Reversible inactivation of wheat leaf nitrate reductase by NADH, involving superoxide ions generated by the oxidation of thiols and FAD

Biochimica et Biophysica Acta 827 (1985) 215-220 Elsevier

215

BBA32107

R e v e r s i b l e inactivation of w h e a t leaf n i t r a t e r e d u c t a s e b y N A D H , involving s u p e r o x i d e i o n s generated by the o x i d a t i o n o f thiols and F A D Arun Prakash Aryan and William Wallace Agricultural Biochemistry Department, Waite Agricultural Research Institute, University of Adelaide, Glen Osmon~ SA 5064 (Australia)

(Received June 29th, 1984)

Key words: Nitrate reductase inactivation; FAD; Superoxide anion; Sulfhydryl group; (Triticum aesticum)

The conversion of wheat leaf NADH-nitrate reductase (NADH:nitrate oxidoreductase, EC 1.6.6.1) to a reduced inactive form, by preincubation with NADH (in the absence of nitrate), occurred in the presence of either dithiothreitol and/or FAD but not with cysteine. This inactivation of nitrate reductase, unlike that by cyanide, was dependent on aerobic conditions and was prevented by EDTA and superoxide dismutase. Superoxide ions were produced by the auto-oxidation of dithiothreitoi but not cysteine, at pH 7.5. Thus, superoxide ions can mediate the inactivation of NADH-reduced nitrate reductase in higher plants. Pretreatment of nitrate reductase with NADH alone (over-reduction) did not inactivate the enzyme. A nudeophilic agent, i.e., cyanide or superoxide is necessary to inhibit electron transfer by the enzyme to nitrate. Nitrite or azide were not effective. Data in the literature which suggest that NADH stabilizes nitrate reductase, rather than resulting in its inactivation, can now be explained. In these cases, cysteine was used as thiol and FAD was not added in the extraction or incubation media so that no superoxide ion was produced.

Introduction Nitrate reductase (NADH:nitrate oxidoreductase, EC 1.6.6.1) from microorganisms and higher plants can be reversibly inactivated by pretreatmerit with NADH, in the absence of nitrate [1]. This inactivation is greatly enhanced by cyanide ions, which bind at the molybdenum site of NADH-reduced enzyme [1,2]. However, NADHinactivation of nitrate reductase occurs in the absence of added cyanide, even with the relatively purified enzyme. In this case, it is proposed that inactivation results from an over-reduction of the enzyme [3,4]. It has also been suggested that superoxide ions are involved in the NADH-mediated inactivation of nitrate reductase from Chlorella fusca [3] and Ankistrodesmus braunii [5]. Auto-oxidation of some thiols [6] and flavins, especially in the presence of N A D H [8], can pro0167-4838/85/$03.30 © 1985 Elsevier Science Publishers B.V.

duce superoxide and other free radicals. Since thiols and FAD are usually included in the extraction and incubation media for nitrate reductase, we have investigated their effects on NADH-pre~ treatment of wheat leaf nitrate reductase. We have also examined the reports that under certain circumstances NADH-pretreatment of nitrate reductase does not inactivate the enzyme but stabilizes it in vitro [9-11]. Experimental procedure Plant materials. Wheat seeds ( Triticum aestivum L cv Bindawarra) were grown as described previously [12] and leaf samples harvested at 12-13 days after sowing. Extraction of nitrate reductase (method A). Leaf tissues were ground in an ice-chilled mortar and pestle with 100 mM .potassium phosphate (pH 7.5)

216 (4 m l . g -1 tissue) containing casein (1%, w/v), insoluble poly(vinyl pyrrolidone) (2.5%, w/v) and thiol or FAD as specified. The crude extract was centrifuged at 15 000 × g for 15 rain and the supernatant desalted on a Sephadex G-25 column (preequilibrated with 50 mM potassium phosphate (pH 7.5)) to remove nitrate [12]. Whenever thiol or FAD was used in the extraction medium, the column buffer also contained thiol (100 #M) and/or FAD (10 #M). In some cases, the fraction precipitated by saturation with 40% ammonium sulphate was prepared as described previously [12] and a desalted sample used for NADH preincubation studies. Extraction and purification of nitrate reductase (method B). The Amicon blue-A affinity chromatography technique of Somerset al. [13] was followed, except that the extraction buffer was 0.25 M Tris-phosphate (pH 8.5) (0.2 M TrisHCI/0.05 M potassium phosphate)/insoluble poly(vinyl pyrrolidone) (1% w/v)/phenylmethylsulphonylflouride (250 #M)/antipain (10 # M ) / leupeptin (10 #M)/pepstatin (5 #g.ml-1). The fractions eluted by N A D H from the blue-A column were concentrated on precipitation by 60% saturation with ammonium sulphate and passed through a Sephacryl-300 column (50 x 3 cm, preequilibrated with 50 mM Tris-phosphate (pH 8.5)/10 #M FAD/100 #M dithiothreitol/10 #M EDTA) to further remove the contaminating proteins. Nitrate reductase samples were desalted on a Sephadex G-25 column (pre-equilibrated with 50 mM potassium phosphate (pH 7.5)/100 #M dithiothreitol/10 #M FAD) and then used for NADH pretreatment. All enzyme isolation steps were undertaken at 0-4°C. Preincubation, reactivation and assay of nitrate reductase. Nitrate reductase samples were preincubated for 30 rain at 25°C with treatments as described in each experiment. The aliquots were then incubated with the assay mixture (described previously, Ref. 12) for 30 rain at 25°C to determine the enzyme activity. To reactivate nitrate reductase,, the sample aliquots were first treated with ferricyanide (300 #M) for 5 rain prior to enzyme assay. Detection of superoxide ions. The production of superoxide ions from various thiols was detected by the Nitro-blue tetrazoliurn reduction method

[6], except that the buffer was potassium phosphate (100 mM, pH 7.5), and EDTA was omitted from assay. The increase in absorbance was measured in 1-cm cuvettes in a Perkin-Elmer ~,-5 spectrophotometer. Protein was determined by the method of Lowry et al. [14] using bovine serum albumin (Sigma A 4378) as standard. Results

Inactivation of nitrate reductase by NADH, in crude extracts and in purified fractions Nitrate reductase from wheat leaves, when isolated in the presence of FAD, dithiothreitol and proteinase inhibitors, was relatively stable to preincubation at 25°C for 30 min (Table I, control). However, when N A D H (100 #M) was included there was 60% loss in enzyme activity of crude extracts as well as purified preparations (Table I). In all cases, the inactivation was enhanced by cyanide ions (1 #M). If the inactivation of nitrate reductase by NADH was mediated by endogenous cyanide, then it follows that either this nucleophile was present in the enzyme sample or else generated during the preincubation period. The latter mechanism would appear to be unlikely for the purified enzyme, since cyanide-generating systems [15-17] should be removed from this preparation. Alternatively, the partial inactivation of nitrate reductase by N A D H could either involve a nucleophile other than cyanide or be independent of this type of reaction. Effects of thiols and~or FAD on the N A D H inactivation of nitrate reductase The data in Table II indicate that the extent of nitrate reductase inactivation by N A D H was affected by the type of thiol compound added (10 #M FAD included in all samples). Inactivation of nitrate reductase was substantially enhanced by dithiothreitol and mercaptoethanol but negligible inactivation occurred with cysteine. With glutathione, the percentage loss of activity was also less than that with no thiol. A crude extract of nitrate reductase prepared and incubated without thiol and FAD was very labile (Table III), but NADH instead of enhancing nitrate reductase inactivation increased the stabil-

217 TABLE I EFFECTS OF PREINCUBATION OF WHEAT LEAF NITRATE REDUCTASE WITH NADH AND KCN AT VARIOUS STAGES OF ENZYME PURIFICATION Nitrate reductase was isolated from 12-day wheat leaves with FAD (10/~M), dithiothreitol (1 raM) and proteinase inhibitors in the extraction medium (method B.). After desalting, the enzyme sample at various stages of purification was preincubated with NADH (100/tM) or 50 mM potassium phosphate Ph 7.5 (control) and KCN (I/~M) as indicated. Enzyme sample

Nitrate reductase activity initial ~

Crude extract Ammonium sulphate fraction (20-50%) Purified enzyme blue A affinity column

after 30 rain preincubation at 25°C (%) control

KCN

NADH

322

98

98

40

8

140

94

93

33

4

90

87

40

10

81 b

NADH + KCN

" nmol NO~-/min per g fresh wt. b spec. act. = 6.4/~mol NOr/rain per mg protein.

ity of the enzyme. A d d i t i o n s of F A D a n d / o r dithiothreitol e n h a n c e d b o t h initial enzyme activity a n d its stability at 25°C, b u t u n d e r these c o n d i t i o n s p r e i n c u b a t i o n with NADH resulted i n e n h a n c e d i n a c t i v a t i o n of the enzyme. W i t h cysteine, however, N A D H provided a n almost complete protection of the enzyme, while this thiol with F A D prevented a n y effect of N A D H o n n i t r a t e reductase stability (Table III).

Production of superoxide ions by auto-oxidation of thiols A u t o - o x i d a t i o n of flavins [7,8] a n d thiols, e.g., dithiothreitol, m e r c a p t o e t h a n o l a n d G S H have b e e n reported to generate superoxide ions a n d other free radicals in the presence of metal ions a n d oxygen [6]. W e f o u n d that dithiothreitol b u t n o t cysteine or G S H generated superoxide ions o n i n c u b a t i o n at p H 7.5 (Fig. 1). T h e rate of Nitroblue tetrazolium r e d u c t i o n b y thiol was m a r k e d l y i n h i b i t e d b y superoxide dismutase. A t higher p H (at least 9.0), cysteine a n d G S H also generated

TABLE II EFFECTS OF THIOLS ON NADH PRETREATMENT OF WHEAT LEAF NITRATE REDUCTASE IN A CRUDE EXTRACT Nitrate reductase was isolated from 12-day wheat leaves with 100 mM potassium phosphate (pH 7.5) containing FAD (10/~M), casein (1%, w/v) and the thiols (1 mM) as specified(method A). The crude extract after desalting into 50 mM potassium phosphate (pH 7.5) and 0.1 mM of respective thiol was preincubated with 50 mM potassium phosphate (pH 7.5) (control) or NADH (200 #M) as indicated. Thiols in extraction medium

Nitrate reductase activity a

None Dithiothreitol Mercaptoethanol GSH Cysteine

295 373 380 402 410

initial

after 30 rain preincubation at 25°C control

+NADH

loss

231 333 350 395 395

75 37 32 180 384

156 (68) b 296 (89) 318 (91) 215 (54) 11 (3)

" nmol NOr/rain per g fresh wt. b Values in brackets are the precentage loss due to NADH.

218

T A B L E III EFFECTS O F T H I O L C O M P O U N D S A N D F A D O N N A D H P R E T R E A T M E N T O F N I T R A T E R E D U C T A S E Nitrate reductase was extracted as described in Table II except that F A D was omitted. During preincubation of the desalted sample, F A D (10 # M ) and N A D H (300 # M ) were added as indicated. Thiol in extraction medium

FAD (10 ~,M)

Nitrate reduetase activity initial a

after 30 in at 25°C (%) -NADH

+NADH

None None Dithiothreitol Dithiothreitol Cysteine Cysteine

+ + +

53 89 207 246 205 279

31 63 47 77 56 69

88 40 38 38 92 68

a nmol N O ~ - / m i n per g fresh wt.

superoxide ions and the rate of superoxide ion production by dithiothreitol was substantially enhanced as reported earlier [6]. Evidence for superoxide ion mediated NADH inactivation of nitrate reductase Anaerobic conditions during N A D H preincubation of nitrate reductase markedly restricted the NADH inactivation observed with FAD plus dithiothreitol (Table IV), whereas NADH plus cyanide mediated inactivation was not affected. In each case, the inactivation of the enzyme by

NADH was reversed by ferricyanide (Table IV). The inactivation of nitrate reductase by NADH in the presence of FAD or dithiothreitol was also prevented by adding superoxide dismutase (0.5 rag-m1-1) and EDTA (Table V) thus indicating the involvement of superoxide ions. Catalase (2000 units) and bovine serum albumin (0.5 mg. m1-1) were ineffective in overcoming this NADH inactivation of nitrate reductase (data not shown). Nitrite (10 #M) and azide (10 #M), also nucleophilic agents, did not mediate the inactivation of NADH-reduced nitrate reductase.

T A B L E IV EFFECTS O F A N A E R O B I C C O N D I T I O N S O N N A D H - I N A C T I V A T I O N OF N I T R A T E R E D U C T A S E Nitrate reductase was isolated as described in Table II except that F A D and thiols were omitted from the extraction buffer. The enzyme samples were preincubated with F A D (10 #M), dithiothreitol (200 #M), K C N (1 # M ) and N A D H (100 # M ) as indicated. Simultaneously, another set of tubes was evacuated (twice, 3 m m H g ) followed by sparging with oxygen-free nitrogen to produce anaerobic conditions. Additions

None F A D + dithiothreitol F A D + dithiothreitol KCN KCN

Anaerobiosis

+ +

Nitrate reductase activity initial a

after 30 min preincubation at 25°C (~) -NADH

+NADH

176 210 210 170 170

43 88 88 44 45

42 19(79) b 67 5(70) 5(69)

a nmoi N O 2 / m i n per g fresh wt. b Values in brackets are activities after reactivation with ferricyanide.

219 TABLE V

,-~ E

PREVENTION OF NADH-INACTIVATION OF NITRATE REDUCTASE BY SUPEROXIDE DISMUTASEAND EDTA Nitrate reductase was isolated as described in Table IV. An ammonium sulphate fraction (0-40%) was desalted and preincuhation with FAD (10 itM), dithiothreitol (200 jaM), EDTA (10 raM), KCN (1/tM), superoxidedismutase (SOD, 760 units, 0.25 mg protein) and NADH (100/tM) as indicated.

O.E

0

0.5

~

(3.4

Additions

Nitrate reductase activity initial a after 30 rain preincubation at 25°C (%)

None FAD FAD + SOD FAD + EDTA Dithiothreitol Dithiothreitol + SOD Dithiothreitol + EDTA KCN KCN + EDTA

27 48 39 39 31 27 31 27 27

g ..~

0.3

(3.2

0.1

0.0 t

0

5

t

I =0

t5

i

2=0

2=5

~0

Time ( rain )

Fig. 1. Generation of superoxide ions by auto-oxidation of thiols. The incubation mixture (1.5 ml) contained 0.5 ml of various thiols (1 mM), 0.5 ml potassium phosphate (100 mM, pH 7.5), 0.3 ml Nitro-blue tetrazolium (125 ~tM) and 0.2 ml superoxide dismutase (200 units) or H20. Incubation at 25°C and increase in absorbance recorded. Dithiothreitol (z~), cysteine (O), GSH (n) and dithiod'treitol+ superoxide dismutase (-)

Discussion

The different effects of various thiols on N A D H inactivation of nitrate reductase (Table II), can be related to their rate of superoxide ion generation (Fig. 1). Further, the prevention of enzyme inactivation by anaerobiosis (Table IV) and superoxide dismutase (Table V) indicate that superoxide ions are involved in N A D H inactivation of the enzyme by dithiothreitol a n d / o r FAD. Thus, inclusion of dithiothreitol and F A D throughout the enzyme purification procedure explains why N A D H inactivation of the enzyme was observed at all stages of purification (Table I). The ineffectiveness of catalase against this inactivation indicates that H202 which could also be generated during auto-oxidation of thiols [6] was not involved. The inhibitory effect of E D T A against N A D H

-NADH

+NADH

48 81 92 95 55 63 55 41 57

52 67 90 92 3 41 48 4 6

a nmol NO2/min per g fresh wt.

inactivation in the presence of dithiothreitol and F A D is probably associated with the chelation of metal ions required for the auto-oxidation of thiols and F A D [6,8]. It could also act as a scavenger for oxidising radicals produced in the reaction mixture [6,18]. This protective effect of E D T A on nitrate reductase however is distinct from its protection against the metal ion dependent inactivation of squash leaf nitrate reductase [19]. In this case, the dehydrogenase component of the enzyme was inactivated by heavy metal ions while the terminal nitrate reducing moiety was unaffected. This inactivation was also irreversible [19]. Our data indicate that as in Ankistrodesmus braunii [5], both cyanide and superoxide ions mediate the inactivation of wheat leaf nitrate reductase in the presence of N A D H . Since N A D H alone did not inactivate nitrate reductase from wheat leaves, we consider that inactivation of nitrate reductase is not simply due to over-reduction of the enzyme by N A D H as proposed by others [3,4]. In order to produce the inactive complex, some nucleophile, e.g., cyanide or superoxide ion must be present to bind at the molybdenum site of the N A D H - r e d u c e d enzyme. Other ions

220 tested, i.e., nitrite and azide, did not replace cyanide or superoxide ions for the inactivation by NADH. The results (Tables III, V) which show that addition of F A D resulted in N A D H inactivation of nitrate reductase, via superoxide ion generation, explains earlier reports that exogenous F A D (100 /~M) increased the sensitivity of the enzyme towards N A D H inactivation in Chlorella fusca [3] and A. braunii [5]. Absence of N A D H inactivation without F A D or dithiothreitol (Tables III, V) implies that the native F A D in the wheat enzyme was not involved in the production of superoxide ions. The generation of superoxide ions in plant tissues is well documented [20], as is the role of superoxide dismutase in protecting cell components from these radicals [21]. Thus, it is uncertain whether superoxide ions are involved in an in vivo inactivation of nitrate reductase by N A D H . However, for in vitro studies on the enzyme, where thiols and F A D are commonly employed, its stability in the reduced state will be greatly influenced by the thiol used. A higher rate of 2-mercaptoethanol oxidation has also been shown with Tris buffer [22]. We can now explain the conflicting data in the literature for the effect of preincubation of nitrate reductase with N A D H ; inactivation of the enzyme [1,3,12,19] or its stabilization [9-11]. In the latter studies, the incubation media (pH 7.5) did not contain FAD, and cysteine was u s e d as thiol. Thus, inhibitory superoxide ion would not have been produced and as shown in Table III, under these conditions N A D H would increase the stability of nitrate reductase.

Acknowledgements We thank Professor D.J.D. Nicholas for helpful discussion during the course of the work and his guidance during the preparation of the manuscript. We also thank R.G. Batt for technical assistance and A.P.A. acknowledges the receipt of a postgraduate research scholarship from the University of Adelaide.

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