564
J. Smarrelli, Jr. and D. Castignetti: Ferriphytosiderophore reduction by nitrate reductase from squash I
j
I
I
I
I
I
I
[
10o
8o
~. 6 0
I-
,~4o 2O
pH
Fig. 1. Activity-versus-pH profile for reduction of a ferriphytosiderophore from barley by nitrate reductase from squash cotyledons. All assays were performed as described in the text and 100% activity equals 7.8 nmol ferriphytosiderophore reduced. min- 1. (ml enzyme) - 1
tion of the method of Dailey and Lascelles (1977). The 1-ml assay mixture contained 25 mM oxalate-maleate buffer at the appropriate pH, 0.l mM NADH, 1.2mM ferrozine, and 0.14 mM ferriphytosiderophore. The formation of the ferrous iron-ferrozine complex was monitored at 562 nm. Control assays were performed by omitting enzyme or substrate from the reaction mixture. Protein was determined according to Lowry et al. (1951) using bovine serum albumin as the standard. Kinetic analyses were performed with 0.1 mM NADH at the optimum pH for the reduction of the siderophore. Michaelis constants were determined using the Direct Linear Plot with a computer program to locate median intersections (Campbell and Smarrelli 1978). All chemicals were reagent grade or better. Immunoglobulins raised in rabbits, monospecific against squash nitrate reductase, were a gift from Wilbur H. Campbell, Michigan Technological University, Houghton, MI, USA. The ferriphytosiderophore isolated in pure form from barley (Hordeum vulgare L. cv. Europa) was obtained from V. R6hmheld, Universitfit Hohenheim, Stuttgart, FRG, and was 100% ferrated as determined by its absorption spectrum (data not shown). An aqueous 1.4 mM stock solution was prepared and diluted appropriately for the enzymatic analyses.
Our previous results have demonstrated that NADH:nitrate reductase from squash cotyledons reduced ferrisiderophores of microbial origin, generally with maximum activities between pH 4 and 5, and showing kinetic constants for these iron chelates in the micromolar range (Castignetti and Smarrelli 1984; Smarrelli and Castignetti 1986). Figure I depicts the pH dependence for the reduction of the pure barley ferriphytosiderophore by squash nitrate reductase. Unlike previously examined ferrisiderophores, maximum activity occurred at pH 6, resembling the pH profile for the ferric citrate-reductase activity of nitrate reductase (Redinbaugh and Campbell 1983), but clearly different
from the pH 7.5 activity maximum with nitrate (Campbell and Smarrelli 1986). In addition, marked ferriphytosiderophore-reductase activity is still found at both pH 7 and 8 which is different from the pH-versus-activity profiles for the previously examined ferrisiderophores (Castignetti and Smarrelli 1986). By varying the ferriphytosiderophore concentration from 0.014 to 0.21 mM while keeping the concentration of N A D H at 0.2 mM during the assay for ferriphytosiderophore-reductase activity, and analyzing the data using direct linear plots, apparent K,, and VmaX values were obtained (Table 1). The K,, of 76 gM is intermediate with respect to other ferrisiderophores studied, while a Vmax of 21 nmol" rain- 1. (mg protein)- 1 for a bluesepharose-purified nitrate reductase preparation was found. The ferriphytosiderophore-reductase activity is markedly inhibited by nitrate-reductasespecific antibodies (data not shown). To examine the competition between nitrate and the ferriphytosiderophore for electrons from nitrate reductase, the inhibition by the ferriphytosiderophore on nitrate reduction, and the inhibition by nitrate on ferriphytosiderophore reduction were examined (Table 2). Although the rate of ferriphytosiderophore reduction is markedly lower than that for nitrate reduction, the ferriphytosiderophore was a better inhibitor of nitrate reduction than nitrate was of ferriphytosiderophore reduction. An explanation may be that each assay was performed under optimal conditions for each respective substrate, and at pH 6 nitrate reacts poorly with nitrate reductase. The reduction of the ferriphytosiderophore from barley by squash cotyledon NADH:nitrate reductase is, to our knowledge, the first demonstration of such an activity by a plant enzyme. With respect to the kinetic parameters of Vm~X and Km (Table 1), the enzyme reduced this ferriphytosiderophore with an efficiency similar to that previously noted for the reduction of microbial ferrisiderophores. The pH optimum of ferriphytosiderophore reduction is somewhat higher than those noted for the microbial ferrisiderophores but pronounced reduction of the ferriphytosiderophore (approx. 40-50%) still occurred at a pH of 4-5. NADH : nitrate reductase thus displays a broad catalytic activity, being able to reduce nitrate (for a review, see Campbell and Smarrelli 1986), six different ferrisiderophores (Castignetti and Smarrelli 1986), ferric citrate (Redinbaugh and Campbell 1983), and at least one ferriphytosiderophore. In addition, the lack of appreciable inhibition of ferriphytosiderophore reduction by nitrate coupled with
564
J. Smarrelli, Jr. and D. Castignetti: Ferriphytosiderophore reduction by nitrate reductase from squash I
j
I
I
I
I
I
I
[
10o
8o
~. 6 0
I-
,~4o 2O
pH
Fig. 1. Activity-versus-pH profile for reduction of a ferriphytosiderophore from barley by nitrate reductase from squash cotyledons. All assays were performed as described in the text and 100% activity equals 7.8 nmol ferriphytosiderophore reduced. min- 1. (ml enzyme) - 1
tion of the method of Dailey and Lascelles (1977). The 1-ml assay mixture contained 25 mM oxalate-maleate buffer at the appropriate pH, 0.l mM NADH, 1.2mM ferrozine, and 0.14 mM ferriphytosiderophore. The formation of the ferrous iron-ferrozine complex was monitored at 562 nm. Control assays were performed by omitting enzyme or substrate from the reaction mixture. Protein was determined according to Lowry et al. (1951) using bovine serum albumin as the standard. Kinetic analyses were performed with 0.1 mM NADH at the optimum pH for the reduction of the siderophore. Michaelis constants were determined using the Direct Linear Plot with a computer program to locate median intersections (Campbell and Smarrelli 1978). All chemicals were reagent grade or better. Immunoglobulins raised in rabbits, monospecific against squash nitrate reductase, were a gift from Wilbur H. Campbell, Michigan Technological University, Houghton, MI, USA. The ferriphytosiderophore isolated in pure form from barley (Hordeum vulgare L. cv. Europa) was obtained from V. R6hmheld, Universitfit Hohenheim, Stuttgart, FRG, and was 100% ferrated as determined by its absorption spectrum (data not shown). An aqueous 1.4 mM stock solution was prepared and diluted appropriately for the enzymatic analyses.
Our previous results have demonstrated that NADH:nitrate reductase from squash cotyledons reduced ferrisiderophores of microbial origin, generally with maximum activities between pH 4 and 5, and showing kinetic constants for these iron chelates in the micromolar range (Castignetti and Smarrelli 1984; Smarrelli and Castignetti 1986). Figure I depicts the pH dependence for the reduction of the pure barley ferriphytosiderophore by squash nitrate reductase. Unlike previously examined ferrisiderophores, maximum activity occurred at pH 6, resembling the pH profile for the ferric citrate-reductase activity of nitrate reductase (Redinbaugh and Campbell 1983), but clearly different
from the pH 7.5 activity maximum with nitrate (Campbell and Smarrelli 1986). In addition, marked ferriphytosiderophore-reductase activity is still found at both pH 7 and 8 which is different from the pH-versus-activity profiles for the previously examined ferrisiderophores (Castignetti and Smarrelli 1986). By varying the ferriphytosiderophore concentration from 0.014 to 0.21 mM while keeping the concentration of N A D H at 0.2 mM during the assay for ferriphytosiderophore-reductase activity, and analyzing the data using direct linear plots, apparent K,, and VmaX values were obtained (Table 1). The K,, of 76 gM is intermediate with respect to other ferrisiderophores studied, while a Vmax of 21 nmol" rain- 1. (mg protein)- 1 for a bluesepharose-purified nitrate reductase preparation was found. The ferriphytosiderophore-reductase activity is markedly inhibited by nitrate-reductasespecific antibodies (data not shown). To examine the competition between nitrate and the ferriphytosiderophore for electrons from nitrate reductase, the inhibition by the ferriphytosiderophore on nitrate reduction, and the inhibition by nitrate on ferriphytosiderophore reduction were examined (Table 2). Although the rate of ferriphytosiderophore reduction is markedly lower than that for nitrate reduction, the ferriphytosiderophore was a better inhibitor of nitrate reduction than nitrate was of ferriphytosiderophore reduction. An explanation may be that each assay was performed under optimal conditions for each respective substrate, and at pH 6 nitrate reacts poorly with nitrate reductase. The reduction of the ferriphytosiderophore from barley by squash cotyledon NADH:nitrate reductase is, to our knowledge, the first demonstration of such an activity by a plant enzyme. With respect to the kinetic parameters of Vm~X and Km (Table 1), the enzyme reduced this ferriphytosiderophore with an efficiency similar to that previously noted for the reduction of microbial ferrisiderophores. The pH optimum of ferriphytosiderophore reduction is somewhat higher than those noted for the microbial ferrisiderophores but pronounced reduction of the ferriphytosiderophore (approx. 40-50%) still occurred at a pH of 4-5. NADH : nitrate reductase thus displays a broad catalytic activity, being able to reduce nitrate (for a review, see Campbell and Smarrelli 1986), six different ferrisiderophores (Castignetti and Smarrelli 1986), ferric citrate (Redinbaugh and Campbell 1983), and at least one ferriphytosiderophore. In addition, the lack of appreciable inhibition of ferriphytosiderophore reduction by nitrate coupled with
J. Smarrelli, Jr. and D. Castignetti: Ferriphytosiderophore reduction by nitrate reductase from squash
565
Table 1. Comparative kinetic constants for selected iron-reductase activities of NADH: nitrate reductase. The ferriphytosiderophore
reductase assay was performed using the enzyme preparation and assay conditions described in the text. Kinetic constants were determined at optimum pH. Substrate
pH optimum
K,, (gM)
Vmax (nmol- min- 1. (mg protein)- 1
Reference
Ferriphytosiderophore Ferrirhodotorulic acid Ferrischizokinen Ferric citrate
6 5 4 6.2
76 170 10 20
21 200 82 -
This study (1) (1) (2)
(1) Smarrelli and Castignetti 1986 (2) Redinbaugh and Campbell 1983
Table 2. Inhibition of nitrate-reductase activity by the ferriphy-
tosiderophore and inhibition of ferriphytosiderophore reductase activity by nitrate
Nitrate reduction (pH 7.5) Nitrate (gM) 50 50 100 100 200 200 1000
Ferriphytosiderophore (gM) 140 210 140 210 140 210 140
Inhibition (%) 79 100 67 78 75 83 64
Ferriphytosiderophore reduction (pH 6.0) Ferriphytosiderophore (gM) 56 56 70 70 210 210
Nitrate (gM) 200 1000 200 1000 200 1000
Inhibition (%) 7 9 10 20 0 0
the marked inhibition of nitrate reduction by the ferriphytosiderophore (Table 2), indicates that ferriphytosiderophore reduction is similar, with respect to electron flow through NADH:nitrate reductase, to microbial ferrisiderophore reduction (Castignetti and Smarrelli 1986). The reduction of the ferriphytosiderophore by nitrate reductase demonstrates that squash has the potential to assimilate the Fe 3 + from a ferriphytosiderophore synthesized by another plant. Since most higher plant nitrate reductases display biochemical characteristics similar to squash nitrate reductase (Campbell and Smarrelli 1986), it may be suggested that other higher-plant nitrate reductases show a similar ferrisiderophore-reductase activity. The diverse catalytic activity of the enzyme with a number of different Fe 3+ substrates may
be of value to squash by permitting it to assimilate iron from a number of different compounds. The mechanism of ferrisiderophore and ferriphytosiderophore transport into the plant roots, however, is unknown and requires elucidation. The iron-assimilation model of R6mheld and Marschner (1986 a, b) includes the possibility that ferrisiderophores enter the cytoplasm of plant cells intact and are subsequently reduced. Once these compounds gain access to the cell, however, it is clear that cytoplasmically located squash cotyledon NADH: nitrate reductase may function to release the Fe 3 + of these compounds such that the metal may participate in plant metabolism. This study was funded in part by grants from the National Science Foundation (DMB 8404243, J.S.), by Loyola University of Chicago Research Stimulation Grants (D.C., J.S.), and by the Biomedical Research Support Grant (1S07 RR07210, D.C). We wish to thank V. Romheld for the gift of the phytosiderophore Cpi-3-hydroxy-mugineic acid, and Drs. Mori (Tokyo), Kawai (Iwate), and Nomoto (Suntory) for its positive identification.
References Becker, J.O., Messens, E., Hedges, R.W. (1985) The influence of agrobactin on the uptake of ferric ion by plants. FEMS Microbiol. Ecol 31, 171-175 Campbell, W.H., Smarrelli, J., Jr. (1978) Purification and kinetics of higher plant NADH: nitrate reductase. Plant Physiol. 61,611-616 Campbell, W.H., Smarrelli, J., Jr. (1986) Nitrate reductase: biochemistry and regulation. In: Biochemical basis of plant breeding, pp. 1-39, Neyra, C.A., ed. CRC Press, Boca Raton, FL., USA Castignetti, D., Smarrelli, J., Jr. (1984) Siderophore reduction catalyzed by higher plant NADH :nitrate reductase. Biochem. Biophys. Res. Commun. 125, 52-58 Castignetti, D., Smarrelli, J., Jr. (1986) Siderophores, the iron nutrition of plants, and nitrate reductase. FEBS Lett. 209, 147-151 Cline, G.R., Reid, C.P., Powell, P.E., Szaniszlo, P.J. (1984) Effects of a hydroxamate siderophore on iron absorption by sunflower and sorghum. Plant Physiol. 76, 36~39
566
J. Smarrelli, Jr. and D. Castignetti: Ferriphytosiderophore reduction by nitrate reductase from squash
Dailey, H.A., Lascelles, J. (1977) Reduction of iron in synthesis of protoheme by Spirillum itersoni and other organisms. J. Bacteriol. 129, 815-820 Duss, F., Mozafar, A., Oertli, J.J., Jaeggi, W. (1986) Effect of bacteria on the iron uptake of axenically-cultured roots of Fe-efficient and Fe-inefficient tomatoes (Lyeopersicon esculentum Mill). J. Plant Nutr. 9, 587-598 Emery, T.E. (1982) Iron metabolism in humans and plants. Am. Sci. 70, 626-632 Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193, 265-270 Miller, G.W., Pushnik, J.C., Brown, J.C., Emery, T.E., Jolley, V.D., Warnick, K.Y. (1985) Uptake and translocation of iron from ferrated rhodotorulic acid in tomato. J. Plant Nutr. 8, 249-264 Orlando, J.A., Neilands, J.B. (1982) Ferrichrome compounds as a source of iron for higher plants. In: Chemistry and biology of hydroxamic acids, pp. 123-129, Kehl, H., ed. S. Karger, Basel Page, E.R. (1966) Sideramines in plants and their possible role in iron metabolism. Biochem. J. 100, 34 Powell, P.E., Szaniszlo, P.J., Cline, G.R., Reid, C.P. (1982) Hydroxamate siderophores in the iron nutrition of plants. J. Plant Nutr. 5, 653-673
Redinbaugh, M.G., Campbell, W.H. (1983) Reduction of ferric citrate catalyzed by NADH:nitrate reductase. Biochem. Biophys. Res. Commun. 114, 1182-1188 Reid, C.P., Crowley, D.E., Kim, H.J., Powell, P.E., Szaniszlo, P.J. (1984) Utilization of iron by oat when supplied as ferrated synthetic chelate or as ferrated hydroxamate siderophore. J. Plant Nutr. 7, 437M47 R6mheld, V., Marschner, H. (1986a) Evidence for a specific uptake system for iron phytosiderophores in roots of grasses. Plant Physiol. 80, 175-180 R6mheld, V., Marschner, H. (1986b) Mobilization of iron in the rhizosphere of different plant species. Adv. Plant Nutr. 2, 155-204 Smarrelli, J. Jr., Campbell, W.H. (1979) NADH dehydrogenase .activity of higher plant nitrate reductase (NADH). Plant Sci. Lett. 16, 139-147 Smarrelli, J., Jr., Castignetti, D. (1986) Iron acquisition by plants: the reduction of ferrisiderophores by higher plant NADH:nitrate reductase. Biochim. Biophys. Acta. 882, 337-342 Stutz, E. (1964) Aufnahme von Ferrioxamine B durch Tomatenpflanzen. Experientia 20, 43~431 Received 20 August; accepted 13 November 1987
J. Smarrelli, Jr. and D. Castignetti: Ferriphytosiderophore reduction by nitrate reductase from squash I
j
I
I
I
I
I
I
[
10o
8o
~. 6 0
I-
,~4o 2O
pH
Fig. 1. Activity-versus-pH profile for reduction of a ferriphytosiderophore from barley by nitrate reductase from squash cotyledons. All assays were performed as described in the text and 100% activity equals 7.8 nmol ferriphytosiderophore reduced. min- 1. (ml enzyme) - 1
tion of the method of Dailey and Lascelles (1977). The 1-ml assay mixture contained 25 mM oxalate-maleate buffer at the appropriate pH, 0.l mM NADH, 1.2mM ferrozine, and 0.14 mM ferriphytosiderophore. The formation of the ferrous iron-ferrozine complex was monitored at 562 nm. Control assays were performed by omitting enzyme or substrate from the reaction mixture. Protein was determined according to Lowry et al. (1951) using bovine serum albumin as the standard. Kinetic analyses were performed with 0.1 mM NADH at the optimum pH for the reduction of the siderophore. Michaelis constants were determined using the Direct Linear Plot with a computer program to locate median intersections (Campbell and Smarrelli 1978). All chemicals were reagent grade or better. Immunoglobulins raised in rabbits, monospecific against squash nitrate reductase, were a gift from Wilbur H. Campbell, Michigan Technological University, Houghton, MI, USA. The ferriphytosiderophore isolated in pure form from barley (Hordeum vulgare L. cv. Europa) was obtained from V. R6hmheld, Universitfit Hohenheim, Stuttgart, FRG, and was 100% ferrated as determined by its absorption spectrum (data not shown). An aqueous 1.4 mM stock solution was prepared and diluted appropriately for the enzymatic analyses.
Our previous results have demonstrated that NADH:nitrate reductase from squash cotyledons reduced ferrisiderophores of microbial origin, generally with maximum activities between pH 4 and 5, and showing kinetic constants for these iron chelates in the micromolar range (Castignetti and Smarrelli 1984; Smarrelli and Castignetti 1986). Figure I depicts the pH dependence for the reduction of the pure barley ferriphytosiderophore by squash nitrate reductase. Unlike previously examined ferrisiderophores, maximum activity occurred at pH 6, resembling the pH profile for the ferric citrate-reductase activity of nitrate reductase (Redinbaugh and Campbell 1983), but clearly different
from the pH 7.5 activity maximum with nitrate (Campbell and Smarrelli 1986). In addition, marked ferriphytosiderophore-reductase activity is still found at both pH 7 and 8 which is different from the pH-versus-activity profiles for the previously examined ferrisiderophores (Castignetti and Smarrelli 1986). By varying the ferriphytosiderophore concentration from 0.014 to 0.21 mM while keeping the concentration of N A D H at 0.2 mM during the assay for ferriphytosiderophore-reductase activity, and analyzing the data using direct linear plots, apparent K,, and VmaX values were obtained (Table 1). The K,, of 76 gM is intermediate with respect to other ferrisiderophores studied, while a Vmax of 21 nmol" rain- 1. (mg protein)- 1 for a bluesepharose-purified nitrate reductase preparation was found. The ferriphytosiderophore-reductase activity is markedly inhibited by nitrate-reductasespecific antibodies (data not shown). To examine the competition between nitrate and the ferriphytosiderophore for electrons from nitrate reductase, the inhibition by the ferriphytosiderophore on nitrate reduction, and the inhibition by nitrate on ferriphytosiderophore reduction were examined (Table 2). Although the rate of ferriphytosiderophore reduction is markedly lower than that for nitrate reduction, the ferriphytosiderophore was a better inhibitor of nitrate reduction than nitrate was of ferriphytosiderophore reduction. An explanation may be that each assay was performed under optimal conditions for each respective substrate, and at pH 6 nitrate reacts poorly with nitrate reductase. The reduction of the ferriphytosiderophore from barley by squash cotyledon NADH:nitrate reductase is, to our knowledge, the first demonstration of such an activity by a plant enzyme. With respect to the kinetic parameters of Vm~X and Km (Table 1), the enzyme reduced this ferriphytosiderophore with an efficiency similar to that previously noted for the reduction of microbial ferrisiderophores. The pH optimum of ferriphytosiderophore reduction is somewhat higher than those noted for the microbial ferrisiderophores but pronounced reduction of the ferriphytosiderophore (approx. 40-50%) still occurred at a pH of 4-5. NADH : nitrate reductase thus displays a broad catalytic activity, being able to reduce nitrate (for a review, see Campbell and Smarrelli 1986), six different ferrisiderophores (Castignetti and Smarrelli 1986), ferric citrate (Redinbaugh and Campbell 1983), and at least one ferriphytosiderophore. In addition, the lack of appreciable inhibition of ferriphytosiderophore reduction by nitrate coupled with
564
J. Smarrelli, Jr. and D. Castignetti: Ferriphytosiderophore reduction by nitrate reductase from squash I
j
I
I
I
I
I
I
[
10o
8o
~. 6 0
I-
,~4o 2O
pH
Fig. 1. Activity-versus-pH profile for reduction of a ferriphytosiderophore from barley by nitrate reductase from squash cotyledons. All assays were performed as described in the text and 100% activity equals 7.8 nmol ferriphytosiderophore reduced. min- 1. (ml enzyme) - 1
tion of the method of Dailey and Lascelles (1977). The 1-ml assay mixture contained 25 mM oxalate-maleate buffer at the appropriate pH, 0.l mM NADH, 1.2mM ferrozine, and 0.14 mM ferriphytosiderophore. The formation of the ferrous iron-ferrozine complex was monitored at 562 nm. Control assays were performed by omitting enzyme or substrate from the reaction mixture. Protein was determined according to Lowry et al. (1951) using bovine serum albumin as the standard. Kinetic analyses were performed with 0.1 mM NADH at the optimum pH for the reduction of the siderophore. Michaelis constants were determined using the Direct Linear Plot with a computer program to locate median intersections (Campbell and Smarrelli 1978). All chemicals were reagent grade or better. Immunoglobulins raised in rabbits, monospecific against squash nitrate reductase, were a gift from Wilbur H. Campbell, Michigan Technological University, Houghton, MI, USA. The ferriphytosiderophore isolated in pure form from barley (Hordeum vulgare L. cv. Europa) was obtained from V. R6hmheld, Universitfit Hohenheim, Stuttgart, FRG, and was 100% ferrated as determined by its absorption spectrum (data not shown). An aqueous 1.4 mM stock solution was prepared and diluted appropriately for the enzymatic analyses.
Our previous results have demonstrated that NADH:nitrate reductase from squash cotyledons reduced ferrisiderophores of microbial origin, generally with maximum activities between pH 4 and 5, and showing kinetic constants for these iron chelates in the micromolar range (Castignetti and Smarrelli 1984; Smarrelli and Castignetti 1986). Figure I depicts the pH dependence for the reduction of the pure barley ferriphytosiderophore by squash nitrate reductase. Unlike previously examined ferrisiderophores, maximum activity occurred at pH 6, resembling the pH profile for the ferric citrate-reductase activity of nitrate reductase (Redinbaugh and Campbell 1983), but clearly different
from the pH 7.5 activity maximum with nitrate (Campbell and Smarrelli 1986). In addition, marked ferriphytosiderophore-reductase activity is still found at both pH 7 and 8 which is different from the pH-versus-activity profiles for the previously examined ferrisiderophores (Castignetti and Smarrelli 1986). By varying the ferriphytosiderophore concentration from 0.014 to 0.21 mM while keeping the concentration of N A D H at 0.2 mM during the assay for ferriphytosiderophore-reductase activity, and analyzing the data using direct linear plots, apparent K,, and VmaX values were obtained (Table 1). The K,, of 76 gM is intermediate with respect to other ferrisiderophores studied, while a Vmax of 21 nmol" rain- 1. (mg protein)- 1 for a bluesepharose-purified nitrate reductase preparation was found. The ferriphytosiderophore-reductase activity is markedly inhibited by nitrate-reductasespecific antibodies (data not shown). To examine the competition between nitrate and the ferriphytosiderophore for electrons from nitrate reductase, the inhibition by the ferriphytosiderophore on nitrate reduction, and the inhibition by nitrate on ferriphytosiderophore reduction were examined (Table 2). Although the rate of ferriphytosiderophore reduction is markedly lower than that for nitrate reduction, the ferriphytosiderophore was a better inhibitor of nitrate reduction than nitrate was of ferriphytosiderophore reduction. An explanation may be that each assay was performed under optimal conditions for each respective substrate, and at pH 6 nitrate reacts poorly with nitrate reductase. The reduction of the ferriphytosiderophore from barley by squash cotyledon NADH:nitrate reductase is, to our knowledge, the first demonstration of such an activity by a plant enzyme. With respect to the kinetic parameters of Vm~X and Km (Table 1), the enzyme reduced this ferriphytosiderophore with an efficiency similar to that previously noted for the reduction of microbial ferrisiderophores. The pH optimum of ferriphytosiderophore reduction is somewhat higher than those noted for the microbial ferrisiderophores but pronounced reduction of the ferriphytosiderophore (approx. 40-50%) still occurred at a pH of 4-5. NADH : nitrate reductase thus displays a broad catalytic activity, being able to reduce nitrate (for a review, see Campbell and Smarrelli 1986), six different ferrisiderophores (Castignetti and Smarrelli 1986), ferric citrate (Redinbaugh and Campbell 1983), and at least one ferriphytosiderophore. In addition, the lack of appreciable inhibition of ferriphytosiderophore reduction by nitrate coupled with
J. Smarrelli, Jr. and D. Castignetti: Ferriphytosiderophore reduction by nitrate reductase from squash
565
Table 1. Comparative kinetic constants for selected iron-reductase activities of NADH: nitrate reductase. The ferriphytosiderophore
reductase assay was performed using the enzyme preparation and assay conditions described in the text. Kinetic constants were determined at optimum pH. Substrate
pH optimum
K,, (gM)
Vmax (nmol- min- 1. (mg protein)- 1
Reference
Ferriphytosiderophore Ferrirhodotorulic acid Ferrischizokinen Ferric citrate
6 5 4 6.2
76 170 10 20
21 200 82 -
This study (1) (1) (2)
(1) Smarrelli and Castignetti 1986 (2) Redinbaugh and Campbell 1983
Table 2. Inhibition of nitrate-reductase activity by the ferriphy-
tosiderophore and inhibition of ferriphytosiderophore reductase activity by nitrate
Nitrate reduction (pH 7.5) Nitrate (gM) 50 50 100 100 200 200 1000
Ferriphytosiderophore (gM) 140 210 140 210 140 210 140
Inhibition (%) 79 100 67 78 75 83 64
Ferriphytosiderophore reduction (pH 6.0) Ferriphytosiderophore (gM) 56 56 70 70 210 210
Nitrate (gM) 200 1000 200 1000 200 1000
Inhibition (%) 7 9 10 20 0 0
the marked inhibition of nitrate reduction by the ferriphytosiderophore (Table 2), indicates that ferriphytosiderophore reduction is similar, with respect to electron flow through NADH:nitrate reductase, to microbial ferrisiderophore reduction (Castignetti and Smarrelli 1986). The reduction of the ferriphytosiderophore by nitrate reductase demonstrates that squash has the potential to assimilate the Fe 3 + from a ferriphytosiderophore synthesized by another plant. Since most higher plant nitrate reductases display biochemical characteristics similar to squash nitrate reductase (Campbell and Smarrelli 1986), it may be suggested that other higher-plant nitrate reductases show a similar ferrisiderophore-reductase activity. The diverse catalytic activity of the enzyme with a number of different Fe 3+ substrates may
be of value to squash by permitting it to assimilate iron from a number of different compounds. The mechanism of ferrisiderophore and ferriphytosiderophore transport into the plant roots, however, is unknown and requires elucidation. The iron-assimilation model of R6mheld and Marschner (1986 a, b) includes the possibility that ferrisiderophores enter the cytoplasm of plant cells intact and are subsequently reduced. Once these compounds gain access to the cell, however, it is clear that cytoplasmically located squash cotyledon NADH: nitrate reductase may function to release the Fe 3 + of these compounds such that the metal may participate in plant metabolism. This study was funded in part by grants from the National Science Foundation (DMB 8404243, J.S.), by Loyola University of Chicago Research Stimulation Grants (D.C., J.S.), and by the Biomedical Research Support Grant (1S07 RR07210, D.C). We wish to thank V. Romheld for the gift of the phytosiderophore Cpi-3-hydroxy-mugineic acid, and Drs. Mori (Tokyo), Kawai (Iwate), and Nomoto (Suntory) for its positive identification.
References Becker, J.O., Messens, E., Hedges, R.W. (1985) The influence of agrobactin on the uptake of ferric ion by plants. FEMS Microbiol. Ecol 31, 171-175 Campbell, W.H., Smarrelli, J., Jr. (1978) Purification and kinetics of higher plant NADH: nitrate reductase. Plant Physiol. 61,611-616 Campbell, W.H., Smarrelli, J., Jr. (1986) Nitrate reductase: biochemistry and regulation. In: Biochemical basis of plant breeding, pp. 1-39, Neyra, C.A., ed. CRC Press, Boca Raton, FL., USA Castignetti, D., Smarrelli, J., Jr. (1984) Siderophore reduction catalyzed by higher plant NADH :nitrate reductase. Biochem. Biophys. Res. Commun. 125, 52-58 Castignetti, D., Smarrelli, J., Jr. (1986) Siderophores, the iron nutrition of plants, and nitrate reductase. FEBS Lett. 209, 147-151 Cline, G.R., Reid, C.P., Powell, P.E., Szaniszlo, P.J. (1984) Effects of a hydroxamate siderophore on iron absorption by sunflower and sorghum. Plant Physiol. 76, 36~39
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J. Smarrelli, Jr. and D. Castignetti: Ferriphytosiderophore reduction by nitrate reductase from squash
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