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Role of the diaphorase moiety on the reversible inactivation of the Chlamydomonas reinhardii nitrate reductase complex
8
Biochimica et Biophvsica Acta 827 (1985) 8 13 Elsevier
BBA32084
Role of the diaphorase moiety on the reversible inactivation of the Chlamydomonas reinhardii nitrate reductase complex Francisco C6rdoba, Jacobo Cfirdenas * and Emilio Fernandez Departamento de Bioqu'trniea, Facultad de Ciencias, Unit,ersidad de Cbrdoba, Cbrdoba (Spain) (Received June 25th, 1984)
Key words: Nitrate reductase; Redox inactivation; (C. reinhardii).
Nitrate reductase (NAD(P)H: nitrate oxidoreductase, EC 1.6.6.2) of Chlamydomonas reinhardii mutant 305, lacking NAD(P)H-cytochrome c reductase activity, became rapidly and reversibly inactivated in vitro upon incubation with dithionite and reactivated by oxidation with ferricyanide. Nitrate protected against the inactivation. Unlike the native nitrate reductase complex of its parental wild strain, the enzyme of 305 was not inactivated by reduced pyridine nucleotides. In contrast to that of wild-type cells, the in vivo reversible inactivation of mutant 305 nitrate reductase was observed only after complete elimination of nitrate from the media and subsequent transfer to ammonium medium or darkness conditions. The inactive enzyme was reactivated by addition of nitrate to the media without previous removal of ammonium, which indicates that, unlike in the wild-type cells, ammonium does not prevent nitrate uptake by 305 cells. This different regulation pattern is due to the structural modification of 305 nitrate reductase. We conclude that in vitro an active diaphorase moiety is required for the inactivation by reduced pyridine nucleotides of the nitrate reductase of C. reinhardii, and that in vivo the absence of nitrate rather than the presence of ammonium is the triggering event for nitrate reductase inactivation, which can be also achieved by reductants other than NAD(P)H.
Introduction
NAD(P)H-nitrate reductase of eukaryotes (NAD(P)H: nitrate oxidoreductase EC 1.6.6.2) consists of two partial activities, namely NAD(P)H-cytochrome c reductase or diaphorase and reduced benzyl viologen-nitrate reductase, which can be distinguished by chemical and physical treatments and act sequentially in the reduction of nitrate to nitrite [1-3]. In green algae and higher plants, nitrate reductase is regulated in vivo either by redox interconversion [1,2] or by reversible inactivation under reducing conditions with cyanide [4-6] or with superoxide radicals [7,8] this inactivation being * To whom correspondence should be addressed. 0167-4838/85/$03.30 © 1985 Elsevier Science Publishers B.V.
centered on the terminal nitrate reductase moiety of the enzyme complex [8-10]. In some instances ammonium or darkness promotes the in vivo inactivation of nitrate reductases [6,9,11], which seems to depend on the availability of intracellular nitrate controlled by the uptake system [12]. Inactive enzyme can be reactivated in vitro by oxidation with nitrate or ferricyanide [5,9,13]. Requirement of diaphorase activity for the reversible inactivation process remains controversial in spite of many published data in this respect [9,10,14,15]. Chlamydomonas reinhardii mutants deficient in nitrate reductase have been isolated [16] and used to study in vivo the regulation of the activities of the nitrate reductase complex [17]. In the present work we compare the in vitro and in vivo reversible redox inactivations of nitrate reductases of
wild-type and mutant 305 which lacks NAD(P)Hnitrate reductase and NAD(P)H-diaphorase [18]. The results indicate that in vitro an active diaphorase is required for terminal nitrate reductase inactivation by NAD(P)H, whereas in vivo inactivation by ammonia occurs in the absence of nitrate and is a redox process which can be performed by reductants other than NAD(P)H. In addition, reactivation in vivo of inactive terminal nitrate reductase is achieved by reoxidation following the entrance of nitrate within the cells. Experimental Cells of C. reinhardii, wild-type 6145c and mutant 305 [16], were grown with 8 mM NH4CI and derepressed with 4 mM KNO 3 for 5.5 h under reported conditions [17,19]. Cells were disrupted by freezing in liquid nitrogen and thawing in a 50 mM Tris-HC1 buffer (pH 7.5)/0.1 mM EDTA/0.1 mM dithioerythritol (4 m l / g wet weight) as previously described [17]. The suspension was centrifuged at 27000 × g, 15 min, and the supernatant was used as the source of enzyme. NAD(P)H-nitrate reductase and reduced benzyl viologen-nitrate reductase activities were determined in vitro by measuring the nitrite formed in the enzymatic reduction of nitrate [20,21]. Reduced benzyl viologen-nitrate reductase activity was assayed in situ by using 1 ml of cell suspensions previously treated with 20 ~1 of commercial toluene [12]. Reactivation of nitrate reductase was achieved by preincubating extracts or toluenized cell suspensions with 0.3 ~mol ferricyanide for 1 min and doubling the concentration of reductant in the standard assay mixture [22]. Nitrite was determined colorimetrically by the diazotization method of Snell and Snell [23]. Protein was estimated colorimetrically by the method of Bradford [24], using bovine serum albumin as standard, and chlorophyll by its absorbance at 652 nm according to Arnon [25]. The nitrate uptake was measured by determining the disappearance of nitrate from the medium according to Cawse [26]. Results In vitro inactivation of nitrate reductase NADH, N A D P H or sodium dithionite in-
activated terminal nitrate reductase of C. reinhardii wild cells up to 30% of its original activity after 60 min incubation (Fig. 1A). NAD(P)Hnitrate reductase activity responded similarly to the same treatments (results not shown). By contrast, nitrate reductase of mutant 305 was inactivated to the same extent only by sodium dithionite (Fig. 1B). NAD(P)H concentrations up to 3 mM did not inactivate terminal nitrate reductase nor reduced enzymatically nitrate in 305 even after a 2 h assay at 30°C. Added flavins or viologens increased neither the rate nor the extent of inactivation exerted by dithionite.
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Fig. 1. In vitro inactivation course of wild-type (A) and mutant 305 (B) nitrate reductases from C. reinhardii. Extracts from wild-type and mutant 305 were incubated at 4°C with 0.3 m M N A D H (D), 0.3 m M N A D P H (m), 4.6 m M Na2S204 (A), 10 m M K N O 3 +4.6 m M Na2S204 (zx), or without addition (control) (e). At the indicated times, the reduced benzyl viologennitrate reductase activity was determined. 1005 of nitrate reductase activity in wild-type and mutant 305 corresponded to 34 and 75 m U / m g protein, respectively.
10 wild and m u t a n t enzymes up to the original levels of untreated controls (results not shown). Nitrate always protected against the in vitro inactivation by dithionite (Fig. 1A and B) a n d N A D ( P ) H (results not shown). Thus, terminal nitrate reductase of m u t a n t 305, like that of the wild type, is reversibly inactivated u n d e r reducing conditions in the absence of nitrate and reactivated by oxidation. Reducing conditions seemed to be the sole requirement for nitrate reductase inactivation, since extracts of wild-type and m u t a n t 305, either treated previously or not with ferricyanide and filtered through a Sephadex G-25 c o l u m n (27 × 1.6 cm), were still reversibly inactivated by dithionite to the same extent (results not shown). I n vivo inactiuation o f n i t r a t e r e d u c t a s e
A m m o n i a inactivated terminal nitrate reductase of C. reinhardii wild-type cells even in the presence
of nitrate (Figs. 2B and 2C). This inactivation followed a course similar to that found in vitro a n d was also a reduction process, since ferricyanide reactivated the inactive enzyme in the in vitro assays up to the control values of Fig. 2A (results not shown). Likewise, reversible inactivation took place in wild cells grown on nitrate and transferred either to illuminated N-free media (Fig. 3B) or to nitrate media in the dark (Fig. 3C). By contrast, nitrate reductase of m u t a n t 305 was inactivated only by a m m o n i a in the light (Figs. 2 a n d 3), and in N-free media in the dark (results not shown). Unlike in the wild cells, nitrate protected the nitrate reductase of m u t a n t 305 against inactivation by a m m o n i a (Fig. 2B) and was also able to reactivate it, even in the presence of amm o n i a (Table I), which indicates that nitrate entered into the m u t a n t cells in presence of a m m o n i a or dark conditions.
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TiME (min) Fig. 2. In vivo inactivation by ammonia of wild-type and mutant 305 nitrate reductases from C. reinhardii. Cells of wild type (O) or mutant 305 (O), grown on ammonia and derepressed with nitrate, were transferred to media containing 8 mM KNO3 (A), 8 mM KNO3 +8 mM NH4CI (B), or 8 mM NH4CI (C). At the indicates times, the reduced benzyl viologen-nitrate reductase activity was determined in 1 ml aliquots of the culture, previously treated with 20 ~1 toluene/ml culture. 100% of nitrate reductase activity in wild-type and mutant 305 corresponded to 62 and 130 mU/mg Chl, respectively.
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Fig. 3. In vivo inactivation in the absence of a nitrogen source or in darkness of wild-type and mutant 305 nitrate reductases from C. reinhardii. Cells of wild-type (O) or mutant 305 (O), grown on ammonia and derepressed with nitrate, were transferred to media containing 8 mM KNO3 (A), no nitrogen (B), or 8 mM KNO 3 in darkness (C). At the indicated times, the reduced benzyl viologen-nitrate reductase activity was determined as in the legend to Fig. 2. 100% of nitrate reductase activity in wild type and mutant 305 corresponded to 78 and 144 mU/mg Chl, respectively.
11 TABLE I IN VIVO REACTIVATION OF THE INACTIVE NITRATE REDUCTASE OF WILD TYPE AND MUTANT 305 FROM C. R E I N H A R D I I Cells of wild type and mutant 305, grown on ammonia and derepressed with nitrate, and containing 63 and 139 m U / m g Chl (100%) of reduced benzyl viologen-nitrate reductase, respectively, were washed and transferred to media containing 8 mM NH4CI. After 2 h, cells were harvested and transferred to the indicated media 8 mM in each of the used nitrogen sources. After 1 h treatment, the activity was measured in situ in toluenized cells. Reduced benzyl viologen nitrate reductase activity (%)
Treatment
Wild type
Mutant 305
10 9 87
8 60 82
NH4CI NH4CI + KNO 3 KNO 3
The reactivation process was independent of the synthesis de novo of protein, since it also occurred in presence of cycloheximide (results not shown).
TABLE II EFFECT OF D I F F E R E N T I N D U C T I O N MEDIA ON THE REGULATION OF NITRATE REDUCTASE FROM WILD A N D MUTANT CELLS OF C. R E I N H A R D l l Cells of wild type and mutant 305, grown on ammonia, were derepressed in media containing 100 #M KNO 3 plus 40 #M KCNO, and 1 mM KNO 2, respectively. After 5.5 h, cells were harvested and transferred to the indicated media 8 mM in each of the used nitrogen sources. After 2 h treatment, the enzyme activity was assayed in situ and the nitrate uptake determined. 100% of reduced benzyl viologen-nitrate reductase activity in wild and mutant cells corresponded to 66 and 137 m U / m g Chl, respectively. 100% of the rate of nitrate uptake corresponded to 9.8 #mol NO 3 per mg Chl per h. n.d., not detectable. Strain
Treatment
Nitrate reductase activity (%)
Rate of nitrate uptake (%)
Wild type
KNO 3 KNO 3 + NH4CI KNO 3, darkness NH4CI
100 8 16 n.d.
100 n.d. 6
Mutant 305
KNO 3 KNO 3 + NH4CI KNO 3, darkness NH4CI
100 87 79 12
n.d. n.d. n.d. -
Mutant 305 can use nitrite but not nitrate for growth (cf. Ref. 17), and induction in nitrate medium leads to a short period of nitrogen starvation. When mutant cells were induced in nitrite media and wild cells in a nitrate non-utilizing media containing nitrate plus cyanate [12], nitrate reductase of both wild-type and mutant 305 showed the same regulatory responses described above irrespective of whether the cells were induced under nitrogen-utilizing or non-utilizing conditions (Table II and Figs. 2 and 3). Discussion
Like many other nitrate reductases of higher plants and green algae [1,2], nitrate reductases of wild type and mutant strain 305 of C. reinhardii are reversibly inactivated in vitro by reduction and reactivated by oxidation. N A D H or N A D P H cannot inactivate mutant 305 nitrate reductase which lacks NAD(P)H-diaphorase activity presumably because they are incapable of reducing directly the terminal moiety of the enzyme. This is consistent with the reported inability of NAD(P)H to reduce the cytochrome b557 component of the mutant 305 nitrate reductase [18]. However, sodium dithionite, a strong chemical reductant, which cannot act as electron donor for enzymatic nitrate reduction [10,18], inactivated reversibly terminal nitrate reductase of wild and mutant cells. Thus, an active diaphorase moiety is required for the in vitro inactivation by NAD(P)H. The same conclusion was achieved in the C. reinhardii mutant nit-A, phenotypically identical to our strain 305 [18,27], although in this case cyanide, dithionite and benzyl viologen or flavin mononucleotide were required to inactivate nitrate reductase in vitro [15]. The reversible inactivation afforded by dithionite, a powerful scavenger of oxygen, suggests that neither superoxide nor other oxygen radicals participate in the in vitro system of nitrate reductase redox interconversion. These results are at variance with those reported for purified nitrate reductase from Ankistrodesmus braunii, where dithionite protected against reversible inactivation of the enzyme by NAD(P)H [8]. Nitrate reductases of either wild-type or mutant 305, filtered through Sephadex G-25, were also susceptible to reversible inactivation by dithionite. This rules out the in-
12
volvement of chelating agents such as cyanide in free or bound form in the redox interconversion process, which has been proposed for nitrate reductases of other green algae [4-6], since bound cyanide is released when the enzyme is oxidized with ferricyanide prior to the filtration step [4]. Although N A D H and N A D P H , the physiological electron donors in enzymatic nitrate reduction, did not inactivate in vitro nitrate reductase of mutant 305, the enzyme resulted reversibly inactivated in vivo in cells incubated with ammonia. This indicates that some endogenous electron donor(s), different from pyridine nucleotides and incapable of supporting nitrate reduction in vivo, should promote inactivation without participation of the diaphorase activity. This is consistent with the low, but detectable, levels of 15N from NO 3 incorporated into insoluble-nitrogen by the nit-A mutant of C. reinhardii [27]. However, an insignificant role has been attributed to reversible inactivation in the regulation of nitrate reductase of this mutant [15]. In C. reinhardii, ammonium inactivates nitrate reductase by preventing the entrance of nitrate into the cells and thus the protection afforded by it [12]. Our results confirm these findings, since nitrate reductase of wild-type cells was inactivated whenever nitrate uptake could not occur, viz. in the presence of ammonia, under conditions of darkness or when cells were placed in N-free media [12,28]. Conversely, nitrate uptake and reversible inactivation are unconnected in mutant 305, since nitrate reductase is not inactivated in cells transferred to media with nitrate plus ammonia or with nitrate in the dark (Figs. 2 and 3, Table II), and nitrate reactivated the inactive enzyme even in the presence of ammonia (Table I). Presumably, the structural mutation of nitrate reductase in 305 leads to deregulation of the transport system of nitrate which passes freely across the membrane barrier, thus keeping active the enzyme under conditions in which nitrate reductase of wild cells became inactivated. The lack of inactivation observed in 305 cells placed in N-free media (Fig. 3B) can be due to accumulation of nitrate inside the cells during the derepression treatment (5.5 h). The different response of 305 nitrate reductase to N-free media (Fig. 3B) and ammonia (Fig. 2C) can
be attributed to a rising in the levels of endogenous electron donors following ammonia assimilation, as proposed in Chlorella fusca [31]. In many green algae, nitrogen starvation changes the regulatory properties of the nitrate uptake system [28-30]. The different behaviour in the regulation of wild and mutant 305 nitrate reductase is not due to nitrogen starvation of mutant 305 (Table II), but should be ascribed mainly to the structure of the corresponding enzymes [18]. Our data suggest a close relationship between the nitrate transport system and nitrate reductase activity. This relationship can determine the level of the intracellular nitrate pool which, in turn, is responsible for the extent of the enzyme activation in both wild and mutant 305 cells.
Acknowledgements This work was supported by Grant no. 1834-82 from C A I C Y T (Spain). One of us (F.C.) thanks the Ministerio de Educaci6n y Ciencia (Spain) for a fellowship.
References 1 Losada, M. Guerrero, M.G. and Vega, J.M. (1981) in Biology of Inorganic Nitrogen and Sulfur (Bothe, H. and Trebst, A., eds.), pp. 30-63, Springer-Verlag, Berlin 2 Guerrero, M.G., Vega, J.M. and Losada, M. (1981) Annu. Rev. Plant Physiol. 32, 169-204 3 Hewitt, E.J. and Notton, B.A. (1980) in Molybdenum and Molybdenum-Containing Enzymes (Coughlan, M.P.. ed.), pp. 275-325, Pergamon Press, Oxford 4 Lorimer, G.H,, Gewith, H-S., V61ker, W., Solomonson, L.P. and Vennesland, B. (1974) J. Biol. Chem. 249, 6074-6079 5 Gewith, H-S., Lorimer, G.H., Solomonson, L.P. and Vennesland, B. (1974) Nature 249, 79-81 6 Pistorius, E.K., Funkhouser, E.A. and Voss, H. (1978) Planta 141,279 282 7 Chaparro, A., De la Rosa, M.A. and Vega, J.M. (1979l Z. Pflanzenphysiol. 95, 77-85 8 De la Rosa, M.A., G6mez-Moreno, C. and Vega, J.M. (1981) Biochim. Biophys. Acta 662, 77-85 9 Herrera, J., Paneque, A., Maldonado, J.M., Barea, J.L. and Losada, M. (1972) Biochem. Biophys. Res. Commun. 48. 996-1003 10 Barea, J.L., Sosa, F.M. and Cardenas, J. (1976) Z. Pflanzenphysiol. 79, 237-245 11 Rigano, C. and Violante, U. (1973) Arch. Mikrobiol. 90, 27-33 12 Florencio, F.J. and Vega, J.M. (1982) Phytochemistry 21, 1195-1200
13 13 G6mez-Moreno, C., Aparicio, P.J., Palacihn, E. and Losada,
14 15 16 17 18 19 20 21
M. (1972) FEBS Lett. 26, 11-14 Solomonson, L.P. (1974) Biochim. Biophys. Acta 334, 297-308 Hipkin, C.R., AI-Bassam, B.A. and Syrett, P.J. (1980) Planta 150, 13-18 Sosa, F.M., Ortega, T. and Barea, J.L. (1978) Plant Sci. Lett. 11, 51-58 Fernb.ndez, E. and Cb.rdenas, J. (1982) Mol. Gen. Genet. 186, 164-169 Fern/~ndez, E. and C~trdenas, J. (1983) Z. Naturforsch. 38c, 439 445 Fernandez, E. and C~rdenas, J. (1981) Biochim. Biophys. Acta 657, 1-12 Paneque, A. and Losada, M. (1966) Biochim. Biophys. Acta 128, 202-204 Paneque, A., Del Campo, F.F., Ramirez, J.M. and Losada, M. (1965) Biochim. Biophys. Acta 109. 79-85
22 Losada, M., Paneque, A., Aparicio, P.J., Vega, J.M., Chrdenas, J. and Herrera, J. (1970) Biochem. Biophys. Res. Commun. 38, 1009-1015 23 Snell, F.D. and Snell, C.T. (1949) in Colorimetric Methods of Analysis, Vol. 2, p. 804, Van Nostrand, New York 24 Bradford, M.M. (1976) Anal. Biochem. 72, 248-256 25 Arnon, D.I. (1949) Plant Physiol. 2~,, 1-15 26 Cawse, P.A. (1967) Analyst 92, 311-315 27 Nichols, G.L., Shehata, A.M. and Syrett, P.J. (1978) J. Gen. Microbiol. 108, 79-88 28 Florencio, F.J. and Vega, J.M. (1983) Planta 158, 288-293 29 Thacker, A. and Syrett, P.J. (1972) New Phytol. 71,423-433 30 Tischner, R. (1981) Ber. Deutsch. Bot. Ges. 94, 635-645 31 Chaparro, A., Maldonado, J.M., Diez, J., Relimpio, A.M. and Losada, M. (1976) Plant Sci. Lett. 6, 335-342
Biochimica et Biophvsica Acta 827 (1985) 8 13 Elsevier
BBA32084
Role of the diaphorase moiety on the reversible inactivation of the Chlamydomonas reinhardii nitrate reductase complex Francisco C6rdoba, Jacobo Cfirdenas * and Emilio Fernandez Departamento de Bioqu'trniea, Facultad de Ciencias, Unit,ersidad de Cbrdoba, Cbrdoba (Spain) (Received June 25th, 1984)
Key words: Nitrate reductase; Redox inactivation; (C. reinhardii).
Nitrate reductase (NAD(P)H: nitrate oxidoreductase, EC 1.6.6.2) of Chlamydomonas reinhardii mutant 305, lacking NAD(P)H-cytochrome c reductase activity, became rapidly and reversibly inactivated in vitro upon incubation with dithionite and reactivated by oxidation with ferricyanide. Nitrate protected against the inactivation. Unlike the native nitrate reductase complex of its parental wild strain, the enzyme of 305 was not inactivated by reduced pyridine nucleotides. In contrast to that of wild-type cells, the in vivo reversible inactivation of mutant 305 nitrate reductase was observed only after complete elimination of nitrate from the media and subsequent transfer to ammonium medium or darkness conditions. The inactive enzyme was reactivated by addition of nitrate to the media without previous removal of ammonium, which indicates that, unlike in the wild-type cells, ammonium does not prevent nitrate uptake by 305 cells. This different regulation pattern is due to the structural modification of 305 nitrate reductase. We conclude that in vitro an active diaphorase moiety is required for the inactivation by reduced pyridine nucleotides of the nitrate reductase of C. reinhardii, and that in vivo the absence of nitrate rather than the presence of ammonium is the triggering event for nitrate reductase inactivation, which can be also achieved by reductants other than NAD(P)H.
Introduction
NAD(P)H-nitrate reductase of eukaryotes (NAD(P)H: nitrate oxidoreductase EC 1.6.6.2) consists of two partial activities, namely NAD(P)H-cytochrome c reductase or diaphorase and reduced benzyl viologen-nitrate reductase, which can be distinguished by chemical and physical treatments and act sequentially in the reduction of nitrate to nitrite [1-3]. In green algae and higher plants, nitrate reductase is regulated in vivo either by redox interconversion [1,2] or by reversible inactivation under reducing conditions with cyanide [4-6] or with superoxide radicals [7,8] this inactivation being * To whom correspondence should be addressed. 0167-4838/85/$03.30 © 1985 Elsevier Science Publishers B.V.
centered on the terminal nitrate reductase moiety of the enzyme complex [8-10]. In some instances ammonium or darkness promotes the in vivo inactivation of nitrate reductases [6,9,11], which seems to depend on the availability of intracellular nitrate controlled by the uptake system [12]. Inactive enzyme can be reactivated in vitro by oxidation with nitrate or ferricyanide [5,9,13]. Requirement of diaphorase activity for the reversible inactivation process remains controversial in spite of many published data in this respect [9,10,14,15]. Chlamydomonas reinhardii mutants deficient in nitrate reductase have been isolated [16] and used to study in vivo the regulation of the activities of the nitrate reductase complex [17]. In the present work we compare the in vitro and in vivo reversible redox inactivations of nitrate reductases of
wild-type and mutant 305 which lacks NAD(P)Hnitrate reductase and NAD(P)H-diaphorase [18]. The results indicate that in vitro an active diaphorase is required for terminal nitrate reductase inactivation by NAD(P)H, whereas in vivo inactivation by ammonia occurs in the absence of nitrate and is a redox process which can be performed by reductants other than NAD(P)H. In addition, reactivation in vivo of inactive terminal nitrate reductase is achieved by reoxidation following the entrance of nitrate within the cells. Experimental Cells of C. reinhardii, wild-type 6145c and mutant 305 [16], were grown with 8 mM NH4CI and derepressed with 4 mM KNO 3 for 5.5 h under reported conditions [17,19]. Cells were disrupted by freezing in liquid nitrogen and thawing in a 50 mM Tris-HC1 buffer (pH 7.5)/0.1 mM EDTA/0.1 mM dithioerythritol (4 m l / g wet weight) as previously described [17]. The suspension was centrifuged at 27000 × g, 15 min, and the supernatant was used as the source of enzyme. NAD(P)H-nitrate reductase and reduced benzyl viologen-nitrate reductase activities were determined in vitro by measuring the nitrite formed in the enzymatic reduction of nitrate [20,21]. Reduced benzyl viologen-nitrate reductase activity was assayed in situ by using 1 ml of cell suspensions previously treated with 20 ~1 of commercial toluene [12]. Reactivation of nitrate reductase was achieved by preincubating extracts or toluenized cell suspensions with 0.3 ~mol ferricyanide for 1 min and doubling the concentration of reductant in the standard assay mixture [22]. Nitrite was determined colorimetrically by the diazotization method of Snell and Snell [23]. Protein was estimated colorimetrically by the method of Bradford [24], using bovine serum albumin as standard, and chlorophyll by its absorbance at 652 nm according to Arnon [25]. The nitrate uptake was measured by determining the disappearance of nitrate from the medium according to Cawse [26]. Results In vitro inactivation of nitrate reductase NADH, N A D P H or sodium dithionite in-
activated terminal nitrate reductase of C. reinhardii wild cells up to 30% of its original activity after 60 min incubation (Fig. 1A). NAD(P)Hnitrate reductase activity responded similarly to the same treatments (results not shown). By contrast, nitrate reductase of mutant 305 was inactivated to the same extent only by sodium dithionite (Fig. 1B). NAD(P)H concentrations up to 3 mM did not inactivate terminal nitrate reductase nor reduced enzymatically nitrate in 305 even after a 2 h assay at 30°C. Added flavins or viologens increased neither the rate nor the extent of inactivation exerted by dithionite.
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Fig. 1. In vitro inactivation course of wild-type (A) and mutant 305 (B) nitrate reductases from C. reinhardii. Extracts from wild-type and mutant 305 were incubated at 4°C with 0.3 m M N A D H (D), 0.3 m M N A D P H (m), 4.6 m M Na2S204 (A), 10 m M K N O 3 +4.6 m M Na2S204 (zx), or without addition (control) (e). At the indicated times, the reduced benzyl viologennitrate reductase activity was determined. 1005 of nitrate reductase activity in wild-type and mutant 305 corresponded to 34 and 75 m U / m g protein, respectively.
10 wild and m u t a n t enzymes up to the original levels of untreated controls (results not shown). Nitrate always protected against the in vitro inactivation by dithionite (Fig. 1A and B) a n d N A D ( P ) H (results not shown). Thus, terminal nitrate reductase of m u t a n t 305, like that of the wild type, is reversibly inactivated u n d e r reducing conditions in the absence of nitrate and reactivated by oxidation. Reducing conditions seemed to be the sole requirement for nitrate reductase inactivation, since extracts of wild-type and m u t a n t 305, either treated previously or not with ferricyanide and filtered through a Sephadex G-25 c o l u m n (27 × 1.6 cm), were still reversibly inactivated by dithionite to the same extent (results not shown). I n vivo inactiuation o f n i t r a t e r e d u c t a s e
A m m o n i a inactivated terminal nitrate reductase of C. reinhardii wild-type cells even in the presence
of nitrate (Figs. 2B and 2C). This inactivation followed a course similar to that found in vitro a n d was also a reduction process, since ferricyanide reactivated the inactive enzyme in the in vitro assays up to the control values of Fig. 2A (results not shown). Likewise, reversible inactivation took place in wild cells grown on nitrate and transferred either to illuminated N-free media (Fig. 3B) or to nitrate media in the dark (Fig. 3C). By contrast, nitrate reductase of m u t a n t 305 was inactivated only by a m m o n i a in the light (Figs. 2 a n d 3), and in N-free media in the dark (results not shown). Unlike in the wild cells, nitrate protected the nitrate reductase of m u t a n t 305 against inactivation by a m m o n i a (Fig. 2B) and was also able to reactivate it, even in the presence of amm o n i a (Table I), which indicates that nitrate entered into the m u t a n t cells in presence of a m m o n i a or dark conditions.
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60
120
TiME (min) Fig. 2. In vivo inactivation by ammonia of wild-type and mutant 305 nitrate reductases from C. reinhardii. Cells of wild type (O) or mutant 305 (O), grown on ammonia and derepressed with nitrate, were transferred to media containing 8 mM KNO3 (A), 8 mM KNO3 +8 mM NH4CI (B), or 8 mM NH4CI (C). At the indicates times, the reduced benzyl viologen-nitrate reductase activity was determined in 1 ml aliquots of the culture, previously treated with 20 ~1 toluene/ml culture. 100% of nitrate reductase activity in wild-type and mutant 305 corresponded to 62 and 130 mU/mg Chl, respectively.
~ ,
i
0
i
60 TIME
i
120 (rnin)
Fig. 3. In vivo inactivation in the absence of a nitrogen source or in darkness of wild-type and mutant 305 nitrate reductases from C. reinhardii. Cells of wild-type (O) or mutant 305 (O), grown on ammonia and derepressed with nitrate, were transferred to media containing 8 mM KNO3 (A), no nitrogen (B), or 8 mM KNO 3 in darkness (C). At the indicated times, the reduced benzyl viologen-nitrate reductase activity was determined as in the legend to Fig. 2. 100% of nitrate reductase activity in wild type and mutant 305 corresponded to 78 and 144 mU/mg Chl, respectively.
11 TABLE I IN VIVO REACTIVATION OF THE INACTIVE NITRATE REDUCTASE OF WILD TYPE AND MUTANT 305 FROM C. R E I N H A R D I I Cells of wild type and mutant 305, grown on ammonia and derepressed with nitrate, and containing 63 and 139 m U / m g Chl (100%) of reduced benzyl viologen-nitrate reductase, respectively, were washed and transferred to media containing 8 mM NH4CI. After 2 h, cells were harvested and transferred to the indicated media 8 mM in each of the used nitrogen sources. After 1 h treatment, the activity was measured in situ in toluenized cells. Reduced benzyl viologen nitrate reductase activity (%)
Treatment
Wild type
Mutant 305
10 9 87
8 60 82
NH4CI NH4CI + KNO 3 KNO 3
The reactivation process was independent of the synthesis de novo of protein, since it also occurred in presence of cycloheximide (results not shown).
TABLE II EFFECT OF D I F F E R E N T I N D U C T I O N MEDIA ON THE REGULATION OF NITRATE REDUCTASE FROM WILD A N D MUTANT CELLS OF C. R E I N H A R D l l Cells of wild type and mutant 305, grown on ammonia, were derepressed in media containing 100 #M KNO 3 plus 40 #M KCNO, and 1 mM KNO 2, respectively. After 5.5 h, cells were harvested and transferred to the indicated media 8 mM in each of the used nitrogen sources. After 2 h treatment, the enzyme activity was assayed in situ and the nitrate uptake determined. 100% of reduced benzyl viologen-nitrate reductase activity in wild and mutant cells corresponded to 66 and 137 m U / m g Chl, respectively. 100% of the rate of nitrate uptake corresponded to 9.8 #mol NO 3 per mg Chl per h. n.d., not detectable. Strain
Treatment
Nitrate reductase activity (%)
Rate of nitrate uptake (%)
Wild type
KNO 3 KNO 3 + NH4CI KNO 3, darkness NH4CI
100 8 16 n.d.
100 n.d. 6
Mutant 305
KNO 3 KNO 3 + NH4CI KNO 3, darkness NH4CI
100 87 79 12
n.d. n.d. n.d. -
Mutant 305 can use nitrite but not nitrate for growth (cf. Ref. 17), and induction in nitrate medium leads to a short period of nitrogen starvation. When mutant cells were induced in nitrite media and wild cells in a nitrate non-utilizing media containing nitrate plus cyanate [12], nitrate reductase of both wild-type and mutant 305 showed the same regulatory responses described above irrespective of whether the cells were induced under nitrogen-utilizing or non-utilizing conditions (Table II and Figs. 2 and 3). Discussion
Like many other nitrate reductases of higher plants and green algae [1,2], nitrate reductases of wild type and mutant strain 305 of C. reinhardii are reversibly inactivated in vitro by reduction and reactivated by oxidation. N A D H or N A D P H cannot inactivate mutant 305 nitrate reductase which lacks NAD(P)H-diaphorase activity presumably because they are incapable of reducing directly the terminal moiety of the enzyme. This is consistent with the reported inability of NAD(P)H to reduce the cytochrome b557 component of the mutant 305 nitrate reductase [18]. However, sodium dithionite, a strong chemical reductant, which cannot act as electron donor for enzymatic nitrate reduction [10,18], inactivated reversibly terminal nitrate reductase of wild and mutant cells. Thus, an active diaphorase moiety is required for the in vitro inactivation by NAD(P)H. The same conclusion was achieved in the C. reinhardii mutant nit-A, phenotypically identical to our strain 305 [18,27], although in this case cyanide, dithionite and benzyl viologen or flavin mononucleotide were required to inactivate nitrate reductase in vitro [15]. The reversible inactivation afforded by dithionite, a powerful scavenger of oxygen, suggests that neither superoxide nor other oxygen radicals participate in the in vitro system of nitrate reductase redox interconversion. These results are at variance with those reported for purified nitrate reductase from Ankistrodesmus braunii, where dithionite protected against reversible inactivation of the enzyme by NAD(P)H [8]. Nitrate reductases of either wild-type or mutant 305, filtered through Sephadex G-25, were also susceptible to reversible inactivation by dithionite. This rules out the in-
12
volvement of chelating agents such as cyanide in free or bound form in the redox interconversion process, which has been proposed for nitrate reductases of other green algae [4-6], since bound cyanide is released when the enzyme is oxidized with ferricyanide prior to the filtration step [4]. Although N A D H and N A D P H , the physiological electron donors in enzymatic nitrate reduction, did not inactivate in vitro nitrate reductase of mutant 305, the enzyme resulted reversibly inactivated in vivo in cells incubated with ammonia. This indicates that some endogenous electron donor(s), different from pyridine nucleotides and incapable of supporting nitrate reduction in vivo, should promote inactivation without participation of the diaphorase activity. This is consistent with the low, but detectable, levels of 15N from NO 3 incorporated into insoluble-nitrogen by the nit-A mutant of C. reinhardii [27]. However, an insignificant role has been attributed to reversible inactivation in the regulation of nitrate reductase of this mutant [15]. In C. reinhardii, ammonium inactivates nitrate reductase by preventing the entrance of nitrate into the cells and thus the protection afforded by it [12]. Our results confirm these findings, since nitrate reductase of wild-type cells was inactivated whenever nitrate uptake could not occur, viz. in the presence of ammonia, under conditions of darkness or when cells were placed in N-free media [12,28]. Conversely, nitrate uptake and reversible inactivation are unconnected in mutant 305, since nitrate reductase is not inactivated in cells transferred to media with nitrate plus ammonia or with nitrate in the dark (Figs. 2 and 3, Table II), and nitrate reactivated the inactive enzyme even in the presence of ammonia (Table I). Presumably, the structural mutation of nitrate reductase in 305 leads to deregulation of the transport system of nitrate which passes freely across the membrane barrier, thus keeping active the enzyme under conditions in which nitrate reductase of wild cells became inactivated. The lack of inactivation observed in 305 cells placed in N-free media (Fig. 3B) can be due to accumulation of nitrate inside the cells during the derepression treatment (5.5 h). The different response of 305 nitrate reductase to N-free media (Fig. 3B) and ammonia (Fig. 2C) can
be attributed to a rising in the levels of endogenous electron donors following ammonia assimilation, as proposed in Chlorella fusca [31]. In many green algae, nitrogen starvation changes the regulatory properties of the nitrate uptake system [28-30]. The different behaviour in the regulation of wild and mutant 305 nitrate reductase is not due to nitrogen starvation of mutant 305 (Table II), but should be ascribed mainly to the structure of the corresponding enzymes [18]. Our data suggest a close relationship between the nitrate transport system and nitrate reductase activity. This relationship can determine the level of the intracellular nitrate pool which, in turn, is responsible for the extent of the enzyme activation in both wild and mutant 305 cells.
Acknowledgements This work was supported by Grant no. 1834-82 from C A I C Y T (Spain). One of us (F.C.) thanks the Ministerio de Educaci6n y Ciencia (Spain) for a fellowship.
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