Immunological studies of ferredoxin-nitrite reductases and ferredoxin-glutamate synthases from photosynthetic organisms

Polyclonal antiserum specific for ferredoxin-nitrite reductase (EC 1.7.7.1) from the green alga Chlamydomonas reinhardii recognized the nitrite reductase from other green algae, but did not cross-react with the corresponding enzyme from different cyanobacteria or higher plant leaves. An analogous situation was also found for ferredoxin-glutamate synthase (EC 1.4.7.1), using its specific antiserum. Besides, the antibodies raised against C. reinhardii ferredoxin-glutamate synthase were able to inactivate the ferredoxin-dependent activity of nitrite reductase from green algae.These results suggest that there exist similar domains in ferredoxin-nitrite reductases and ferredoxin-glutamate synthases from green algae. In addition, both types of enzymes share common antigenic determinants, probably located at the ferredoxin-binding domain. In spite of their physicochemical resemblances, no apparent antigenic correlation exists between the corresponding enzymes from green algae and those from higher plant leaves or cyanobacteria.Abbreviations Fd ferredoxin - GOGAT glutamate synthase - MV⁺ reduced methyl viologen (radical cation) - NiR nitrite reductase - PMSF phenylmethylsulphonyl fluoride - SDS sodium dodecyl sulfate     

Purification and Properties of Nitrite Reductase from Spinach Leaves

Nitrite reductase (EC 1.6.6.4) has been purified 730-fold from spinach leaves. The enzyme catalyzes the reduction of nitrite to ammonia, with the use of reduced form of methyl viologen and ferredoxin. A stoichiometry of one molecule of nitrite reduced per molecule of ammonia formed has been found. KCN at 2.5×10^-4 m inhibited nitrite reductase activity almost completely. Purified enzyme was almost homogeneous by disk electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 61,000 from gel filtration. Nitrite reductase, in the oxidized form, has absorption maxima at 276, 388 and 573 mμ. Both methyl viologen and ferredoxin linked nitrite reductase activities of the enzyme were inactivated on exposure to low ionic strength.     

Purification and characterization of ferredoxin–nitrite reductase from the eukaryotic microalga Monoraphidium braunii

Nitrite reductase (NiR; EC 1.7.7.1) from the eukaryotic microalga Monoraphidium braunii has been purified to electrophoretic homogeneity, resulting in a preparation with a specific activity of 3574 nkat mg^–1 and a purification factor of 2553-fold. The enzyme is a single polypeptide chain with a molecular mass of 63 kDa, and absorption maxima at 690, 573, 385 and 280 nm. Kinetic data indicate K_m values of 0.7 mM for nitrite, 10 μM for M. braunii ferredoxin (Fd) and 0.26 mM for methyl viologen. The enzyme showed an optimum pH of 7.5 in 100 mM Tris–HCl buffer and an optimum temperature of 40 °C. NiR activity was inhibited by the sulfhydryl reagent p-hydroxymercuribenzoate and the chelating reagent KCN. Immunological studies revealed the presence of common antigenic determinants, at the Fd-binding domain, in NiR and glutamate synthase (EC 1.4.7.1) from M. braunii.     

Nitrate assimilation during the anaerobic germination of rice: expression of ferredoxin-dependent glutamate synthase

Ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) is the last enzyme involved in the pathway of nitrate assimilation in higher plants. This paper describes the synthesis and expression of the enzyme in anaerobic coleoptiles of rice (Oryza sativa L.) and its regulation by exogenous nitrate. The activity of Fd-GOGAT was strongly inhibited by cycloheximide between 4 and 9 d of anaerobic germination. The addition of nitrate slightly increased, in the first 5 h, the specific activity of Fd-GOGAT as well as the amount of a 160-kDa protein specifically immunoprecipitated with anti-Fd-GOGAT serum. Northern blot analysis, performed with a specific riboprobe, showed the presence of mRNA of the expected size and the inductive effect of nitrate. The role of Fd-GOGAT is discussed in relation to the anaerobic assimilation of nitrate by rice coleoptiles.Abbreviations CHX cycloheximide - Fd ferredoxin - GOGAT glutamate synthase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase The authors wish to thank Dr. J. Turner (Rothamsted Experimental Station, Harpenden, UK) for providing Fd-GOGAT antibody and Dr. H. Sakakibara (Nagoya University, Nagoya, Japan) for Fd-GOGAT clone. This research was supported by the National Research Council of Italy, special project RAISA, sub-projekt N. 2, paper N. 2174.     

Influence of light on the ferredoxin-dependent glutamate synthase in maize leaves

The ferredoxin (Fd)-dependent glutamate synthase (EC 1.4.7.1) and NADH-dependent glutamate synthase (EC 1.4.1.14) activities are carried out by two immunochemically distinct enzyme proteins in maize leaves (Zea mays W64A and W182E). Continuous irradiation of etiolated tissue at 75 micro einsteins per square meter per second for 24 hours resulted in a 3-fold increase on a fresh weight basis in the activity of the Fd-dependent glutamate synthase and a slight decrease in the activity of the NADH-dependent enzyme. There was also a significant increase of the Fd-glutamate synthase protein during greening of etiolated tissue.     

Further Characterization of Ferredoxin Nitrite Reductase and the Relationship between the Enzyme and Methyl Viologen-dependent Nitrite Reductase

Ferredoxin-dependent nitrite reductase of spinach has been further characterized and the relationship between this enzyme and methyl viologen-dependent nitrite reductase studied.

Purified ferredoxin nitrite reductase, having a molecular weight of 86,000, showed 2.5 times higher ferredoxin-dependent activity than methyl viologen-linked activity. Besides 4 mol of labile sulfide the enzyme contained about 2 mol of siroheme per mol. When dithionite, methyl viologen and nitrite were added, ESR signals of a heme nitrosyl complex at g = 2.14, 2.07 and 2.02 were observed. Moreover, hyperfine splitting of the signal due to ¹⁴N nuclear spin was also observed at 2.033, 2.023 and 2.013. The sole addition of hydroxylamine to the ferric enzyme also caused the same but much less intense signals with the hyperfine splitting.

On treatment of the ferredoxin nitrite reductase (native enzyme) with DEAE-Sephadex A-50 chromatography, a modified nitrite reductase having a molecular weight of 61,000 and a protein fraction having an apparent molecular weight of 24,000 were separated. The modified enzyme contained about one mol of siroheme and 4 mol of labile sulfide per mol and showed essentially the same heme ESR signals as the native enzyme. Contrary to the native enzyme, this modified enzyme accepted electrons more efficiently from methyl viologen than ferredoxin and the reduction of nitrite to ammonia catalyzed by the modified enzyme was not stoichiometric. The observed nitrite to ammonia ratio was 1 to less than 0.6. Cyanide at concentrations between 0.02 to 0.2 mm inhibited the activity of the native enzyme almost completely but the modified enzyme was inhibited only partially.

From the results obtained, it is suggested that the native ferredoxin-linked nitrite reductase consists of two components (or subunits) and removal of the light component results in formation of a modified enzyme with increased relative affinity to methyl viologen.     

The localisation of enzymes of nitrogen assimilation in maize leaves and their activities during greening

The activities of nitrate reductase (EC1.6.6.1), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC6.3.1.2), glutamate synthase (EC1.4.7.1) and NAD(P)H-dependent glutamate dehydrogenase (EC 1.4.1.3) were investigated in mesophyll and bundle sheath cells of maize leaves (Zea mays L.). Whereas nitrate and nitrite reductase appear to be restricted to the mesophyll and GDH to the bundle sheath, glutamine synthetase and glutamate synthase are active in both tissues.During the greening process, the activities of nitrate and nitrite reductase increased markedly, but glutamine synthetase, glutamate synthase and glutamate dehydrogenase changed little.Abbreviations BDH British Drug Houses - EDTA Ethylene diamine tetra-acetic acid - GDH Glutamate dehydrogenase - NADH Nicotinamide-adenine dinucleotide reduced form - NADPH Nicotnamide-adenine dinucleotide phosphate reduced form - PMSF Phenylmethyl sulphonyl fluoride     

Purification and some properties of the nitrite reductase from the cyanobacterium Phormidium laminosum

Assimilatory ferredoxin-nitrite reductase (EC 1.7.7.1, ammonia: ferredoxin oxidoreductase) has been purified 5300-fold with a specific activity of 625 units/mg protein from the filamentous non-heterocystous cyanobacterium Phormidium laminosum. The enzyme was soluble and consisted of a single polypeptidic chain of 54 kDa. It catalyzed the reduction of nitrite to ammonia using ferredoxin or flavodoxin as electron donor. Methyl and benzyl viologens were also effective as electron donors but neither flavins nor NAD(P)H were. The apparent Michaelis constants for nitrite, ferredoxin and methyl viologen were 40, 22 and 215 microM, respectively. Nitrite reductase activity was inhibited effectively by cyanide and thiol reagents. The enzyme exhibited absorption maxima at 281, 391 (Soret), 570 (alpha) and 695 nm, with epsilon 391 of 4.3 x 10(4) M-1 cm-1, and an absorbance ratio A281/A391 of 1.95, suggesting the presence of siroheme as prosthetic group. These results show that this enzyme is similar to those of eukaryotic organisms.     

Investigation of the site of synthesis of chIcoroplastic enzymes of nitrogen metabolism by the use of heat-treated 70S ribosomedeficient rye leaves

The activities of the enzymes nitrate reductase (EC 1.6.6.1), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC 6.3.1.2), glutamate synthase (GOGAT; EC 1.4.7.1), glutamate-oxaloacetate aminotransferase (EC 2.6.1.1), and glutamate dehydrogenase (EC 1.4.1.2) were compared in light-grown green or etiolated leaves of rye seedlings ( Secale cereale L. cv. Halo) raised at 22°C, and in the bleached 70S ribosome-deficient leaves of rye seedlings grown at a non-permissive high temperature of 32°C. Under normal permissive growth conditions the activities of most of the enzymes were higher in light-grown, than in dark-grown, leaves. All enzyme activities assayed were also observed in the heat-treated 70S ribosome-deficient leaves. Glutamine synthetase, glutamate synthase, and glutamate-oxaloacetate aminotransferase occurred in purified ribosome-deficient plastids separated on sucrose gradients. For glutamate-oxaloacetate aminotransferase four multiple forms were separated by polyacrylamide gel electrophoresis from leaf extracts. The chloroplastic form of this enzyme was also present in 70S ribosome-deficient leaves. It is concluded that the chloroplast-localized enzymes nitrite reductase, glutamine synthetase, glutamate synthase and glutamate-oxaloacetate aminotransferase, or their chloroplast-specific isoenzyme forms, are synthesized on cytoplasmic 80S ribosomes.     

Intracellular distribution of enzymes associated with nitrogen assimilation in roots

The cellular distribution of enzymes involved in nitrogen assimilation: nitrate reductase (EC 1.6.6.2), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) has been studied in the roots of five plants: maize (Zea mays L. hybrid W 64A x W 182E), rice (Oryza sativa L. cv. Delta), bean (Phaseolus vulgaris L. cv. Contender), pea (Pisum sativum L. cv. Demi-nain), and barley (Hordeum vulgare L.). Initially, cell organelles were separated from soluble proteins by differential centrifugation. Cell organelles were also subjected to sucrose density gradients. The results obtained by these two methods indicate that nitrite reductase and glutamate synthase are localized in plastids, nitrate reductase and glutamine synthetase are present in the cytosol, and glutamate dehydrogenase is a mitochondrial enzyme.     

Localization of Nitrogen-Assimilating Enzymes in the Chloroplast of Chlamydomonas reinhardtii

The specific activities of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase were determined in intact protoplasts and intact chloroplasts from Chlamydomonas reinhardtii. After correction for contamination, the data were used to calculate the portion of each enzyme in the algal chloroplast. The chloroplast of C. reinhardtii contained all enzyme activities for nitrogen assimilation, except nitrate reductase, which could not be detected in this organelle. Glutamate synthase (NADH- and ferredoxin-dependent) and glutamate dehydrogenase were located exclusively in the chloroplast, while for nitrite reductase and glutamine synthetase an extraplastidic activity of about 20 and 60%, respectively, was measured. Cells grown on ammonium, instead of nitrate as nitrogen source, had a higher total cellular activity of the NADH-dependent glutamate synthase (+95%) and glutamate dehydrogenase (+33%) but less activity of glutamine synthetase (−10%). No activity of nitrate reductase could be detected in ammonium-grown cells. The distribution of nitrogen-assimilating enzymes among the chloroplast and the rest of the cell did not differ significantly between nitrate-grown and ammonium-grown cells. Only the plastidic portion of the glutamine synthetase increased to about 80% in cells grown on ammonium (compared to about 40% in cells grown on nitrate).     

Occurrence of ferredoxin-dependent glutamate synthase in plant cell fraction of soybean root nodules (Glycine max)

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) and NADH-dependent glutamate synthase (EC 1.4.1.14) have been identified in the plant cells of soybean nodules. Ferredoxin-dependent glutamate synthase is 2-fold more active than NADH-dependent enzyme in vitro. Ferredoxin-dependent glutamate synthase cross-reacts with IgG against ferredoxin-dependent glutamate synthase of rice green leaves, whereas NADH-dependent glutamate synthase does not recognize the IgG, indicating that there are two distinct enzyme proteins. Ferredoxin-dependent glutamate synthase is composed of polypeptide chain(s) of 165 kDa and has a high affinity to spinach leaf ferredoxin as an electron carrier.     

The interaction of ferredoxin and glutamate synthase: cross-linking and immunological studies

The water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) serves as an effective reagent for cross-linking spinach leaf ferredoxin and the ferredoxin-dependent spinach leaf enzyme, glutamate synthase. The cross-linked complex was functional in the absence of added ferredoxin, suggesting that ferredoxin is cross-linked to glutamate synthase at the physiological binding site on the enzyme for this iron-sulfur protein electron donor. The ferredoxin:glutamate synthase stoichiometry of the cross-linked complex was estimated to be 2:1. The absorbance spectrum of the oxidized, cross-linked complex was very similar to that of an electrostatically stabilized, noncovalent, 2:1 complex of the two proteins. An antibody raised against spinach NADP+ reductase, which recognizes a ferredoxin-binding site on glutamate synthase, does not recognize the cross-linked ferredoxin-glutamate synthase complex. This implies that the ferredoxin-binding sites on the two enzymes are structurally similar enough so that an antibody raised against one of these ferredoxin-dependent enzymes recognizes an epitope at the ferredoxin-binding site of the second enzyme. Cross-linking of ferredoxin to its binding site on glutamate synthase renders this epitope inaccessible to the antibody.     

Complex formation between ferredoxin and Synechococcus ferredoxin: nitrate oxidoreductase

The ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 has been shown to form a high-affinity complex with ferredoxin at low ionic strength. This complex, detected by changes in both the absorbance and circular dichroism (CD) spectra, did not form at high ionic strength. When reduced ferredoxin served as the electron donor for the reduction of nitrate to nitrite, the activity of the enzyme declined markedly as the ionic strength increased. In contrast, the activity of the enzyme with reduced methyl viologen (a non-physiological electron donor) was independent of ionic strength. These results suggest that an electrostatically stabilized complex between Synechococcus nitrate reductase and ferredoxin plays an important role in the mechanism of nitrate reduction catalyzed by this enzyme. Treatment of Synechococcus nitrate reductase with either an arginine-modifying reagent or a lysine-modifying reagent inhibited the ferredoxin-dependent activity of the enzyme but did not affect the methyl viologen-dependent activity. Treatment with these reagents also resulted in a large decrease in the affinity of the enzyme for ferredoxin. Formation of a nitrate reductase complex with ferredoxin prior to treatment with either reagent protected the enzyme against loss of ferredoxin-dependent activity. These results suggest that lysine and arginine residues are present at the ferredoxin-binding site of Synechococcus nitrate reductase. Results of experiments using site-specific, charge reversal variants of the ferredoxin from the cyanobacterium Anabaena sp. PCC 7119 as an electron donor to nitrate reductase were consistent with a role for negatively charged residues on ferredoxin in the interaction with Synechococcus nitrate reductase.     

Spinach ferredoxin-nitrite reductase: characterization of catalytic activity and interaction of the enzyme with substrates

The steady-state kinetic parameters of the enzymatic reduction of nitrite by spinach ferredoxin-nitrite reductase [EC 1.7.7.1] were measured under anaerobic conditions. The maximum velocity of ferredoxin-linked activity was essentially the same as for the methyl viologen-linked activity of the enzyme. The initial velocity patterns of the oxidation of reduced ferredoxin suggested a sequential reaction scheme by which nitrite and reduced ferredoxin bind to the free enzyme. The binding of nitrite and ferredoxin to the enzyme was also investigated by different spectra produced by the complex formed by the enzyme with the substrates. Nitrite and ferredoxin each gave a 1: 1 complex with the enzyme. The dissociation constant (Kd) of the enzyme-nitrite complex agreed well with the Km value for the ferredoxin-linked activity, whereas the Kd of the enzyme-ferredoxin complex differed from the Km value for the enzyme activity. It was concluded that our preparation of spinach ferredoxin-nitrite reductase differs from both the complex (Mr = 85,000) and the modified (Mr = 61,000) forms of the enzyme reported by Hirasawa et al. [J. Biol. Chem. 262, 12428-12433 (1987)].     

Different Characteristics of the Two Glutamate Synthases in the Green Leaves of Lycopersicon esculentum

The two glutamate synthases, NAD(P)H- and ferredoxin-dependent, from the green leaves of tomato plants (Lycopersicon esculentum L. cv Hellfrucht frühstamm) differed in their chemical properties and catalytic behavior. Gel filtration of NAD(P)H enzyme gave an apparent molecular size of 158 kilodalton, whereas the ferredoxin enzyme molecular size was 141 kilodalton. Arrhenius plots of the activities of the two enzymes showed that the NAD(P)H enzyme had two activation energies; 109.6 and 70.5 kilojoule per mole; the transition temperature was 22°C. The ferredoxin enzyme however, had only one activation energy; 56.1 kilojoule per mole. The respective catalytic activity pH optima for the NAD(P)H- dependent and the ferredoxin dependent enzymes were around 7.3 and 7.8. In experiments to evaluate the effects of modulators aspartate enhanced the NAD(P)H-linked activity, with a K_a value of 0.25 millimolar, but strongly inhibited that of the ferredoxin-dependent glutamate synthase with a K_i of 0.1 millimolar. 3-Phosphoserine was another inhibitor of the ferredoxin dependent enzyme with a K_i value of 4.9 millimolar. 3-Phosphoglyceric acid was a potent inhibitor of the ferredoxin-dependent form, but hardly affected the NAD(P)H-dependent enzyme. The results are discussed and interpreted to propose different specific functions that these activities may have within the leaf tissue cell.     

Protein modulase appears to be a complex of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin

Protein modulase and ferredoxin/thioredoxin reductase are soluble proteins that have been suggested to catalyze the light-dependent modulation of enzyme activity in the stromal compartment of the chloroplast. Protein modulase is active in vitro without additional ferredoxin and thioredoxin, whereas ferredoxin/thioredoxin reductase requires additional ferredoxin and thioredoxin. We hypothesize that protein modulase is a complex protein composed of ferredoxin/thioredoxin reductase, ferredoxin, and thioredoxin. In reconstituted chloroplast systems, antiserum directed against ferredoxin, at concentrations sufficient to inhibit the photoreduction of NADP, had no effect on light modulation. Antiserum directed against thioredoxin gave variable results: one batch of polyclonal antibodies inhibited light modulation, another was stimulatory, and another was without effect. These results suggest that the ferredoxin and thioredoxin active in light modulation are not free in solution. Furthermore, molecular sieve chromatography of stromal proteins results in the elution of four species that catalyze light modulation. Based on whether or not ferredoxin and/or thioredoxin must be added for activity, these four species have been tentatively identified as protein modulase, a complex of ferredoxin/thioredoxin reductase and ferredoxin, a complex of ferredoxin/thioredoxin reductase and thioredoxin, and ferredoxin/thioredoxin reductase. That is, the four correspond to all the possible combinations of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin. We suggest that buffer ionic strength affects the interactions among these proteins and in part determines the fate of the protein modulase complex in vitro.     

Studies on ammonium-assimilating enzymes of Platymonas striata Butcher (Prasinophyceae)

Platymonas striata Butcher displays significant levels of glutamate synthase (GS) (EC 2.6.1.53) and glutamine synthetase (GOGAT) (EC 6.3.1.2.), but very low levels of glutamate dehydrogenase (GDH) (EC 1.4.1.4). This suggests that the GS/GOGAT pathway is important for nitrogen assimilation. The in vitro rates of enzyme activity can however only account for about 10% of the in vivo rates of nitrogen assimilation. Nitrogen-starvation reduced GS activity to undetectable levels. On nitrate or ammonium ion refeeding the cellular GS activity was rapidly restored, and reached levels of 56% and 91% greater than the unstarved values 24h after refeeding nitrate or ammonium respectively.Abbreviations NAR nitrate reductase - NIR nitrate reductase     

The effect of low osmotic potential on nitrite reduction in intact spinach chloroplasts

The effect of water stress (reduced osmotic potential) on photosynthetic nitrite reduction was investigated using intact, isolated spinach (Spinacia oleracea) chloroplasts. Nitrite-dependent O₂ evolution was inhibited 39% at −29.5 bars osmotic potential, relative to a control at −11 bars. In the presence of an uncoupler of photophosphorylation this inhibition was not seen. Reduced osmotic potential did not inhibit either methyl viologen reduction or photosynthetic O₂ reduction. These results indicate that an inhibition of electron transport to ferredoxin cannot account for the observed inhibition of nitrite-dependent O₂ evolution. In vitro assay of nitrite reductase activity showed that the interaction of the enzyme with nitrite was not affected by changes in the concentrations of ions or molecules that might be caused by water stress conditions. These results indicate that the most likely site for the effect of water stress on chloroplastic nitrite reduction is the interaction of ferredoxin with nitrite reductase.     

Glutamate synthase in rice roots. Studies on the electron donor specificity

Rice root glutamate synthase activity was assayed with various reducing systems. Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) and pyridine nucleotide-dependent glutamate synthase (NADH, EC 1.4.1.14; or NADPH, EC 1.4.1.13) exhibited a strict specificity for the electron donor. The ferredoxin-dependent glutamate synthase from rice roots could accept electrons from photoreduced ferredoxin in an illuminated reconstituted spinach chloroplast system. Thioredoxin, a potent electron carrier, was not able to provide either ferredoxin-dependent or pyridine nucleotide-dependent glutamate synthase with electrons as no glutamate formation was detected in the presence of reduced thioredoxin f or m.