NADH dehydrogenase activity of higher plant nitrate reductase (NADH)

Plant Science Letters, 16 (1979) 139--147

139

O Elsevier/North-HollandScientific Publishers Ltd.

NADH DEtIYDROGENASE ACTIVITY OF HIGHER PLANT NITRATE REDUCTASE (NADH)

JOHN SMARRELLI, JR., and WILBUR H. CAMPBELL Department of Chemistry, SUNY College of Environmental Science and Forestry, Syracuse, New York 13210 (U.S.A.)

(Received February 5th, 1979) (Revision received and accepted May 25th, 1979)

SUMMARY Squash nitrate reductase (NADH) was purified on blue-Sepharose and used to study the NADH dehydrogenase activity. In the presence of nitrate reductase, NADH will reduce: nitrate, ferricyanide, methylene blue, benzoquinone and menadione. Coenzyme Qi0 and plastoquinone were not reduced. Reduction of nitrate and menadione were shown to require EDTA. Yeast glutathione reductase and lipoamide dehydrogenase differed in their ability to transfer electrons to menadione. The comparison of these three enzymes and other NAD(P)H dehydrogenases with respect to electron acceptor specificity and requirement for activators or protectors has led to the suggestion of two classes of NAD(P)H flavodehydrogenases: (1) soluble; (2) membrane bound or complexed. While glutathione reductase is a class I type, lipoamide dehydrogenase and nitrate reductase share the properties of class 2 types. INTRODUCTION Higher plant NADH: nitrate oxido reductase (NR) (EC 1.6.6.1) is generally accepted to be of large molecular size (tool. wt 200 000) and to contain heine-iron, flavin adenine dinucleotide (FAD), and molybdenum [1]. These components appear to act as carriers in the transfer of electrons between the NADH oxidation site and the nitrate reduction site [ 1,2]. A nitrate reductase NADH dehydrogenase activity has mso been described, which catalyzes the reduction of ferricyanide and other acceptors [1,3]. Blue-Sepharose affinity chromatography has been successfully used to purify higher plant NRs. The Abbreviations: DCPIP, dichlorophenolindophenol;FAD, flavin adenine dinueleotide;

NR, nitrate reductase;PAGE, polyacrylamidegel electrophoresis,

140 study of the steady state kinetics led to the suggestion of a two-site pingpong mechanism for the NRs of squash and corn [2]. Since the NADH dehydrogenase activity of NR appears to be associated with the enzyme's FAD containing portion [1], other flavoprotein dehydrogenases, which can accept electrons from pyridine nucleotides may share properties with NR [4]. Approximately 30 enzymes are classed as oxidoreductases acting on NADH or NADPH (EC 1.6~.x), although not all are considered flavoproteins [5]. Many of these enzymes were isolated and purified by monitoring the reduction of artificial electron acceptors. For some of these enzymes, many years may have passed between the original isolation and the identification of the biological function and the natural acceptor [4,5]. Since many of these enzymes exist as components of larger complexes, especially bound into membranes, the same enzyme may have been isolated with different substrate specificities and molecular properties as a result of the preparation procedures [ 4,5]. Mitochondrial inner membrane NADH dehydrogenase (EC 1.6.99.3), chloroplastic ferredoxin-NADP ÷ reductase (EC 1.6.7.1), NAD(P)H dehydrogenase (quinone) (EC 1.6.99.2) and lipoamide (NADH) (EC 1.6.4.3) are representative examples of flavoenzymes the biological functions of which have only recently been established [4,5]. These enzymes have been known by many names. An exception is glutathione reductase (EC 1.6.4.2) which is known by no other name [5]. The experiments described here had their genesis in a comparison of the dehydrogenase properties of NAD(P)H dehydrogenase of rat liver and the NR of plants [1--3,6]. Both enzymes must be assayed in the presence of activators for maximal activity and can catalyze the reduction of a variety of electron acceptors [1--3,6,7]. NKD(P)H dehydrogenase is probably the vitamin K reductase of rat liver and, when in the isolated form free of microsomal membranes, can transfer electrons from NAD(P)H to vitamin K3 (menadione) [6 ]. The current studies of NR extend the list of electron acceptors to include quinones. MATERIALSAND METHODS NR was purified from the green cotyledons of 10
141 Pharmacia GE-4 apparatus with the method of Davis [9], but omitting sample and stacking gels. Before polymerization, the acrylamide solution was adjusted to either pH 6.7 or 8.9. The electrophoresis were performed with the running buffer as Tris-glycine (pH 8.3) [ 9]. The electrophoresed gels were stained for protein with Coomassie blue and for enzymatic activit~: as described by Lebowitz and Campbell (unpublished results). Separate gels were frozen, sliced into 4 mm sections, extracted with 100 mM potassium phosphate (pH 7.5) with 1 mM EDTA-10% glycerol and assayed for NR and menadione reductase. Glutathione reductase (Type III) from baker's yeast, lipoamide dehydrogenase (Type IV) from Torula, malate dehydrogenase from pig heart and all biochemicals were obtained from Sigma Chemical Co. AnalaR potassium dihydrogen phosphate, EDTA, potassium nitrate and potassium hydroxide were purchased from BDH Chemicals Ltd. Acrylamide, BIS and TEMED were from Bio-Rad Labs. The quinones were a kind gift of Professor A.T. Jagendorf, Cornell University. RESULTS Since many NADH dehydrogenase activities are demonstrable in plant leaf crude extracts [ 10], only those activities which co-purify with NR will be described. Affinity chromatography using blue-Sepharose was used as the initial step for purification of NR [2]. In Fig. 1, the co~lution of NR and menadione reductase from blue-Sepharose by NADH is shown. The copurification of NR and several dehydrogenase activities is summarized in Table I. Only 10--30% of the NADH dehydrogenase activities found in the crude extract are associated with the NR blue-Sepharose fraction. A higher percent of menadione reductase activity is recovered. Since PAGE revealed that this NR fraction was a mixture of several proteins, further purification of NR seemed essential to establish that NR could transfer electrons to menadione. Malate dehydrogenase has been established to be a contaminant of blue-Sepharose purified NR both by enzymatic activity and gel stains for dehydrogenase activity (Lebowitz and Campbell, unpublished results). Malate dehydrogenase obtained from Sigma had no activity for reduction of menadione with 200 ~M NADH at pH 7.5. The blue-Sepharose NR fraction contained no NADH or NADPH glutathione reductase nor NADH lipoamide dehydrogenase activities. PAGE gels were stained for protein and NR activity, revealed that NR was well separated from other proteins at pH 8.9 but, at pH 6.7, NR electrophoresed at the front even when bromophenol blue was omitted (Fig. 2). When gels were sliced into sections, NR activity was recovered in the region of the protein band which stained for NR activity. This region also re. presented the only peak of menadione reductase activity in the gel extracts (Fig. 2). NR is concluded to be the menadione reductase catalyst present in the NADH fraction from the blue-Sepharose column. In addition, NR can

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Fig. 1 (/eft). Elution of nitrate reduetase and menadione reduetase activities from blueSepharose using 0.1 mM NADH (2). (e---e) NADH nitrate reductase (43% recovered). (o---o) NADH menadione reduetase (46% recovered). Fig. 2 (fight). Nitrate reduetase, menadione reduetase and protein staining after gel electrophoresis. Protein (20 0zg)of a blue-Sepharosepurified NR fraction was applied to each gel. (Spec. act., 2.9 units/ms for NR and 19.4 units/ms for memmdionereductue). Enzyme activitieswere determined after the gelswereelectrophoresed,sliced and extracted (see text). Gel representations show the position of Coomamie blue4tained protein bands. Arrow indicates the position of the bromophenol blue tracker dye. NR acti~tie~ are shown as 10 times the actual value. (,----e) NADH nitrat~ reductase (16% recovered at pH 6.7 and 16% at pH 8.9). (o---o) NADH menadione reductase (54% recovered at pH 6.7 and 12% at pH 8.9).

be purified to a single PAGE protein staining band by these procedures. With this two step method, NR can be purified to apparent homogeneity in 6--8 h. The NADH dehydrogenase activity of the blue-Sepharose purified NR was found to have a pH optimum of 7 with menadione as acceptor. The apparent Kmswere: 7 pM NADH and 275 pM menadione. NADPH will not reduce menadione, although a slow NADPH ferricyanide reduction was catalyzed by squash and spinach Nits [ 11]. p-Hydroxymercufibenzoate (50 ~M) inhibits both nitrate and ~lenadione reduction, as expected [1,2]. Sodium azide at I mM gave 90% inhibition of NR while stimulating menadione reduction by 50%. Since the nitzate reduction site is physically separated from the NADH oxidation site [ 2], the azide results suggest nitrate and menadione are reduced at different sites on NR. Dicoumarol, a vitamin K antagonist, is a competitive inhibitor toward NADH witha K i of 13 ~M when NR is assayed. The rat liver NAD(P)H dehydrogenase has a K i of 0.005 pM dicoumarol indicating that NR's vitamin K reductive capacity is not like that of the suspec~:; rat liver vitamin K reductase but more like the other rat liver dia-

143 TABLE I SUMMARY OF T H E P U R I F I C A T I O N OF N I T R A T E R E D U C T A S E AND NADH: DEHYDROGENASE ACTIVITIES

Activity is expressed as ~mollmin and spec. act. as ~mol/minlmg of protein. Concentrations for the acceptors are as described (in refs. 2,3 and 7). Purification procedure

Electron acceptors Nitrate

Ferricyanide

Cytochrome ¢

DCPIP

units

units

units mg

units units mg

0.14

48

0.18

36

0.13

0.1

24

0.13

17

0.1

8

16

units

units mg

units

units mg

Centrifuged crude extract

5.4

0.02

350

1.3

38

Not bound to blue-Sepha-

0.4

0.002

250

1.4

9

3.8

3.2

units mg

Menadione

,,

rose

NADH elution

38

31

13

11

9

14

of blueSepharose

phorases [12]. When NR activity was assayed by observing the appearance of nitrite, the NR activity was inhibited 30% by the addition of 500/~M menadione. The control here contained an equal concentration of ethanol which may rapidly alter NR act'm'ties [13]. The acceptor specificity of the quinone reductase activity of NR is shown in Table II. Although both benzo- and naphthoquinones are reduced, the biologically-impo~t CoQt0 and plastoquinone were not. NAD(P)H and NADH dehydrogenases which have quinones as natural acceptors have similar properties to NR when solubilized [4,5,12]. Thus, in the green cell, NR might reduce the biological quinones despite the absence of this ability in vitro. The dependence of the NR and menadione reductase activities on the presence of EDTA was tested by gel filtering away the EDTA [7]. While this treatment may not remove all the EDTA, it does render NR totally dependent on added EDTA (Fig. 3). Menadione reductase activity is stimulated approx. 10-fold by 50 ~M EDTA. The r~,duction of ferricytocl~ome e is also EDTA dependent while ferricyanide reduction is less so [7]. The; NR reaction is stimulated both for NADH oxidation and nitrite production by a wide variety of agents including the 20 amino acids, oxidized and reduced glutatione, serum albumin and chelators such as o-phenanthroline ~7]. Several other flavoprotein dehydrogenases have similar properties [ 7,13,15]. The

144 TABLE H ELECTRON ACCEPTORS FOR NITRATE REDUCTASE ~ae quinone acceptors w~.~:edissolved in absolute ethanol and 0.1 ml of each was added to a 1.9 ml reaction mixt,~e. Change in A3~0 was memmred except for ¢ytoehrome c (Ass0; emM = 21) and DCPIP (A,00; emM = 21). The nitrate concentration was chosen to match the other aeceptors ~mdwas, therefore, suboptimal [2 ]. Acceptor

Assay concentration (~M)

NADH dehydrogenase (units/mg)

KNOs (no ethanol) KNO3 menadione potassium ferricyanide ferricytochrome c (horse heart) DCPIP methylene blue benzoquinone 2,3 dichloro 1,4 naphthoquinone CoQj0 plastoquinone chloranil sodium anthroquinone ~sulfonate dehydroaseorbate

200 200 200 200 40 200 200 200 200 100 100 200 200 200

0.6 0.5 2.6

9.6 2.2 3.1 4.9 4.9 1.6 0.0 0.0 0.0 0.0 0.0

significance of these activators or protectors is both poorly understood and controversial [ 6,7,12,14,,, 5 ]. For most flavoprotein dehydrogenases solubilization can lead to variation in enzymatic and physical properties with a few notable exceptions [4]. Nitrate reductase, glutathione r e d u ~ , lipoamide dehydrogenase and thioredoxin reductase can be easily solubflized [1,2,12,16]. Since no literature references could be found for glutathione reductase NADPH dehydrogenase activity, Sigma yeast glutathione reductase was tested for menadione reductase activity and found to be virtually inactive. Furthermore, treatment of the enzyme by gel filtration, in the same way as NR (see Fig. 3) led to little change in glutathione reductase activity. Although 80% of the glutathione reductase was recovered from the G-25 column without EDTA in the assay, the addition of EDTA lengthened the linear phase of the assay and enhanced recovery by 10%. On the o t h e r hand, lipoamide dehydrogenase is known to reduce a variety of electron acceptors a n d to require EDTA for demonstration of the native activity [ 14,17]. Sigma yeast lipoamide dehy"drogenase can reduce menadione and has almost th e identical response to EDTA removal as is shown for NR in Fig. 3.

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Fig. 8. Nitrate reductase inactivation by gel filtration. NR, blue-Sepharose purified, had specific activities of 6 units/mg for NR and 13 units/mg for menadione reductase. The protein mixture was applied to a Sephadex G-25 column and eluted with 0.1 M potassium phosphate (pH 7.5). Enzymes were assayed by observing the change in A340 with and without 50 ~M EDTA added to the assays. EDTA had no effect on activities of the applied sample, which was in 1 mM EDTA. ( o - - - ~ ) NR ( - E D T A ) -- 0% recovered. (e-----~) NR (+ EDTA) -- 62% recovered. (o----c) Menadione reductase ( - E D T A ) -- 6% recovered. ( , - - - i ) Menadione reductase (+ EDTA) -- 56% recovered.

DISCUSSION

The ability of nitrate reductase to catalyze the reduction of quinones, ferricyanide and mammalian cytochrome c appears to be a function of the flavin dehydrogenase component of the enzyme. This dehydrogenase activity of nitrate reductase seems to be a non.physiological activity similar in character to that observed for other flavoprotein oxidoreductases [ 4,6]. However, the NADH dehydrogenase activities of flavoprotein oxidoreductases may provide clues about the environment of the flavin. Glutathione reductase and lipoamide dehydrogenase appear to represent extremes of flavin environment. Lipoamide dehydrogenase has menadione reductase activity and probably has a flavin environment which allows solutes access to the site of reduction. Glutathione reductase does not measurably reduce menadione and probably has a flavin environment quite different from that of lipoamide dehydrogenase, with respect to solution access. Since these enzymes have been found to function in a catalytically-similar manner in accepting and transferring electrons [18], we suggest that the functional location of these enzymes dictates the accessibility of the flavin to solutes. Glutathione reductase, being a soluble enzyme, must protect its electrons from less desirable solutes. This enzyme confers specificity to the flavin electron transfer sites which excludes many viable electron acceptors that may be present in the cytosol or organellar

146

solution. Glutathione reductase has a narrow substrate specificity for disultides [18]. Conversely, lipoamide dehydrogenase is bound in the pyruvate dehydrogenase complex, or in another similar complex, and has little need for acceptor specificity as the electrons are transferred to the flavin internally via bound lipoamide [18]. When lipoamide dehydrogenase is solubilized from the complex, EDTA must be provided to protect the enzyme from inactivation by copper [16--18]. Lipoamide dehydrogenase requires EDTA for activity while glutathione reductase has virtually no requirement for EDTA. This difference can also be viewed from the standpoint of flavin and solute interactions. Simply stated, glutathione reductase requires little protection from the solution's heavy metals because their access to the enzyme's active sites is Hmited. In fact, the glutathione binding site has been suggested to be between the two subunits of glutathione reductase [19]. Nitrate reductase is generally viewed as a soluble protein of the green cell [1], but there are dissenters from this point of view [20]. Experience has shown that NR is an enzyme easily solubilized during cell disruption and NR behaves as a soluble protein [1,2]. However, we have found that NR is EDTA~lependent and others have also found activation of NR important for maximum activity [7]. In the studies presented here, NR and lipoamide dehydrogenase have both responded reversibly to EDTA removal. We find that when the flavoproteins are reduced by NADH with no electron acceptor available, they are inhibited irreversibly in the absence of EDTA [7]. Although this inhibition of reduced NR probably results from trace metal ion contaminants, Chelex pretreatment of the assay solutions did not prevent inhibition [ 7 ]. The mechanism of EDTA activation remains to be determined and is under further investigation. NR appears to have a solute accessible flavin as shown by EDTA activation and the long established NADH dehydrogenase activities of the NR, which now include quinones as acceptors [1--3,7,13 and this papcr]. Does the solute accessibility of NR's flavin reflect a second physiologically useful site for Nit in addition to the nitrate reduction site? Or is NR bound in a complex in the cell which is disrupted upon lysis? Comparison of glutathione reductase, lipoamide dehydrogenase, and NR, using as a basis the EDTh~ effects and the NADH dehydrogenase acceptor specificity, suggests that NF. may be part of a larger complex. The important difference between the physiological acceptors of oxidoreductases acting on NADH or NADPH with a disulfide as acceptor (glutathione reductase and iipoamide dehydrogenase) and with a nitrogenous compound as acceptor (NR), should be noted. The comparison described here is based on the artificial acceptors for these enzymes and not limited to the natural acceptors. Nit has more in common with lipoamide dehydrogenase than with glutathione reductase in regard to flavin accessibility. Therefore, NR seems more likely to be complexed in some form in the cell rather than as a soluble protein.

147 ACKNOWLEDGEMENTS We t h a n k Drs. J.K. Bryan and W.H. O r m e ~ o h n s o n for helpful discussions concerning this research. The support of the National Science F o u n d a t i o n (PCM 7 6 1 8 8 0 3 ) is gratefully acknowledged. REFERENCES

1 2 3 4 5

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