (Bedford, U.S.A.). Glutamate dehydrogenase and gel filtration molecular weight markers were obtained from Boehringer-Mannheim (Mannheim, F.R.G.). Sulfanilamide was from Fluka (Buchs, Switzerland). The protein assay dye-reagent was from Bio-Rad (Miinchen, F.R.G.). All other chemicals were reagents of analytical grade supplied by Merck (Darmstad, F.R.G.).
Cell culture and cell free extracts P. laminosum (strain OH-l-p. Cll) cells were grown photoautotrophically at 45 ° C in the mineral medium D [19] in 60 1 glass containers. Cultures were stirred by an air stream and continuous illumination was provided by white fluorescent lamps (Tungsram F7 Daylight) with a photon flux density at the surface of the vessels of about 100/~mol m -2. s -1. Cells were harvested in the early stationary phase with a continuous flow rotor in a Kubota centrifuge, washed once with 50 mM Tris-HC1 (pH 8.0) (standard buffer) and suspended in the same buffer. Aliquots of 80 ml of this suspension were disrupted by sonication (MSE) for 10 min at 14/~m setting in an ice-bath. The sonicate was centrifuged at 110000 x g for 1 h at 4 ° C and the supernatant was used as crude extract.
Purification of nitrite reductase All steps except those of fast-protein liquid chromatography (FPLC) were carried out at 4 ° C. Chromatographies were done at a flow rate of 1 m l / m i n unless otherwise stated. (1) First DEAE-cellulose chromatography. The crude extract was passed through a DE-52 column (2.2 x 30 cm) equilibrated with standard buffer (50 mM Tris-HC1 (pH 8)). After washing the column with two bed volumes of standard buffer, nitrite reductase was eluted with 500 ml of 200 mM KCI in standard buffer. (2) Acetone fractionation. Chilled acetone ( - 20 ° C) was added slowly to the protein solution to 50% (v/v). The preparation was centrifuged at 10000 x g for 10 min, and the pellet was resuspended in 90 ml of standard buffer. This preparation had to be dialysed against the standard buffer before assaying the enzyme activity. (3) Second DEAE-cellulose chromatography. The above enzyme preparation was loaded onto a DE-52 column (2.2 x 30 cm) equilibrated with standard buffer. The column was washed with 120 ml of 20 mM KC1 in
standard buffer and nitrite reductase was eluted from the column with 200 mM KC1 in standard buffer. (4) Phenyl-Sepharose chromatography. The active fractions were pooled and solid ammonium sulphate was added up to 415 mM. The resulting solution was adsorbed to a Phenyl-Sepharose column (2.5 x 20 cm) equilibrated with 415 mM ammonium sulfate in standard buffer. The column was washed sequentially with 120 ml of equilibration buffer and 200 ml of 150 mM ammonium sulfate in standard buffer. Finally, nitrite reductase was eluted with standard buffer containing 75 mM ammonium sulfate, while most of the contaminant phycobiliproteins remained adsorbed to the column. The active fractions were pooled, concentrated to 30 ml with immersible CX-10 filters and dialysed against 5 mM KC1 in standard buffer. (5) Mono-Q chromatography. The dialysed preparation was applied to a Mono-Q H R 5 / 5 column equilibrated with 5 mM KC1 in standard buffer using an FPLC apparatus (Pharmacia, Uppsala, Sweden). The column was washed sequentially with 40 ml of the same buffer and with 36 ml of 100 mM KC1 in standard buffer. Nitrite reductase was then eluted with a linear gradient of 10 ml from 100-250 mM KC1 in standard buffer, followed by an isocratic elution with 10 ml of the last buffer. (6) Phenyl-Superose chromatography. The above enzyme preparation was adjusted to 1 M ammonium sulfate and loaded at a flow rate of 0.5 m l / m i n to an FPLC Phenyl-Superose H R 5 / 5 column equilibrated with 1 M ammonium sulfate in standard buffer. After washing with 10 ml of equilibration buffer, nitrite reductase was eluted applying a linear gradient of 24 ml from 1 - 0 M of ammonium sulfate in standard buffer and the active fractions were pooled. (7) Superose 12 chromatography. The resulting protein preparation was concentrated to 200 /~l by ultrafiltration (Amicon MPS-1) and loaded onto a Superose 12 H R 10/30 gel filtration column equilibrated with 100 mM KC1 in standard buffer. Elution was done with the same buffer at a flow rate of 0.5 m l / m i n and the active fractions were pooled. The purified nitrite reductase was stored at - 2 0 ° C until used.
Purification of ferredoxin and flavodoxin Ferredoxin and flavodoxin from P. laminosum were purified by a method consisting of DEAE-cellulose chromatography, ammonium sulfate fractionation, FPLC anionic exchange chromatography (Mono-Q H R 5 / 5 column) and Sephadex G-50 chromatography. After the last step of purification the absorbance ratios for ferredoxin (Aa23//A275) and flavodoxin (A465//A274) were 0.6 and 0.17, respectively. Ferredoxin and flavodoxin were estimated from their molar absorption coefficient of 9.2 mM - 1c m - 1, at 423 nm [20] and 9.7 mM - 1. c m - l, at 465 nm [21], respectively.
239
Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis were carried out in a PhastSystem equipment (Pharmacia, Uppsala, Sweden). SDS-electrophoresis was performed in 8-25% ( w / v ) acrylamide gradient gels (PhastGel 8-25) with PhastGel SDS Buffer Strips. Native-electrophoresis were carried out in both 8-25% ( w / v ) acrylamide gradient gels (PhastGel 8-25) and homogeneous gels made as described in the PhastSystem Separation Technique File No. 121 (Pharmacia) with PhastGel Native Buffer Strips. The electrophoretic conditions were those described in the PhastSystem separation Technique Files No. 110, 120 and 121 (Pharmacia), and the SDS- and native-electrophoresis in homogeneous gels were stopped when the bromophenol blue had reached the anode. Proteins were stained using Coomassie brilliant blue [22] or silver staining (PhastSystem Development Technique File No. 210, Pharmacia). Nitrite reductase was located in the gels by methyl viologen-dependent activity staining [11]. Enzyme assays Nitrite reductase activity was routinely assayed in a similar way to that previously described [23]. The reaction mixture contained in a 1 ml final volume: 90 m M Tris-HC1 (pH 7.5), 2.5 m M K N O 2, 3 m M methyl viologen, 20 m M sodium dithionite (freshly dissolved in 0.3 M NaHCO3) and an appropriate amount of enzyme. After 5 min preincubation, the reaction was carried out for 10 rain at 30 o C, and stopped by vigorous-shaking to obtain the complete oxidation of the excess of reductant. Nitrite disappearance was determined after a 100-fold dilution of the reaction mixture [24]. Blank tubes without enzyme were also included. When the K m for ferredoxin, methyl viologen and nitrite were estimated, assays were carried out in sealed test-tubes saturated with He or N 2 to diminish the oxidation of dithionite. The K m for nitrite was also estimated by progresscurve analysis. This assay was carried out in sealed test-tubes saturated with He, as in the routine assay
described before, but 0.25 m M KNO2 was used in a reaction mixture of 2 ml. The reactions were stopped at different times and 10-fold dilution was done before determining nitrite. The non-enzymatic oxidation of methyl viologen caused an interference always lower than 5%. One unit of enzyme activity (U) was defined as the amount that catalyzed the reduction of 1/~mol of nitrite per min.
Absorption spectra Absorption spectra were recorded at room temperature against a buffer blank in a Shimadzu UV-260 spectrophotometer with cuvettes of 1 cm pathlength. Analytical determinations Protein was determined by the method of Bradford [25] using bovine serum albumin as standard. Ammonium was determined enzymatically by following the oxidation of N A D P H catalyzed by glutamate dehydrogenase [26]. lsoelectric point determination Isoelectric point was determined by FPLC-chromatofocusing in a Mono-P H R 5 / 2 0 column. A flow rate of 0.5 m l / m i n and a p H gradient from 7 to 4 were used. After equilibration of the column with 25 m M Bistris-iminodiacetic (pH 7.1), a partially purified sample of nitrite reductase was, injected onto the column and the p H gradient was formed by applying 50 ml of 1 : 10 diluted Polybuffer 74-iminodiacetic buffer ( p H 4). The nitrite reductase activity and p H were determined in the collected fractions of 1 ml. Results
lntracellular location of nitrite reductase When cells of P. laminosum were broken with lysozyme followed by osmotic shock [27] and the homogenate was centrifuged at 110000 x g for 1 h, 80% of
TABLE I
Purification of nitrite reductasefrom P. laminosum The purification procedure from 80 g of cells (fresh weight) and the protein determination were conducted as described in Materials and Methods. Nitrite reductase activity was measured by the routine assay described in Materials and Methods, but at 50 ° C. Step
Volume (ml)
Activity (U)
Protein (mg)
Specific a c t i v i t y (U/mg protein)
Purification (-fold)
Yield (%)
Crude extract DE-52 I 50% Acetone DE-52 II Phenyl-Sepharose Mono-Q Phenyl-Superose Superose 12
315 256 88 300 29 10.4 2.2 2.2
432 382 285 299 210 194 121 69
3692 2560 396 159 11.3 2.9 0.48 0.11
0.117 0.15 0.72 1.88 19 67 252 627
1 1.3 6.2 16.1 162 572 2155 5359
100 88 66 69 49 45 28 16
240 the total nitrite reductase activity was recovered in the supernatant, indicating the soluble nature of the enzyme. With this method, 85% of the cell rupture obtained by sonication was achieved, the insoluble activity probably being due to the incompletely broken ceils. This result is in agreement with the data described for the enzyme from A. cylindrica [28] and Anabaena sp. 7119 [17], but in A. nidulans [29] nitrite reductase is associated with subcellular particles and a long disruption process is necessary for its solubilization. Purification of nitrite reductase Table I summarizes a typical purification from 80 g of wet cells. Nitrite reductase was purified more than 5300-fold, in 16% yield to a specific activity of 627 units/mg protein at 50 ° C. A similar value of specific activity was obtained when the protein content was determined by the method of Lowry et al. (1951) modified by Peterson [30]. After the last step the enzyme preparation gave a single protein band upon Coomassie blue stained native-electrophoresis, with a positive reaction for methyl viologen-nitrite reductase activity (Fig. 1, lane A). Nevertheless, after silver staining of the same slab, a contaminant representing less than 2% of the total protein could be seen (Fig. 1, lane B). The high purity of the nitrite reductase preparation was also confirmed by the single protein band detected in the silver stained, SDSelectrophoresis (Fig. 1, lane C). Molecular mass The molecular mass of nitrite reductase was estimated by four methods: native-electrophoresis in either acrylamide gradient gels, or homogeneous gels of different acrylamide concentrations, SDS-electrophoresis in acrylamide gradient gels and gel filtration.
A
B
D
94
kDa
66 43 30 20.1 14.4
Fig. 1. Polyacrylamide gel electrophoresis of the purified nitrite reductase from P. larninosum. Lane A. Activity staining of 200 ng of purified nitrite reductase after native-electrophoresis in an 8-25 (w/v) acrylamide gradient gel. Lane B. Silver staining of the electrophoresis of lane A. Lane C. Silver staining of 25 ng of purified nitrite reductase after SDS-electrophoresis in an 8-25 (w/v) acrylamide gradient gel. Lane D. Silver staining of 300 ng of standard proteins after SDS-electrophoresis in an 8-25 (w/v) acrylamide gradient gel. The standard proteins used were as follows: phosphorylase b (94 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), carbonic anhidrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa).
The molecular mass obtained by native-electrophoresis in a 8-25% (w/v) acrylamide gradient gel was 69 kDa, which was similar to that of 64 kDa calculated from the Ferguson plot (Fig. 2). However, when the
A 2OO
B
180
I: e~
C
12
,\
160
NiR
r~
J
O
140 em
_e 120 4 ¸
1
v-4
100 5 80
•
0
I
lS
~
Ni !
20
•
Ill
2•
25
GEL CONCENTRATION (%)
i
30
40
,
I
80
•
I
120
m (kDa)
Fig. 2. Ferguson plot analysis of the molecular mass of nitrite reductase. (A) Effect of different gel concentrations on the relative mobilities (R m) of nitrite reductase (NiR) and standard proteins in native-electrophoresis. (B) Slope/m relation of nitrite reductase and standard proteins. The standard proteins used were as follows: 1, a-lactalbumin (14.2 kDa); 2, carbonic anhydrase (29 kDa); 3, chicken egg albumin (45 kDa); 4, monomer of bovin serum albumin (66 kDa); and 5, dimer of bovin serum albumin (132 kDa).
241 same enzyme preparation was injected into a gel filtration Superose 12 column, a value of 54 kDa was obtained. In the same way, a single protein band corresponding to 54 kDa was detected by SDS-electrophoresis in 8-25% (w/v) acrylamide gradient gel (Fig. 1, lanes C and D). Nitrite reductase bands in both nativepolyacrylamide gel electrophoresis were rather wide, and these methods are not as generally accepted as the SDS-electrophoresis method, so 54 kDa seems to be the most reliable value for the molecular mass of nitrite reductase. On the other hand, when a crude extract was either dialysed against standard buffer or was kept at 4 °C for 4 days, two bands of methyl viologen-dependent nitrite reductase activity appeared after a native-electrophoresis in an acrylamide gradient gel. One band corresponded to the molecular mass of the purified nitrite reductase and the second minor band corresponded to a 14 kDa smaller molecular mass. These results could suggest the heterodimeric nature of the enzyme, with both the dimer and the biggest monomer having methyl viologen-dependent activity, in a similar way to that reported in spinach [7], etiolated bean shoots [8] and the green alga Chlamydomonas reinhardtii [9]. Nevertheless, the addition to the crude extract of 2.8 mM iodoacetamide (a thiol-proteinase inhibitor) prevented the appearance of the second activity band without affecting the whole nitrite reductase activity. Moreover, when a purified enzyme preparation was used in these experiments, the second activity band did not appear. These results could be explained by assuming that a proteolysis of the enzyme in the crude extract occurs. So, it is concluded that the native nitrite reductase from P. laminosum consists of a single polypeptidic chain of 54 kDa. A Stokes radius of 3.14 nm was determined by gel filtration in Superose 12 and a frictional coefficient of 1.26 was calculated for nitrite reductase.
lsoelectric point The isoelectric point of the enzyme determined by chromatofocusing was 5.6. This value is between those of 4.3-4.5 described for the enzymes from leaves, scuteUa, roots of corn [31], and leaves and roots of barley [32], and that of 6.2 described for the enzyme from C. reinhardtii [33]. Catalytic properties Nitrite reductase from P. laminosum catalyzed the stoichiometric reduction of nitrite to ammonia, one ammonium being formed for each nitrite disappeared (data not shown). A K m for ferredoxin of 22/tM was calculated from a Hanes plot of the initial velocities of nitrite reduction at different concentrations of ferredoxin reduced by dithionite. This value corresponds well with those ob-
2O
16 o
12
8
m
•
4
0
-50
0
50
100
150
200
S.-S / In (So/S) (ltlVl) Fig. 3. Progress curve analysis of the effect of nitrite concentration on nitrite reductase activity. Nitrite was determined at different incubation times from the reaction-mixture described in Materials and Methods. The reaction was carried out under He atmosphere at 30 o C. SO is the initial substrate concentration (0.25 mM), and S the concentration at time t. The a m o u n t of enzyme used was 0.06 units.
tained for the enzymes from cyanobacteria [14,17,18] and some photosynthetic organisms [9,34-36], but is higher than the value of 0.4 #M reported for the enzyme from barley [3]. The K m for reduced methyl viologen obtained from a Hanes plot of the initial velocities of nitrite reduction at different concentrations of methyl viologen reduced by dithionite, at saturating concentration of nitrite, was 215 #M. This value is in the range from 60-400 #M of previous data for the enzyme from cyanobacteria [14,17,18] and eukaryotic organisms [2,3,6]. The K m for nitrite determined by progress curve analysis of nitrite was 40 #M (Fig. 3), but the initial velocities method gave a considerably higher value (about 0.6 mM). Fry et al. [37] also showed variations on the K m value for nitrite for the enzyme from vegetable marrow leaves, depending on the aerobic or anaerobic system used. Table II shows the ability of various electron donors to transfer electrons to the nitrite reductase. Methyl viologen was the most effective electron donor, while benzyl viologen was less effective. Both ferredoxin and flavodoxin were also partially effective, although the concentrations used here were not saturating. Neither NAD(P)H nor dithionite-reduced flavins were active as electron donors for nitrite reductase, in contrast to the enzyme from non-photosynthetic organisms [1]. The effect of temperature from 25-65 °C on nitrite reductase activity was examined. The activity increased up to 50 °C and the activation energy calculated from an Arrhenius plot was 50 kJ/mol. These results are similar to those obtained for the enzyme from C. reinhardtii [38].
242 TABLE II
TABLE III
Electron donors for the purified nitrite-reductase from P. laminosum
Effect of inhibitors on nitrite reductase activity from P. laminosum
Nitrite reductase activity was determined by the routine assay described in Materials and Methods, except that the indicated electron donors were added instead of methyl viologen and dithionite. 100% activity corresponds to 6.5 U / m g protein.
Nitrite reductase activity was determined by the routine assay described in Materials and Methods, except that the indicated compounds were added to a final reaction mixture of 2 ml.
Electron donor
Concentration (mM)
NADH NADPH Dithionite methyl viologen benzyl viologen + FAD + FMN + flavodoxin + ferredoxin
5 5 20 5 5 5 5 0.005 0.01
++
Activity (%)
Inhibitor
Concentration (mM)
Inhibition (%)
NaCN
0.005 0.05 0.1
30 78 92
p-Hydroxymercuribenzoate
0.1
35 68
0 0 100 34 0 0 25 25
The pH dependence of the nitrite reductase activity was studied in potassium phosphate buffer in the p H range of 6.8-7.8, and in Tris-HC1 in the range of 7.5-8.5. The optimum p H for nitrite reduction was 7.3 and 7.6 with phosphate and Tris-HCl buffers, respectively. These values are in the range from 7-8, which includes the optimum pH values reported for most nitrite reductases [2,9,14,17,18,35,38]. The P. laminosum nitrite reductase was fully thermostable until 40°C, but more than 95% of the activity was lost when the enzyme was heated for 10 rain at 60 o C. The thermal inactivation reaction was of second order and the activation energy calculated was 167 kJ/mol. So, this enzyme is slightly less thermostable than other enzymes involved in the nitrate assimilation in P. laminosum, such as glutamate dehydrogenase [39] and glutamine synthetase [40]. The enzyme was stable for a week at 4 ° C and for several months at - 2 0 o C, even without the addition of thiol reagents (dithiothreitol) or chelating agents (EDTA). Table III shows the inhibitory effect of several compounds on the nitrite reductase activity. Cyanide was a very powerful inhibitor of the enzyme activity, while azide and bathophenanthroline were weak inhibitors. Thiol reagents, such as p-hydroxymercuribenzoate and N-ethylmaleimide, also caused a significant inhibition. Sulfite and hydroxylamine inhibited only at high concentrations.
Absorption spectra The absorption spectrum of the purified nitrite reductase from P. laminosum showed maxima at 281,391 (Soret), 570 (a) and 695 nm (Fig. 4). The A281/A391 absorbance ratio was 1.95, and the Soret to a band ratio was 3.33. The molar absorption coefficients were: 8.4. 104 M -1 . c m -], at 281 nm; 4 . 3 . 1 0 a M -1 . c m -1, at 391 nm; and 1.3-104 M - 1 . c m -], at 570 nm. This
1
N-Ethylmaleimide
0.1 1
16 47
NaN 3
1
15
Bathophenanthroline
1
14
Sulfite
1 10
5 24
Hydroxylamine
1 10
16 34
spectrum is very similar to that of the enzyme from eukaryotic organisms, and only the enzymes from spinach leaves [6] and C. reinhardtii [9] showed a lower A280/A39 o absorbance ratio. Therefore, the little differences in the spectrum of the enzyme from the cyanobacterium S. platensis [18] would not represent different characteristics of the cyanobacterial enzyme but they would probably be due to some contaminants of the preparation.
0"101 o C
0.05
o
250
450 Wavelength
650 (nm)
Fig. 4. Absorption spectrum of the purified nitrite reductase from P. laminosum. The absorption spectrum of the enzyme solution (50 /~g/ml) in 100 mM KC1 in 50 mM Tris-HC! (pH 8) was recorded against a buffer blank.
243
0.050
iC
C
0.025
,¢
0
t
" I
I
350
I
i
550 Wavelength
-
~
750
(rim)
Fig. 5. Absorption spectra of the purified nitrite reductase in the presence of dithionite and nitrite. (A) Absorption spectrum of nitrite reductase (50 /~g/ml) in 100 mM KCI in 50 mM Tris-HC1 (pH 8) ( ). (B) Absorption spectrum of nitrite reductase after the addition of solid dithionite ( . . . . . . ). (C) Absorption spectrum of nitrite reductase after the addition of a few crystals of nitrite to the sample previously reduced by dithionite ( . . . . . ).
Addition of dithionite to nitrite reductase resulted in the bleaching of the 570 and 695 nm peaks, with the appearance of a broad shoulder at 580 nm and a shoulder at 410 nm. The subsequent addition of nitrite to the dithionite-reduced enzyme restored the absortion maximum at 570 nm with a shift to 580 nm, but the absorption at 695 was not restored (Fig. 5). Similar spectral changes have also, been reported for the enzymes from spinach leaves [11] and C. reinhardtii [9]. Discussion
The purification procedure reported here allows almost the complete purification of the P. laminosum nitrite reductase. The purified preparation showed a specific activity of 625 U / m g protein at 50 °C which corresponded to 225 U / m g protein at 30 ° C. This value is much higher than those reported for the enzyme from A. cylindrica (10.75 U / m g protein at 25°C) [14], Anabaena sp. 7119 (21.5 U / m g protein at 30°C) [16] and higher than that of S. platensis (195 U / m g protein at 35 ° C) [18]. The best specific activity reported for the enzyme from an eukaryotic source (spinach leaves) is 207 U / m g protein [6]. The high specific activity ob-
tained shows that the purification procedure yields a good enzyme preparation, not only from the point of view of the purity but also from that of the little damage caused to the enzyme. The molecular mass of our enzyme (54 kDa) is similar to those reported for other cyanobacterial enzymes (52-54 kDa) [15,16,18]. However, a value of 68 kDa was reported for A. cylindrica nitrite reductase [14]. In contrast with the enzyme from some higher plants [7,8] or C. reinhardtii [9], where the native nitrite reductase seems to consist of two subunits of different size, the cyanobacterial enzyme appears as a monomeric protein in all cases. This subunit composition agrees with that showed by most higher plant nitrite reductases [2-6]. During the revision of this manuscript, a new paper, confirming the two-subunit composition of the spinach nitrite reductase, was published [41]. According to that, the use of fl-mercaptoethanol could be the main factor to explain the apparent absence of the small subunit in a number of plant nitrite reductase preparations, this effect being increased by low ionic strengh buffers or by the absence of proteinase inhibitors. Despite the fact that this procedure involves the use of buffers of low ionic strengh and the absence of proteinase inhibitors, fl-mercaptoethanol is not used in the purification steps. Moreover, native-electroph6resis of both the crude extract and the purified enzyme shows that the molecular mass of the enzyme does not change through the purification procedure. Finally, SDS-electrophoresis of the purified enzyme shows a single protein band after silver staining, in contrast to the two equally stained bands obtained by Hirasawa et al. [41]. The affinities of P. laminosum nitrite reductase for its substrates (i.e., nitrite, ferredoxin and methyl viologen) were similar to those previously reported for the enzyme from photosynthetic organisms. In this study different values of K m for nitrite have been found depending on the assay method (initial velocities or progress-curve analysis) used. The different results obtained due to the assay method, the utihzation of dithionite as reductant [42] and the use of aerobic or anaerobic conditions [37] could explain the diversity of data concerning the affinities for substrates, especially for nitrite, reported in the literature [2]. All nitrite reductases isolated from eukaryotic sources were inhibited by thiol reagents [3,11,33,36], whereas these compounds did not affect the cyanobacterial enzyme previously-studied [15,17,29]. However, the P. laminosum nitrite reductase was strongly inhibited by p-hydroxymercuribenzoate and N-ethylmaleimide, denoting the presence of thiols involved in the catalysis. The other inhibitors tested affected the P. laminosum enzyme in a similar way to that reported for the enzyme from other sources. The absorption spectrum of our purified enzyme and the differences in the spectrum caused by the reduction
244 and the addition of nitrite are similar to that of eukaryotic organisms [2,6,9,43], suggesting the presence of a siroheme as the prosthetic group involved in the binding and reduction of nitrite. According to the molecular, catalytic and spectral properties showed by the P. laminosum nitrite reductase and the available data on other cyanobacteria, we can conclude that the enzyme of these prokaryotes is similar, with the possible exception of the subunit composition, to that characterized from photosynthetic eukaryotic organisms.
Acknowledgments This work was partially supported by grants from Comisi6n Asesora de Investigaci6n Cientifica y T&nica (928/84) and the Basque Country Government (X86.049).
References 1 Guerrero, M.G., Vega, J.M. and Losada M. (1981) Annu. Rev. Plant Physiol. 32, 169-204. 2 Vega, J.M., Chrdenas, J. and Losada, M. (1980) Methods Enzymol. 69, 255-270. 3 Serra, J.L., Ibarlucea, J.M., Arizmendi, J.M. and Llama, M.J. (1982) Biochem. J. 201, 167-170. 4 Small, I.S. and Gray, J.C. (1984) Eur. J. Biochem. 145, 291-297. 5 Gupta, S.C. and Beevers, L. (1985) Planta 166, 89-95. 6 Ida, S. and Mikami, B. (1986) Biochim. Biophys. Acta 871, 167176. 7 Hirasawa, M. and Tamura, G. (1980) Agric. Biol. Chem. 44, 749-758. 8 Ishiyama, Y. and Tamura, G. (1985) Plant Sci. Lett. 37, 251-256. 9 Romero, L.C., Galvhn, F. and Vega, J.M. (1987) Biochim. Biophys. Acta 914, 55-63. 10 Murphy, M.J., Siegel, L.M., Tove, S.R. and Kamin, H. (1974) Proc. Natl. Acad. Sci. USA 71, 612-616. 11 Vega, J.M. and Kamin, H. (1977) J. Biol. Chem. 252, 896-909. 12 Hirasawa, M., Shaw, R.W., Palmer, G. and Knaff, D.B. (1987) J. Biol. Chem. 262, 12428-12433. 13 Lancaster, J.C., Vega, J.M., Kamin, H., Orme-Johnson, N.R., Orme-Johnson W.H., Krueger, R.J. and Siegel L.M. (1979) J. Biol. Chem. 254, 1268-1272. 14 Hattori, A. and Uesugi, I. (1968) Plant Cell Physiol. 9, 689-699.
15 Manzano, C. (1977) Ph. D. Thesis, University of Seville, Seville, Spain. 16 M6ndez, J.M. and Vega, J.M. (1981) Physiol. Plantarum 52, 7-14. 17 M6ndez, J.M., Herrero, A. and Vega, J.M. (1981) Z. Pflanzenphysiol. 103, 305-315. 18 Yabuki, Y., Moil, E. and Tamura, G. (1985) Agric. Biol. Chem. 49, 3061-3062. 19 Castenholz, R.W. (1970) Schewiz. Z. Hydrol. 32, 538-551. 20 Hall, D.O., Rao, K.K. and Cammack, R. (1972) Biochem. Biophys. Res. Commun. 47, 798-802. 21 Smillie, R.M. and Entsch, B. (1971) Methods Enzymol. 23, 504514. 22 Heukeshoven, J. and Dernick, R. (1988) Electrophoresis 9, 60-61. 23 Arizmendi, J.M., Fresnedo, O., Martinez-Bilbao, M., Alaha, A. and Serra, J.L. (1987) Physiol. Plantarum 70, 703-707. 24 Snell, F.D. and Snell, C.T. (1949) Colorimetric Methods of Analysis, Vol. 3, pp. 804-805, D. van Nostrand, Princeton. 25 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 26 Guerrero, M.G. (1982) in Techniques in Bioproductivity and Photosynthesis, (Coombs, J. and Hall, D.O., eds.), pp. 124-130, Pergamon, Oxford. 27 Stewart, A.C. and Bendall, D.S. (1980) Biochem. J. 188, 351-361. 28 Hattoil, A. and Myers, J. (1966) Plant Cell Physiol. 41, 1031-1036. 29 Guerrero, M.G., Manzano, C. and Losada, M. (1974) Plant Sci. Lett. 3, 273-278. 30 Peterson, G.L. (1983) Methods Enzymol. 91, 95-119. 31 Dalling, M.J., Hucklesby, D.P. and Hageman R.H. (1973) Plant Physiol. 51,481-484. 32 Ida, S., Moil, E. and Morita, Y. (1974) Planta 121,213-224. 33 Romero, L.C. (1988) Ph.D. Thesis, University of Seville, Seville. 34 Hirasawa, M. and Tamura, G. (1981) Agric. Biol. Chem. 45, 1615-1620. 35 Ide, T. and Tamura, G. (1987) Agric. Biol. Chem. 51, 3391-3393. 36 Zumft, W.G. (1972). Biochim. Biophys. Acta 276, 363-375. 37 Fry, I.V., Cammack, R., Hucklesby, D.P. and Hewitt, E.J. (1982) Biochem. J. 205, 235-238. 38 C6rdoba, F., C/trdenas, J. and Fern~ndez E. (1986) Plant Physiol. 82, 904-908. 39 Martlnez-Bilbao, M., Martinez, A., Urkijo, I., Llama, M.J. and Serra, J.L. (1988) J. Bacteriol. 170, 4897-4902. 40 Blanco, F., Alai~a, A., Llama, M.J. and Serra, J.L. (1989) J. Bacteriol. 171, 1158-1165. 41 Hirasawa, M., Gray, K.A., Sung, J-D. and Knaff, D.B. (1989) Arch. Biochem. Biophys. 275, 1-10. 42 Hirasawa-Soga, M., Tamura, G. and Horie, S. (1983) J. Biochem. 94, 1833-1840. 43 Bowsher, C.G., Emes, M.J., Cammack, R. and Hucklesby D.P. (1988) Planta 175, 334-340.
237
Elsevier BBAPRO33728
Purification and some properties of the nitrite reductase from the cyanobacterium Phormidium laminosum Jesfis M. A r i z m e n d i a n d J u a n L. S e r r a Departamento de Bioqulmica y Biologla Molecular, Facultad de Ciencias, Universidaddel Pals Vasco, Bilbao (Spain)
(Received12 October 1989) (Revised manuscriptreceived24 April 1990)
Key words: Nitritereductase;Enzymepurification; ( P. laminosum) Assimilatory ferredoxin-nitrite reductase (EC 1.7.7.1, ammonia: ferredoxin oxidoreductase) has been purified 5300-fold with a specific activity of 625 units/mg protein from the filamentous non-heterocystous cyanobacterium Phormidium laminosum. The enzyme was soluble and consisted of a single polypeptidic chain of 54 kDa. It catalyzed the reduction of nitrite to ammonia using ferredoxin or flavodoxin as electron donor. Methyl and benzyl viologens were also effective as electron donors but neither flavins nor NAD(P)H were. The apparent Michaelis constants for nitrite, ferredoxin and methyl viologen were 40, 22 and 215 FM, respectively. Nitrite reductase activity was inhibited effectively by cyanide and thiol reagents. The enzyme exhibited absorption maxima at 281, 391 (Sorer), 570 (a) and 695 nm, with ¢~391 of 4.3.104 M -I cm -t, and an absorbance ratio A2s,/A39, of 1.95, suggesting the presence of siroheme as prosthetic group. These results show that this enzyme is similar to those of eukaryotic organisms.
Introduction The assimilatory nitrate reduction is catalyzed by the sequential action of nitrate reductase and nitrite reductase. Assimilatory nitrite reductase catalyzes the six-electron reduction of nitrite to ammonia. In photosynthetic organisms the physiological electron donor is ferredoxin, whereas in non-photosynthetic organisms the donor is NAD(P)H [1]. Ferredoxin-nitrite reductase (ammonia: ferredoxin oxidoreductase, EC 1.7.7.1) has been purified to electrophoretic homogeneity from several higher plants and eukaryotic algae. The enzyme is located in the chloroplast and composed of one polypeptide of 60-63 kDa [2-6], although it has been also reported as a heterodimer composed of two subunits of 61-64 kDa and 24-35 kDa [7-9]. The spinach nitrite reductase is a heme-protein with an iron-sulfur duster. The heme moiety has been identified as siroheme, an iron tetrahydroporphyrin of the isobacteriochlorin type with eight carboxylic acid-containing side chains, which is also present in both assimilatory and dissimilatory sulfite reductases [10]. The presence of one mol siroheme/mol enzyme was classi-
cally reported [6,11], although, recently, a ratio of 2 mol siroheme/mol enzyme has been calculated for the native heterodimer of the spinach enzyme [12]. In addition to siroheme, nitrite reductase also contains an [Fe4-S4] cluster as a prosthetic group [13]. The available information about nitrite reductase from photosynthetic prokaryotes is very scarce. In this group of organisms, cyanobacteria have a special interest because of their phylogenetic situation and their relationship with the origin of the chloroplast. The nitrite reductase from the cyanobacteria Anabaena cylindrica [14], Anacystis nidulans [15], Anabaena sp. 7119 [16,17] and Spirulina platensis [18] have been partially characterized, although only the enzyme from S. platensis has been highly purified. In the present work, we report the purification of nitrite reductase from the filamentous non-heterocystous cyanobacterium Phormidium laminosum, as well as some of its molecular, catalytic and spectral properties. Results show that this enzyme is similar to those of eukaryotic organisms. Materials and Methods Materials
Correspondence: J.M. Afizmendi, Departamento de Bioquimica y BiologlaMolecular,Facultadde Ciencias,Universidaddel Pals Vasco, Apartado 644, 48080-Bilbao, Spain.
Mono-Q, Mono-P, Phenyl-Superose and Superose 12 columns, Sephadex G-50, PhastGel 8-25, SDS and native buffer strips, Coomassie PhastGel Blue R, Poly-
0167-4838/90/$03.50 © 1990 ElsevierSciencePublishersB.V. (BiomedicalDivision)
238 buffer 74 and SDS-polyacrylamide gel electrophoresis markers were obtained from Pharmacia (Uppsala, Sweden). DEAE-cellulose (DE-52) was from Whatman (Maidstone, U.K.). Phenyl-Sepharose, acrylamide, N,N'-methylenebisacrylamide, SDS, methyl viologen, sodium dithionite, N-l-naphthylethylenediamine and native-electrophoresis markers were purchased from Sigma (St. Louis, U.S.A.). Immersible CX-10 ultrafiltration units were from Millipore (Bedford, U.S.A.). Glutamate dehydrogenase and gel filtration molecular weight markers were obtained from Boehringer-Mannheim (Mannheim, F.R.G.). Sulfanilamide was from Fluka (Buchs, Switzerland). The protein assay dye-reagent was from Bio-Rad (Miinchen, F.R.G.). All other chemicals were reagents of analytical grade supplied by Merck (Darmstad, F.R.G.).
Cell culture and cell free extracts P. laminosum (strain OH-l-p. Cll) cells were grown photoautotrophically at 45 ° C in the mineral medium D [19] in 60 1 glass containers. Cultures were stirred by an air stream and continuous illumination was provided by white fluorescent lamps (Tungsram F7 Daylight) with a photon flux density at the surface of the vessels of about 100/~mol m -2. s -1. Cells were harvested in the early stationary phase with a continuous flow rotor in a Kubota centrifuge, washed once with 50 mM Tris-HC1 (pH 8.0) (standard buffer) and suspended in the same buffer. Aliquots of 80 ml of this suspension were disrupted by sonication (MSE) for 10 min at 14/~m setting in an ice-bath. The sonicate was centrifuged at 110000 x g for 1 h at 4 ° C and the supernatant was used as crude extract.
Purification of nitrite reductase All steps except those of fast-protein liquid chromatography (FPLC) were carried out at 4 ° C. Chromatographies were done at a flow rate of 1 m l / m i n unless otherwise stated. (1) First DEAE-cellulose chromatography. The crude extract was passed through a DE-52 column (2.2 x 30 cm) equilibrated with standard buffer (50 mM Tris-HC1 (pH 8)). After washing the column with two bed volumes of standard buffer, nitrite reductase was eluted with 500 ml of 200 mM KCI in standard buffer. (2) Acetone fractionation. Chilled acetone ( - 20 ° C) was added slowly to the protein solution to 50% (v/v). The preparation was centrifuged at 10000 x g for 10 min, and the pellet was resuspended in 90 ml of standard buffer. This preparation had to be dialysed against the standard buffer before assaying the enzyme activity. (3) Second DEAE-cellulose chromatography. The above enzyme preparation was loaded onto a DE-52 column (2.2 x 30 cm) equilibrated with standard buffer. The column was washed with 120 ml of 20 mM KC1 in
standard buffer and nitrite reductase was eluted from the column with 200 mM KC1 in standard buffer. (4) Phenyl-Sepharose chromatography. The active fractions were pooled and solid ammonium sulphate was added up to 415 mM. The resulting solution was adsorbed to a Phenyl-Sepharose column (2.5 x 20 cm) equilibrated with 415 mM ammonium sulfate in standard buffer. The column was washed sequentially with 120 ml of equilibration buffer and 200 ml of 150 mM ammonium sulfate in standard buffer. Finally, nitrite reductase was eluted with standard buffer containing 75 mM ammonium sulfate, while most of the contaminant phycobiliproteins remained adsorbed to the column. The active fractions were pooled, concentrated to 30 ml with immersible CX-10 filters and dialysed against 5 mM KC1 in standard buffer. (5) Mono-Q chromatography. The dialysed preparation was applied to a Mono-Q H R 5 / 5 column equilibrated with 5 mM KC1 in standard buffer using an FPLC apparatus (Pharmacia, Uppsala, Sweden). The column was washed sequentially with 40 ml of the same buffer and with 36 ml of 100 mM KC1 in standard buffer. Nitrite reductase was then eluted with a linear gradient of 10 ml from 100-250 mM KC1 in standard buffer, followed by an isocratic elution with 10 ml of the last buffer. (6) Phenyl-Superose chromatography. The above enzyme preparation was adjusted to 1 M ammonium sulfate and loaded at a flow rate of 0.5 m l / m i n to an FPLC Phenyl-Superose H R 5 / 5 column equilibrated with 1 M ammonium sulfate in standard buffer. After washing with 10 ml of equilibration buffer, nitrite reductase was eluted applying a linear gradient of 24 ml from 1 - 0 M of ammonium sulfate in standard buffer and the active fractions were pooled. (7) Superose 12 chromatography. The resulting protein preparation was concentrated to 200 /~l by ultrafiltration (Amicon MPS-1) and loaded onto a Superose 12 H R 10/30 gel filtration column equilibrated with 100 mM KC1 in standard buffer. Elution was done with the same buffer at a flow rate of 0.5 m l / m i n and the active fractions were pooled. The purified nitrite reductase was stored at - 2 0 ° C until used.
Purification of ferredoxin and flavodoxin Ferredoxin and flavodoxin from P. laminosum were purified by a method consisting of DEAE-cellulose chromatography, ammonium sulfate fractionation, FPLC anionic exchange chromatography (Mono-Q H R 5 / 5 column) and Sephadex G-50 chromatography. After the last step of purification the absorbance ratios for ferredoxin (Aa23//A275) and flavodoxin (A465//A274) were 0.6 and 0.17, respectively. Ferredoxin and flavodoxin were estimated from their molar absorption coefficient of 9.2 mM - 1c m - 1, at 423 nm [20] and 9.7 mM - 1. c m - l, at 465 nm [21], respectively.
239
Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis were carried out in a PhastSystem equipment (Pharmacia, Uppsala, Sweden). SDS-electrophoresis was performed in 8-25% ( w / v ) acrylamide gradient gels (PhastGel 8-25) with PhastGel SDS Buffer Strips. Native-electrophoresis were carried out in both 8-25% ( w / v ) acrylamide gradient gels (PhastGel 8-25) and homogeneous gels made as described in the PhastSystem Separation Technique File No. 121 (Pharmacia) with PhastGel Native Buffer Strips. The electrophoretic conditions were those described in the PhastSystem separation Technique Files No. 110, 120 and 121 (Pharmacia), and the SDS- and native-electrophoresis in homogeneous gels were stopped when the bromophenol blue had reached the anode. Proteins were stained using Coomassie brilliant blue [22] or silver staining (PhastSystem Development Technique File No. 210, Pharmacia). Nitrite reductase was located in the gels by methyl viologen-dependent activity staining [11]. Enzyme assays Nitrite reductase activity was routinely assayed in a similar way to that previously described [23]. The reaction mixture contained in a 1 ml final volume: 90 m M Tris-HC1 (pH 7.5), 2.5 m M K N O 2, 3 m M methyl viologen, 20 m M sodium dithionite (freshly dissolved in 0.3 M NaHCO3) and an appropriate amount of enzyme. After 5 min preincubation, the reaction was carried out for 10 rain at 30 o C, and stopped by vigorous-shaking to obtain the complete oxidation of the excess of reductant. Nitrite disappearance was determined after a 100-fold dilution of the reaction mixture [24]. Blank tubes without enzyme were also included. When the K m for ferredoxin, methyl viologen and nitrite were estimated, assays were carried out in sealed test-tubes saturated with He or N 2 to diminish the oxidation of dithionite. The K m for nitrite was also estimated by progresscurve analysis. This assay was carried out in sealed test-tubes saturated with He, as in the routine assay
described before, but 0.25 m M KNO2 was used in a reaction mixture of 2 ml. The reactions were stopped at different times and 10-fold dilution was done before determining nitrite. The non-enzymatic oxidation of methyl viologen caused an interference always lower than 5%. One unit of enzyme activity (U) was defined as the amount that catalyzed the reduction of 1/~mol of nitrite per min.
Absorption spectra Absorption spectra were recorded at room temperature against a buffer blank in a Shimadzu UV-260 spectrophotometer with cuvettes of 1 cm pathlength. Analytical determinations Protein was determined by the method of Bradford [25] using bovine serum albumin as standard. Ammonium was determined enzymatically by following the oxidation of N A D P H catalyzed by glutamate dehydrogenase [26]. lsoelectric point determination Isoelectric point was determined by FPLC-chromatofocusing in a Mono-P H R 5 / 2 0 column. A flow rate of 0.5 m l / m i n and a p H gradient from 7 to 4 were used. After equilibration of the column with 25 m M Bistris-iminodiacetic (pH 7.1), a partially purified sample of nitrite reductase was, injected onto the column and the p H gradient was formed by applying 50 ml of 1 : 10 diluted Polybuffer 74-iminodiacetic buffer ( p H 4). The nitrite reductase activity and p H were determined in the collected fractions of 1 ml. Results
lntracellular location of nitrite reductase When cells of P. laminosum were broken with lysozyme followed by osmotic shock [27] and the homogenate was centrifuged at 110000 x g for 1 h, 80% of
TABLE I
Purification of nitrite reductasefrom P. laminosum The purification procedure from 80 g of cells (fresh weight) and the protein determination were conducted as described in Materials and Methods. Nitrite reductase activity was measured by the routine assay described in Materials and Methods, but at 50 ° C. Step
Volume (ml)
Activity (U)
Protein (mg)
Specific a c t i v i t y (U/mg protein)
Purification (-fold)
Yield (%)
Crude extract DE-52 I 50% Acetone DE-52 II Phenyl-Sepharose Mono-Q Phenyl-Superose Superose 12
315 256 88 300 29 10.4 2.2 2.2
432 382 285 299 210 194 121 69
3692 2560 396 159 11.3 2.9 0.48 0.11
0.117 0.15 0.72 1.88 19 67 252 627
1 1.3 6.2 16.1 162 572 2155 5359
100 88 66 69 49 45 28 16
240 the total nitrite reductase activity was recovered in the supernatant, indicating the soluble nature of the enzyme. With this method, 85% of the cell rupture obtained by sonication was achieved, the insoluble activity probably being due to the incompletely broken ceils. This result is in agreement with the data described for the enzyme from A. cylindrica [28] and Anabaena sp. 7119 [17], but in A. nidulans [29] nitrite reductase is associated with subcellular particles and a long disruption process is necessary for its solubilization. Purification of nitrite reductase Table I summarizes a typical purification from 80 g of wet cells. Nitrite reductase was purified more than 5300-fold, in 16% yield to a specific activity of 627 units/mg protein at 50 ° C. A similar value of specific activity was obtained when the protein content was determined by the method of Lowry et al. (1951) modified by Peterson [30]. After the last step the enzyme preparation gave a single protein band upon Coomassie blue stained native-electrophoresis, with a positive reaction for methyl viologen-nitrite reductase activity (Fig. 1, lane A). Nevertheless, after silver staining of the same slab, a contaminant representing less than 2% of the total protein could be seen (Fig. 1, lane B). The high purity of the nitrite reductase preparation was also confirmed by the single protein band detected in the silver stained, SDSelectrophoresis (Fig. 1, lane C). Molecular mass The molecular mass of nitrite reductase was estimated by four methods: native-electrophoresis in either acrylamide gradient gels, or homogeneous gels of different acrylamide concentrations, SDS-electrophoresis in acrylamide gradient gels and gel filtration.
A
B
D
94
kDa
66 43 30 20.1 14.4
Fig. 1. Polyacrylamide gel electrophoresis of the purified nitrite reductase from P. larninosum. Lane A. Activity staining of 200 ng of purified nitrite reductase after native-electrophoresis in an 8-25 (w/v) acrylamide gradient gel. Lane B. Silver staining of the electrophoresis of lane A. Lane C. Silver staining of 25 ng of purified nitrite reductase after SDS-electrophoresis in an 8-25 (w/v) acrylamide gradient gel. Lane D. Silver staining of 300 ng of standard proteins after SDS-electrophoresis in an 8-25 (w/v) acrylamide gradient gel. The standard proteins used were as follows: phosphorylase b (94 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), carbonic anhidrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa).
The molecular mass obtained by native-electrophoresis in a 8-25% (w/v) acrylamide gradient gel was 69 kDa, which was similar to that of 64 kDa calculated from the Ferguson plot (Fig. 2). However, when the
A 2OO
B
180
I: e~
C
12
,\
160
NiR
r~
J
O
140 em
_e 120 4 ¸
1
v-4
100 5 80
•
0
I
lS
~
Ni !
20
•
Ill
2•
25
GEL CONCENTRATION (%)
i
30
40
,
I
80
•
I
120
m (kDa)
Fig. 2. Ferguson plot analysis of the molecular mass of nitrite reductase. (A) Effect of different gel concentrations on the relative mobilities (R m) of nitrite reductase (NiR) and standard proteins in native-electrophoresis. (B) Slope/m relation of nitrite reductase and standard proteins. The standard proteins used were as follows: 1, a-lactalbumin (14.2 kDa); 2, carbonic anhydrase (29 kDa); 3, chicken egg albumin (45 kDa); 4, monomer of bovin serum albumin (66 kDa); and 5, dimer of bovin serum albumin (132 kDa).
241 same enzyme preparation was injected into a gel filtration Superose 12 column, a value of 54 kDa was obtained. In the same way, a single protein band corresponding to 54 kDa was detected by SDS-electrophoresis in 8-25% (w/v) acrylamide gradient gel (Fig. 1, lanes C and D). Nitrite reductase bands in both nativepolyacrylamide gel electrophoresis were rather wide, and these methods are not as generally accepted as the SDS-electrophoresis method, so 54 kDa seems to be the most reliable value for the molecular mass of nitrite reductase. On the other hand, when a crude extract was either dialysed against standard buffer or was kept at 4 °C for 4 days, two bands of methyl viologen-dependent nitrite reductase activity appeared after a native-electrophoresis in an acrylamide gradient gel. One band corresponded to the molecular mass of the purified nitrite reductase and the second minor band corresponded to a 14 kDa smaller molecular mass. These results could suggest the heterodimeric nature of the enzyme, with both the dimer and the biggest monomer having methyl viologen-dependent activity, in a similar way to that reported in spinach [7], etiolated bean shoots [8] and the green alga Chlamydomonas reinhardtii [9]. Nevertheless, the addition to the crude extract of 2.8 mM iodoacetamide (a thiol-proteinase inhibitor) prevented the appearance of the second activity band without affecting the whole nitrite reductase activity. Moreover, when a purified enzyme preparation was used in these experiments, the second activity band did not appear. These results could be explained by assuming that a proteolysis of the enzyme in the crude extract occurs. So, it is concluded that the native nitrite reductase from P. laminosum consists of a single polypeptidic chain of 54 kDa. A Stokes radius of 3.14 nm was determined by gel filtration in Superose 12 and a frictional coefficient of 1.26 was calculated for nitrite reductase.
lsoelectric point The isoelectric point of the enzyme determined by chromatofocusing was 5.6. This value is between those of 4.3-4.5 described for the enzymes from leaves, scuteUa, roots of corn [31], and leaves and roots of barley [32], and that of 6.2 described for the enzyme from C. reinhardtii [33]. Catalytic properties Nitrite reductase from P. laminosum catalyzed the stoichiometric reduction of nitrite to ammonia, one ammonium being formed for each nitrite disappeared (data not shown). A K m for ferredoxin of 22/tM was calculated from a Hanes plot of the initial velocities of nitrite reduction at different concentrations of ferredoxin reduced by dithionite. This value corresponds well with those ob-
2O
16 o
12
8
m
•
4
0
-50
0
50
100
150
200
S.-S / In (So/S) (ltlVl) Fig. 3. Progress curve analysis of the effect of nitrite concentration on nitrite reductase activity. Nitrite was determined at different incubation times from the reaction-mixture described in Materials and Methods. The reaction was carried out under He atmosphere at 30 o C. SO is the initial substrate concentration (0.25 mM), and S the concentration at time t. The a m o u n t of enzyme used was 0.06 units.
tained for the enzymes from cyanobacteria [14,17,18] and some photosynthetic organisms [9,34-36], but is higher than the value of 0.4 #M reported for the enzyme from barley [3]. The K m for reduced methyl viologen obtained from a Hanes plot of the initial velocities of nitrite reduction at different concentrations of methyl viologen reduced by dithionite, at saturating concentration of nitrite, was 215 #M. This value is in the range from 60-400 #M of previous data for the enzyme from cyanobacteria [14,17,18] and eukaryotic organisms [2,3,6]. The K m for nitrite determined by progress curve analysis of nitrite was 40 #M (Fig. 3), but the initial velocities method gave a considerably higher value (about 0.6 mM). Fry et al. [37] also showed variations on the K m value for nitrite for the enzyme from vegetable marrow leaves, depending on the aerobic or anaerobic system used. Table II shows the ability of various electron donors to transfer electrons to the nitrite reductase. Methyl viologen was the most effective electron donor, while benzyl viologen was less effective. Both ferredoxin and flavodoxin were also partially effective, although the concentrations used here were not saturating. Neither NAD(P)H nor dithionite-reduced flavins were active as electron donors for nitrite reductase, in contrast to the enzyme from non-photosynthetic organisms [1]. The effect of temperature from 25-65 °C on nitrite reductase activity was examined. The activity increased up to 50 °C and the activation energy calculated from an Arrhenius plot was 50 kJ/mol. These results are similar to those obtained for the enzyme from C. reinhardtii [38].
242 TABLE II
TABLE III
Electron donors for the purified nitrite-reductase from P. laminosum
Effect of inhibitors on nitrite reductase activity from P. laminosum
Nitrite reductase activity was determined by the routine assay described in Materials and Methods, except that the indicated electron donors were added instead of methyl viologen and dithionite. 100% activity corresponds to 6.5 U / m g protein.
Nitrite reductase activity was determined by the routine assay described in Materials and Methods, except that the indicated compounds were added to a final reaction mixture of 2 ml.
Electron donor
Concentration (mM)
NADH NADPH Dithionite methyl viologen benzyl viologen + FAD + FMN + flavodoxin + ferredoxin
5 5 20 5 5 5 5 0.005 0.01
++
Activity (%)
Inhibitor
Concentration (mM)
Inhibition (%)
NaCN
0.005 0.05 0.1
30 78 92
p-Hydroxymercuribenzoate
0.1
35 68
0 0 100 34 0 0 25 25
The pH dependence of the nitrite reductase activity was studied in potassium phosphate buffer in the p H range of 6.8-7.8, and in Tris-HC1 in the range of 7.5-8.5. The optimum p H for nitrite reduction was 7.3 and 7.6 with phosphate and Tris-HCl buffers, respectively. These values are in the range from 7-8, which includes the optimum pH values reported for most nitrite reductases [2,9,14,17,18,35,38]. The P. laminosum nitrite reductase was fully thermostable until 40°C, but more than 95% of the activity was lost when the enzyme was heated for 10 rain at 60 o C. The thermal inactivation reaction was of second order and the activation energy calculated was 167 kJ/mol. So, this enzyme is slightly less thermostable than other enzymes involved in the nitrate assimilation in P. laminosum, such as glutamate dehydrogenase [39] and glutamine synthetase [40]. The enzyme was stable for a week at 4 ° C and for several months at - 2 0 o C, even without the addition of thiol reagents (dithiothreitol) or chelating agents (EDTA). Table III shows the inhibitory effect of several compounds on the nitrite reductase activity. Cyanide was a very powerful inhibitor of the enzyme activity, while azide and bathophenanthroline were weak inhibitors. Thiol reagents, such as p-hydroxymercuribenzoate and N-ethylmaleimide, also caused a significant inhibition. Sulfite and hydroxylamine inhibited only at high concentrations.
Absorption spectra The absorption spectrum of the purified nitrite reductase from P. laminosum showed maxima at 281,391 (Soret), 570 (a) and 695 nm (Fig. 4). The A281/A391 absorbance ratio was 1.95, and the Soret to a band ratio was 3.33. The molar absorption coefficients were: 8.4. 104 M -1 . c m -], at 281 nm; 4 . 3 . 1 0 a M -1 . c m -1, at 391 nm; and 1.3-104 M - 1 . c m -], at 570 nm. This
1
N-Ethylmaleimide
0.1 1
16 47
NaN 3
1
15
Bathophenanthroline
1
14
Sulfite
1 10
5 24
Hydroxylamine
1 10
16 34
spectrum is very similar to that of the enzyme from eukaryotic organisms, and only the enzymes from spinach leaves [6] and C. reinhardtii [9] showed a lower A280/A39 o absorbance ratio. Therefore, the little differences in the spectrum of the enzyme from the cyanobacterium S. platensis [18] would not represent different characteristics of the cyanobacterial enzyme but they would probably be due to some contaminants of the preparation.
0"101 o C
0.05
o
250
450 Wavelength
650 (nm)
Fig. 4. Absorption spectrum of the purified nitrite reductase from P. laminosum. The absorption spectrum of the enzyme solution (50 /~g/ml) in 100 mM KC1 in 50 mM Tris-HC! (pH 8) was recorded against a buffer blank.
243
0.050
iC
C
0.025
,¢
0
t
" I
I
350
I
i
550 Wavelength
-
~
750
(rim)
Fig. 5. Absorption spectra of the purified nitrite reductase in the presence of dithionite and nitrite. (A) Absorption spectrum of nitrite reductase (50 /~g/ml) in 100 mM KCI in 50 mM Tris-HC1 (pH 8) ( ). (B) Absorption spectrum of nitrite reductase after the addition of solid dithionite ( . . . . . . ). (C) Absorption spectrum of nitrite reductase after the addition of a few crystals of nitrite to the sample previously reduced by dithionite ( . . . . . ).
Addition of dithionite to nitrite reductase resulted in the bleaching of the 570 and 695 nm peaks, with the appearance of a broad shoulder at 580 nm and a shoulder at 410 nm. The subsequent addition of nitrite to the dithionite-reduced enzyme restored the absortion maximum at 570 nm with a shift to 580 nm, but the absorption at 695 was not restored (Fig. 5). Similar spectral changes have also, been reported for the enzymes from spinach leaves [11] and C. reinhardtii [9]. Discussion
The purification procedure reported here allows almost the complete purification of the P. laminosum nitrite reductase. The purified preparation showed a specific activity of 625 U / m g protein at 50 °C which corresponded to 225 U / m g protein at 30 ° C. This value is much higher than those reported for the enzyme from A. cylindrica (10.75 U / m g protein at 25°C) [14], Anabaena sp. 7119 (21.5 U / m g protein at 30°C) [16] and higher than that of S. platensis (195 U / m g protein at 35 ° C) [18]. The best specific activity reported for the enzyme from an eukaryotic source (spinach leaves) is 207 U / m g protein [6]. The high specific activity ob-
tained shows that the purification procedure yields a good enzyme preparation, not only from the point of view of the purity but also from that of the little damage caused to the enzyme. The molecular mass of our enzyme (54 kDa) is similar to those reported for other cyanobacterial enzymes (52-54 kDa) [15,16,18]. However, a value of 68 kDa was reported for A. cylindrica nitrite reductase [14]. In contrast with the enzyme from some higher plants [7,8] or C. reinhardtii [9], where the native nitrite reductase seems to consist of two subunits of different size, the cyanobacterial enzyme appears as a monomeric protein in all cases. This subunit composition agrees with that showed by most higher plant nitrite reductases [2-6]. During the revision of this manuscript, a new paper, confirming the two-subunit composition of the spinach nitrite reductase, was published [41]. According to that, the use of fl-mercaptoethanol could be the main factor to explain the apparent absence of the small subunit in a number of plant nitrite reductase preparations, this effect being increased by low ionic strengh buffers or by the absence of proteinase inhibitors. Despite the fact that this procedure involves the use of buffers of low ionic strengh and the absence of proteinase inhibitors, fl-mercaptoethanol is not used in the purification steps. Moreover, native-electroph6resis of both the crude extract and the purified enzyme shows that the molecular mass of the enzyme does not change through the purification procedure. Finally, SDS-electrophoresis of the purified enzyme shows a single protein band after silver staining, in contrast to the two equally stained bands obtained by Hirasawa et al. [41]. The affinities of P. laminosum nitrite reductase for its substrates (i.e., nitrite, ferredoxin and methyl viologen) were similar to those previously reported for the enzyme from photosynthetic organisms. In this study different values of K m for nitrite have been found depending on the assay method (initial velocities or progress-curve analysis) used. The different results obtained due to the assay method, the utihzation of dithionite as reductant [42] and the use of aerobic or anaerobic conditions [37] could explain the diversity of data concerning the affinities for substrates, especially for nitrite, reported in the literature [2]. All nitrite reductases isolated from eukaryotic sources were inhibited by thiol reagents [3,11,33,36], whereas these compounds did not affect the cyanobacterial enzyme previously-studied [15,17,29]. However, the P. laminosum nitrite reductase was strongly inhibited by p-hydroxymercuribenzoate and N-ethylmaleimide, denoting the presence of thiols involved in the catalysis. The other inhibitors tested affected the P. laminosum enzyme in a similar way to that reported for the enzyme from other sources. The absorption spectrum of our purified enzyme and the differences in the spectrum caused by the reduction
244 and the addition of nitrite are similar to that of eukaryotic organisms [2,6,9,43], suggesting the presence of a siroheme as the prosthetic group involved in the binding and reduction of nitrite. According to the molecular, catalytic and spectral properties showed by the P. laminosum nitrite reductase and the available data on other cyanobacteria, we can conclude that the enzyme of these prokaryotes is similar, with the possible exception of the subunit composition, to that characterized from photosynthetic eukaryotic organisms.
Acknowledgments This work was partially supported by grants from Comisi6n Asesora de Investigaci6n Cientifica y T&nica (928/84) and the Basque Country Government (X86.049).
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