P l a n t a 9 Springer-Verlag 1988
Purification and properties of nitrite reductase from roots of pea (Pisum sativum cv. Meteor) Caroline G. Bowsher *, Michael J. Emes*, R. Cammack **, and Dereck P. Hueklesby *** Department of Agricultural Sciences, University of Bristol, Institute of Arable Crop Research, Long Ashton Research Station, Long Ashton, Bristol BS18 9AF, UK
Abstract. Nitrite reductase (EC 188.8.131.52) prepared from pea roots was found to be immunologically indistinguishable from pea leaf nitrite reductase. Comparisons of the pea root enzyme with nitrite reductase from leaf sources showed a close similarity in inhibition properties, light absorption spectrum, and electron paramagnetic resonance signals. The resemblances indicate that the root nitrite reductase is a sirohaem enzyme and that it functions in the same manner as the leaf enzyme in spite of the difference in reductant supply implicit in its location in a non-photosynthetic tissue. Key words: C u c u r b i t a - Leaf (nitrite reductase) Nitrite reductase - P i s u m (nitrite reductase) - Root (nitrite reductase).
Introduction The assimilation of nitrate by plants may occur in leaves or roots or in both, depending upon species, N supply and developmental stage. Since the process of reduction of nitrate to ammonia is lightregulated and associated in a complex manner with photosynthesis (Beevers and Hageman 1972), the metabolism of nitrate by non-green cells is necessarily different (for review, see Oaks and Hirel 1985). Evidence has accumulated that nitrite reduction, which in leaves is closely coupled to phoPresent addresses: * Department of Cell and Structural Biology, School of Biological Sciences,Universityof Manchester, Manchester M 13 9PL, and ** Department of Biological Sciences, King's College, London SE24 9JF, UK *** To whom correspondence should be addressed Abbreviations: DEAE = diethylaminoethyl; EPR = electron paramagnetic resonance; NIR=nitrite reductase; SDSPAGE = sodium dodecyl sulphate-polyacrylamide gel electrophoresis
tosynthetic electron transport, functions in roots by reception of electrons from the pentose-phosphate pathway (Butt and Beevers 1961; Emes and Fowler 1983). Despite these differences in metabolism, some striking similarities have been noted between leaf and root nitrite reduction. In these tissues the enzyme, nitrite reductase (NIR), is localised in homologous organelles - the chloroplasts of the leaf and the proplastids of the root. The nitrite reductases of green and non-green tissues have many other features in common (Dalling et al. 1973; Ida et al. 1974; Nagaoka et al. 1984). In particular, there is a close similarity in their requirement for electron donors in v i t r o (Hucklesby et al. 1972) in spite of the apparent absence of leaf ferredoxin or other suitable carrier in roots. Recent research has probably resolved this latter problem with the demonstration of a ferredoxinlike compound in cultured non-green cells (Ninomiya and Sato 1984) and roots (Suzuki et al. 1985; Wada et al. 1986). The current paper describes further comparisons of the leaf and root enzymes and presents evidence from spectrophotometry and electron paramagnetic resonance (EPR) studies that the pea root N I R is a sirohaem protein.
Materials and methods Purification of NIR from leaves. Cucurbita pepo L. NIR was purified by modifying a method previously described (Hucklesby et al. 1976). After the first diethylaminoethyl (DEAE)cellulose step, the preparation was further purified by two passes through a Mono-Q column (Pharmacia, Milton Keynes, UK), using a Pharmacia FPLC (fast protein liquid chromatography) apparatus. For the first pass, the enzyme was applied to the column in 0.02 M 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-HC1 and eluted with a gradient of NaC1 in the same buffer, increasing linearly from 0 to 0.35 M NaC1 in 20 ml at 2 ml-min -1. The enzyme from the first pass was diluted with an equal volume of 0.02 M Tris-HC1, pH 7.4 before the
C.G. Bowsher et al. : Purification and properties of nitrite reductase from roots of pea second pass. Enzyme with an absorbance ratio (384/280 rim) of 0.50-0.56 was obtained giving single band on sodium dodecyI sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This enzyme was used for antibody production.
Purification of pea root N1R. All steps were at 0-4 ~ C. Buffer 1 (for extraction) was 0.1 M potassium phosphate containing 0.5 m M EDTA 5 m M cysteine HCI. Buffer 2 (for dialysis and chromatography) was 0.02 M Tris-HC1, pH 7.7, 0.1 M KC1, I0 mM mercaptoethanol and 10% (w/v) glycerol. Buffer 3 (for affinity chromatography) was 5 m M potassium phosphate, pH 7.5, with 10 mM 2-mercaptoethanol and 10% glycerol. Pea (Pisum sativum L. cv. Meteor) roots (0.5-2 kg), either fresh or from storage at - 1 8 ~ C, were homogenised in buffer i using a Waring blender. The homogenate was filtered through two layers of muslin and centrifuged at 5400.g for 40 min in a refrigerated centrifuge (Mistral, 2.8-1 rotor; MSE, Crawley, Sussex, UK). Glycerol (10%) and mercaptoethanoI (I0 mM) were added followed by ammonium sulphate, approx. 48%, added as an equal volume of cold, saturated solution. After standing for 1 h, the solution was centrifuged at 32000.g for 30 rain. The precipitate was discarded and solid, chilled ammonium sulphate added (200 g.1 -~) to the supernatant giving a final saturation of about 80%. The precipitate was removed by centrifugation as for the 48% fractionation and dissolved in a minimum volume of buffer 2. The solution was clarified by centrifugation at 30000.g for 10 rain and desalted by a Sephadex G25 column (50 cm long, 4 cm i.d.) equilibrated with this buffer. Desalting was necessary to prevent spreading of the protein band during the following gel filtration step, which comprised a column of Sephadex G100 (100 cm long, 2.6 cm i.d.) equilibrated against buffer 2. Portions (35 ml) were applied to the column at 20 m l . h ~ at each run o f / 6 - 2 4 h, and 5-ml fractions collected. Nitrite-reductase activity was eluted in a single peak. Active fractions were combined and run at a rate of 20 ml-h -a into a column (20 cm long, 1.6 cm i.d.) of DEAEcellulose (DEAE-Sephacel from Pharmacia) equilibrated with buffer 2. The N I R was eluted from the column with a 400-ml linear concentration gradient, 0.05 M to 0.4 M KC1 in buffer 2. Further purification of the pea root enzyme was performed by affinity chromatography on ferredoxin-Sepharose. Ferredoxin was prepared from acetone-precipitated crude extracts of Cucurbita pepo leaves, based on the method of Tagawa and Arnon (1962) or as a by-product of N I R purification using DEAE-cellulose to collect ferredoxin from the 80%-saturated ammonium-sulphate supernatant (Mayhew 1971). The ferredoxin was purified to an absorbance ratio (E422 rim/E278 nm) of 0.48-0.5 by chromatography on DEAE-cellulose and Sephadex G75 (Scawen et al. 1975) and dialysed against coupling buffer (0.1 M NaHCO3, 0.5 M NaC1 pH 8.3). Ferredoxin-Sepharose was prepared as described by Serra et al. (1982). Cyan-
ogen-bromide-activated Sepharose was swollen in 1 mM HCl (15 rain) and washed with the same solution. The gel was then washed with coupling buffer (5 ml to I g dry weight of gel). About 50 mg of ferredoxin was immediately added to and coupled with 3 g of gel (dry weight) by rotating the gel and ferredoxin end-over-end for 16 h. After treatment at 4 ~ with blocking buffer (0.1 M Tris-HC1, pH 8.0), the gel was washed with four to five changes of 0.1 M acetate buffer, pH 4, containing 0.5 M NaC1, followed by 0.1 M Tris-HC1 pH 8.0, containing 0.5 M NaC1. The gel was loaded into a column and stored at 4 ~ C. Following ion-exchange chromatography, the pea root N I R was dialysed overnight against buffer 3. The protein was applied to a column (10 cm long, I cm i.d.) of ferredoxin-Sepharose equilibrated with buffer 3. Unbound protein was eluted at 10 m l . h ~ with buffer 3 to low absorbance at 280 nm; N I R was eluted with 0.1 M KC1 in buffer 3.
Antibody production. Cucurbita pepo leaf NIR, purified as above, was mixed (1 vol. to 9 vol.) with Freund's complete adjuvant and injected sub-cutaneously into a rabbit. After 10 d, a similar quantity of NIR, mixed with the same ratio of Freund's incomplete adjuvant was injected in the same way. Two further similar injections were made at 10-d intervals; 40 gg of the enzyme were used for each of of the four injections. Serum was collected 40 d after the first injection, refrigerated overnight, centrifuged at 20000.g, and stored at - 1 8 ~ C. Enzyme assays. Methyl viologen reduced by dithionite was used for the assay of N I R as described previously (Hucklesby et al. 1972). In assays with ferredoxin, methyl viologen was replaced in the assay solution by 10 gM ferredoxin. The SDS-PAGE electrophoresis of proteins was on 7.5% acrylamide gels according to Haines (1981). The gels were stained for protein with silver (Bulletin 1200; Bio-Rad Laboratories, Watford, Herts., UK). Western blotting was based on the methods of Campbell and Remmler (1986) and Towbin et al. (1979). Immunodiffusion studies were carried out by the technique of Ouchterlony and Nilsson (1973). Electron-paramagnetic-resonance Studies. The EPR spectra were measured on a Varian Associates (Palo Alto, Cal., USA) E4 spectrometer with an Oxford Instruments (Oxford, UK) helium flow cryostat.
Purification of N1R. The N I R from pea roots was prepared
(Table1) with a specific activity of
Table 1. Purification of nitrite reductase from pea roots
Total activity b (nkat)
Crude extract Ammonium-sulphate ppt. Sephadex G100 DEAE-cellulose Ferredoxin-Sepharose
29 550 16 820 12180 8 530 1600
Protein content (g) 21.0 2.27 0.532 0.117 0.0043
Specific activity (nkat. rag- ~) 1.41 7.41 22.90 72.93 372.09
Yield (%) 86 62 28 5
5 16 52 264
a The starting material was 7 kg of roots. Batches each of 2 kg, were homogenised, purified through the first three stages and then stored. Preparations were combined after the DEAE-cellulose step for final purification on ferredoxin-Sepharose b 1 k a t = i mol N O 2 reduced.s ~ under the standard assay conditions
C,G. Bowsher et al. : Purification and properties of nitrite reductase from roots of pea
372 n k a t . m g - t of protein and showed two bands after SDS-PAGE and silver staining. The molecular weight of the larger of these two proteins was estimated by SDS-PAGE to be 60000-61 500 daltons (Da) and of the smaller 48000. Both reacted positively to the C. pepo leaf nitrite-reductase antibody after electroblotting on to nitrocellulose. Prior to this stage of purification, only one band was observed corresponding to the 60 000-Da polypeptide when SDS gels were electroblotted and challenged with N I R antibody. This implies that the lower-molecular-weight band appearing after the final purification step is a degradation product of nitrite reductase. Immunological studies. An antibody to pure C. pepo leaf N I R was raised in a rabbit and the serum used in Ouchterlony cross-reaction tests with pea leaf and pea root NIR. For these tests, the enzymes were partially purified using the first three steps of the purification schedule (Table 1), i.e. were used after the DEAE-cellulose column. The root and leaf enzymes from pea reacted positively and, as far as could be seen, identically with the antibody to C. pepo NIR, indicating a large degree of similarity to each other. The Ouchterlony reaction of the pea enzymes with the antibody differed to some extent from that of the C. pepo leaf enzyme in that the latter produced a spur (Fig. 1), possibly indicating that the C. pepo leaf enzyme possesses at least one epitope additional to those of the two enzymes from pea. Spinach leaf N I R
Fig. 1. Ouchterlony double diffusion analysis of nitrite reductase purified from C. pepo and P. sativum. Wells (10 gl) contained N I R from: 1, C. pepo leaf; 2, P. sativum leaf; 3, P. sat# r u m root. The centre well contained antibody to C. pepo nitrite reductase. Inset with the diagram is a photocopy of the gel plate
antibody was found to be non-reactive with bean (Phaseolus) root enzyme in Ouchterlony tests (Nagaoka et al. 1984) although it was slightly inhibitory to its activity. Inhibitors. Of several metal-chelating compounds added during N I R assay (Table 2), only 8-hydroxyquinoline and diethyldithiocarbamate gave detectable inhibition. Bathophenanthroline disulphonate and bathocuproine disulphonate, re-
Table 2. Effect of inhibitors on pea root nitrite reductase. Nitrite reductase was purified through the first three stages of the schedule (Table 1). Inhibitors were added immediately before assay. Data given are means from at least three experiments Inhibitor
Concentration of inhibitor (mM)
Activity (% control)
a Concentration of inhibitor (mM)
Activity (% control)
KCN KCN Bathophenanthroline disulphonate Bathocuproine disulphonate 2,2'-Dipyridyl 8-Hydroxyquinoline Diethyldithiocarbamate EDTA A m m o n i u m persulphate Arsenate (Na) Arsenite (Na) Hydrazine sulphate Isonicotinic acid hydrazide o-Phenanthroline Ferrocyanide (K) 4-Chloromercuribenzoate 4-Chloromercuribenzoate+glutathione (2.5 raM)
0.02 0.0085 1 1 I 1 1 1 1 I 1 1 1 1 1 0.5 0.5
0 50 116 116 102 78 82 98 98 79 92 90 87 110 36 38 95
2- 4 34- 48 33- 47 104-106 91- 97 106-115 92-108 94-106 84- 94 96-107
a Hucklesby et al. (1972)
1 1 1 1 1 1 1 1 1 0.5 0.5
( ~ 12 83 85
C.G. Bowsher et al. : Purification and properties of nitrite reductase from roots of pea
ported previously as inhibitors of the maize scutellure enzyme (Hucklesby et al. 1972), were slightly stimulatory to the pea root enzyme. Cyanide was wholly inhibitory at 12 I~M and 50% inhibitory at 8.5 gM. The inhibition was competitive with nitrite at 125 pM KCN. Addition of 0.5 mM 4-chloromercuribenzoate (pCMB) resulted in a 64% inhibition, which was almost wholly prevented by 2.5 mM glutathione. Experiments in which glutathione and pCMB were added prior to assay, showed that inhibition by the latter was suppressed, at least in part, providing that the glutathione was added immediately. Delay resulted in a severe inhibition, indicating that the initial reversible interference, probably with functional sulphydryl groups in the enzyme, is followed by an irreversible reaction. In NIR from C. pepo leaves, similar reversible and irreversible phases have been observed with another mercurial compound, mersalyl, where the irreversible phase was associated with partial loss of the Soret band of the absorption spectrum of the protein, indicating sirohaem breakdown (data not shown).
Fig. 2. Absorption spectrum of P. sativum nitrite reductase, measured in a Pye-Unicam (Cambridge, U K ) 8800 spectrophotometer in cells of 10 mm pathlength. The absorbance at 280 nm was ].24
I ~ ' ' '
' ' '
Absorption spectrum. Figure 2 shows the absorption spectrum of the pea root NIR as purified. Maxima were found at 695, 632, 572, 532, 384 and 278 nm. These may be compared with our previously reported values for the C. pepo leaf enzyme of 697, 635, 572, 532, 384 and 280 nm (Hucklesby et al. 1976). Electron-paramagnetic-resonance spectra. Pea root NIR prepared by the full purification procedure described was examined by low-temperature EPR spectroscopy. Three stable forms of the enzyme which gave characteristic spectra were compared with those of the leaf enzyme. The protein as prepared showed a prominent spectrum, at g=6.9, 5.0 and 1.95. These correspond to gz, gy and gx of a rhombically distorted high-spin ferric haem. The spectrum is indistinguishable from that of the leaf enzyme of C. pepo (Fig. 3; Cammack et al. 1978) and closely similar to those of spinach NIR (Vega and Kamin 1977) and Eseherichia coli sulphite reductase (Peisach et al. 1971; Janick et al. 1983), all of which are considered to be sirohaem-containing proteins. A comparison of the leaf and root enzymes is shown in Fig. 3. As with the C. pepo NIR, contact of the enzyme with nitrite for a few minutes at room temperature eliminated the three above-mentioned peaks from the spectrum. Also visible in the spectra of both leaf and root enzymes (Fig. 3) are small signals at g=2.01 and g=2.06 which correspond
I Pill 200
I Illlltll 300
I Ifltlllll 400
MAGNETIC FIELD (rnT)
Fig. 3. Electron-paramagnetic-resonance spectra of nitrite reductases in the oxidised state. Upper spectrum: C. pepo leaf nitrite reductase. Lower spectrum: P. sativum root nitrite reductase. Conditions of measurement: temperature 10 K; microwave power 20 mW; frequency 9.18 G H z
to a small proportion of the nitrosyl form (compare Fig. 5), which is so stable that it does not dissociate during purification (Fry et al. 1980). The weak signals at intermediate field may represent partially reduced mixed spin states of the enzyme. The EPR spectrum of the [4Fe-4S] clusters in the sirohaem-containing nitrite and sulphite reductases may be observed by reduction in the presence of ligands such as cyanide or carbon monoxide, which convert the haem to a low-spin ferrous form. The spectrum of the reduced cyanide adduct of
C.G. Bowsher et al. : Purification and properties of nitrite reductase from roots of pea g-VALUE g-VALUE 2,1
I l l i l l l l l
I I I I I I
I L I L I I I
l l l l l
[ I I I [ I f l l l l
i l l t l t
20 mW; frequency 9.18 GHz
the enzyme is similar to that of reduced spinach N I R (Aparicio et al. 1975). The E P R spectrum of the reduced P. sativurn N I R is shown in Fig. 4 together with the spectrum of the C. pepo leaf enzyme. Following practice with the leaf enzyme, dithionite and K C N were used to reduce the protein at room temperature in the presence of K C N before freezing and examination in the E P R spectrometer. Reduction of the leaf N I R by dithionite alone is poor, the enzyme requiring attachment of a ligand such as cyanide to the sirohaem to facilitate reduction (Aparicio et al. 1975; Cammack et al. 1978). On reduction, the signals from ferric haem were eliminated and a signal appeared around g=1.94, characteristic of iron sulphur proteins. A third type of E P R signal was observed in turnover conditions. In this case the enzyme was reduced by dithionite with or without methyl viologen in the presence of an excess of nitrite at 0 ~ C. The mixture was then frozen and its spectrum examined at 20 K. Under these conditions an axial spectrum with signals at g = 2.00 and g--2.06 was obtained. The spectrum (Fig. 5) is closely similar to that generated under similar conditions from the leaf enzymes of spinach and marrow and interpreted as representing nitrosyl sirohaem. Hyper-
I I I I I I I I l i l q
[ i l t l
MAGNETIC FIELD CmT)
MAGNETIC FIELD (mT) phur clusters in nitrite reductases, reduced with 5 m M dithionite + 5 m M cyanide. Upper spectrum: C. pepo Leaf nitrite reductase. Lower spectrum : P. sativum root nitrite reductase. Conditions of measurement: temperature 21 K; microwave power
Fig. 4. Electron-paramagnetic-resonance spectra of the iron-sul-
Fig. 5. Electron-paramagnetic-resonance spectra of nitrosyl derivatives of nitrite reductases treated with 5 m M nitrite and 5 m M dithionite. Upper spectrum: C. pepo leaf nitrite reductase. Lower spectrum: P. sativum root nitrite reductase. Conditions of measurement: temperature 20 K ; microwave power 20 mW; frequency 9.18 GHz
fine structure, as a triplet, was visible about g = 2.0, representing an interaction between the iron atom of sirohaem and the nitrogen atom of its nitrosyl ligand. A similar triplet, replaced by a duplet when 1SNO 2 was substituted for t~NO2 as substrate, was also observed with C. pepo nitrite reductase similarly treated (Fry et al. 1980).
Discussion In all aspects of enzyme characterization studied here, the leaf and root nitrite-reductase enzymes were found to be closely similar. Metal-chelating reagents at neutral p H were generally without marked effect upon the activity of the pea root enzyme, a situation noted also for leaf enzymes. The exception to this was the observed sensitivity of the N I R from maize and C. ?epo leaves to bathocuproine disulphonate and bathophenanthroline. These two compounds were also inhibitory to the N I R from maize scutellum (Hucklesby et al. 1972) - another non-chlorophyllous tissue - but were actually slightly stimulatory in the current studies with pea. Inhibition by cyanide of the pea root enzyme was even more severe than of the C. pepo leaf enzyme. Ferrocyanide is an inhibitor of the
C.G. Bowsher et al. : Purification and properties of nitrite reductase from roots of pea
N I R from both green and non-green sources, the cause of this not being understood at present. Immunologically the pea root and pea leaf proteins appeared identical on the basis of the tests carried out. However, an interspecies difference was observed between these two enzymes and the C. pepo leaf NIR, the latter enzyme having probably at least one additional epitope. (The presence of a subunit of 48000 Da, visible on SDS-PAGE electrophoresis and after blotting and antibody reaction, is not seen with the C. pepo or other leaf N I R enzymes and its significance is in doubt. This band may represent a breakdown product of the 61000-Mr protein. So far, we have obtained no evidence from work with leaves or roots that the 61000-Mr N I R protein is part of a larger complex as reported by Hirasawa-Soga et al. (1982).) Two bands of molecular weight 36 500 and 39 500 additional to a band of 63000 Da were observed by Gupta et al. in pea leaf nitrite reductase. The enzyme from spinach leaf gives a single band (63 000 Da) cross-reacting partially with N I R from other plant species (Ida 1987). The absorption spectrum of the pea root N I R possessed maxima at wavelengths virtually identical to those previously recorded for C. pepo leaf enzyme (Hucklesby et al. 1976). The Soret band was placed at a substantially lower wavelength (384 nm) than that reported by Nagaoka et al. (1984) (397 rim), and corresponded very closely to the wavelengths noted for a number of leaf nitritereductase enzymes (384-390 nm). The ratio of absorbances 384/280 rim=0.25. The wavelength of the c~-band (574 rim) also corresponds closely with that reported for leaf nitrite reductases from several species. The band at 532 nm is weak, a common observation with N I R from other sources, unless derivatised e.g. with nitrite or CO. The peaks at approx. 532, 632 and 695 nm are also small, and are close in wavelength to peaks of similar kind in leaf nitrite reductase. In general the spectrum is of the type which has come to be regarded as typical of sirohaem-containing enzymes, following the work of Murphy et al. (1974), in which analytical studies were made of the haem components of E. coli sulphite reductase and spinach NIR. The resemblance in EPR spectra between the C. pepo leaf and P. sativum root N I R is apparent from Figs. 4-6. Spinach nitrite and sulphite reductases appear to be closely similar in spectroscopic properties to the iron sulphur-sirohaem subunit of E. coli sulphite reductase (Krueger and Siegel 1982) and it is likely that they share the same kind of active site. Recent X-ray crystallographic studies of E. coli sulphite reductase (McRee et al. 1986)
have demonstrated that the sirohaem iron and [4Fe-4S] cluster are very close in the protein structure and probably bridged by a cysteine sulphur. This supports the evidence from Mossbauer spectroscopy (Christner et al. 1983) that they are antiferromagnetically coupled. Because of this exchange coupling the characteristic spectrum of the reduced [4Fe-4S] cluster (S= 1/2) is only observed under conditions where the haem is low spin Fe(II), which has electron spin S = 0. Forms of the enzyme in which the haem is high-spin ferric (S = 2) produced coupled species with intermediate spin (Janick et al. 1983). The similarities in structure and properties between nitrite reductases from roots and leaves of pea reported here, for barley (Ida et al. 1974) and for bean (Nagaoka et al. 1984) raise further questions concerning the mode of function of the enzyme in these two metabolic contexts which appear to be very different in the nature of available reductant. It is possible that the ferredoxin-like carrier, found by Ninomiya and Sato (1984) and by Suzuki et al. (1985), might carry a less negative potential than that of leaf ferredoxin which is favoured by a supply of electrons at lower potential than appears to be available in the root. A further question concerns the mode of reduction of nitrite in darkness in leaves. Perhaps the root ferredoxin is also present in small quantity in the leaves. C.G.B. is grateful for the award of a Science and Engineering Research Council studentship. We thank Mr. E.F. Watson, Long Ashton Research Station, for growing plants used in this work.
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Received 2 July 1987; accepted 17 March 1988