Characterization of a ferredoxin:NADP+ oxidoreductase from a nonphotosynthetic plant tissue

A flavoprotein with properties similar to those of ferredoxin:NADP+ oxidoreductases found in the leaves of higher plants has been purified to apparent homogeneity from bean sprouts, a nonphotosynthetic plant tissue. The absorbance and circular dichroism spectra of the bean sprout protein are similar to those of spinach leaf ferredoxin:NADP+ oxidoreductase and an antibody raised against the spinach enzyme recognized the bean sprout enzyme. The bean sprout enzyme catalyzed ferredoxin-dependent electron transfer from NADPH to equine cytochrome c at a high rate but, unlike the spinach enzyme, exhibited little NADPH to 2,6-dichlorophenol indophenol diaphorase activity. The bean sprout enzyme forms a 1:1 electrostatically stabilized complex with ferredoxins isolated from either bean sprouts or spinach leaves.     

Properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense NADPH-dependent enzyme and its isolated alpha subunit

The properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803 were determined by means of kinetic and spectroscopic approaches in comparison to those exhibited by the bacterial NADPH-dependent enzyme form. The ferredoxin-dependent enzyme was found to be similar to the bacterial glutamate synthase alpha subunit with respect to cofactor content (one FMN cofactor and one [3Fe-4S] cluster per enzyme subunit), overall absorbance properties, and reactivity of the FMN N(5) position with sulfite, as expected from the similar primary structure of ferredoxin-dependent glutamate synthase and of the bacterial NADPH-dependent glutamate synthase alpha subunit. The ferredoxin- and NADPH-dependent enzymes were found to differ with respect to the apparent midpoint potential values of the FMN cofactor and of the [3Fe-4S] cluster, which are less negative in the ferredoxin-dependent enzyme form. This feature is, at least in part, responsible for the efficient oxidation of L-glutamate catalyzed by this enzyme form, but not by the bacterial NADPH-dependent counterpart. At variance with earlier reports on ferredoxin-dependent glutamate synthase, in the Synechocystis enzyme the [3Fe-4S] cluster is not equipotential with the flavin cofactor. The present studies also demonstrated that binding of reduced ferredoxin to ferredoxin-dependent glutamate synthase is essential in order to activate reaction steps such as glutamine binding, hydrolysis, or ammonia transfer from the glutamine amidotransferase site to the glutamate synthase site of the enzyme. Thus, ferredoxin-dependent glutamate synthase seems to control and coordinate catalytic activities taking place at its subsites by regulating the reactions of the glutamine amidotransferase site. Association with reduced ferredoxin appears to be necessary, but not sufficient, to trigger the required activating conformational changes.     

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.     

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     

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.     

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.     

Glutamate synthase isoforms in rice: immunological studies of enzymes in green leaf, etiolated leaf, and root tissues

Rabbit antiserum was raised against ferredoxin-dependent glutamate synthase (EC 1.4.7.1) purified from green leaves of Oryza sativa L. cv Delta. Ferredoxin-dependent glutamate synthase, detected in green leaf, etiolated leaf, and root tissues cross-reacted completely with the antiferredoxin glutamate synthase immunoglobulin G. In contrast, the immunoglobulin G did not cross-react with NADH-dependent (EC 1.4.1.14) and NADPH-dependent (EC 1.4.1.13) glutamate synthases found in nonphotosynthetic etiolated leaf and root tissues. In addition, ferredoxin-dependent glutamate synthase was separated and distinguished by its affinity to ferredoxin from NAD(P)H-dependent glutamate synthase on ferredoxin-Sepharose affinity chromatography. Based on the immunological studies, it is suggested that ferredoxin-dependent glutamate synthases in green leaf and etiolated leaf tissues are closely related proteins; in contrast, ferredoxin-dependent glutamate synthase in root tissue is a distinct protein from the leaf enzymes.     

Chemical modification studies of tryptophan,arginine and lysine residues in maize chloroplast ferredoxin:sulfite oxidoreductase

The ferredoxin-dependent sulfite reductase from maize was treated, in separate experiments, with three different covalent modifiers of specific amino acid side chains. Treatment with the tryptophan-modifying reagent, N-bromosuccinimide (NBS), resulted in a loss of enzymatic activity with both the physiological donor for the enzyme, reduced ferredoxin, and with reduced methyl viologen, a non-physiological electron donor. Formation of the 1:1 ferredoxin/sulfite reductase complex prior to treating the enzyme with NBS completely protected the enzyme against the loss of both activities. Neither the secondary structure, nor the oxidation-reduction midpoint potential (E _m) values of the siroheme and [4Fe–4S] cluster prosthetic groups of sulfite reductase, nor the binding affinity of the enzyme for ferredoxin were affected by NBS treatment. Treatment of sulfite reductase with the lysine-modifying reagent, N-acetylsuccinimide, inhibited the ferredoxin-linked activity of the enzyme without inhibiting the methyl viologen-linked activity. Complex formation with ferredoxin protects the enzyme against the inhibition of ferredoxin-linked activity produced by treatment with N-acetylsuccinimide. Treatment of sulfite reductase with N-acetylsuccinimide also decreased the binding affinity of the enzyme for ferredoxin. Treatment of sulfite reductase with the arginine-modifying reagent, phenylglyoxal, inhibited both the ferredoxin-linked and methyl viologen-linked activities of the enzyme but had a significantly greater effect on the ferredoxin-dependent activity than on the reduced methyl viologen-linked activity. The effects of these three inhibitory treatments are consistent with a possible role for a tryptophan residue the catalytic mechanism of sulfite reductase and for lysine and arginine residues at the ferredoxin-binding site of the enzyme.     

The evolution of glutamate synthase

DNA coding for the ferredoxin-dependent glutamate synthase (EC1.4.7.1) of spinach chloroplasts has been cloned and sequenced. It consists of 5015 bp and starts with the codon for the N-terminal cysteine of the mature protein. Ferredoxin-dependent glutamate synthase is one of the key enzymes in the early stages of ammonia assimilation in plants, algae and cyanobacteria. In addition to the ferredoxin-dependent enzyme, there are two other forms of glutamate synthase, one of which uses NADH as the electron donor and a second that uses NADPH. Although all three forms catalyze the reductive transamidation of the amido nitrogen from glutamine to 2-oxoglutarate to form two molecules of glutamate, ferredoxin-dependent glutamate synthases differ from the NADH and NADPH-dependent forms in subunit composition and amino acid sequence. The recent availability of sequence data for glutamate synthases from spinach and from two archael species has produced a clearer and more detailed picture of the evolution of this key enzyme in nitrogen metabolism and the origins of the two subunit/domain structure of the enzyme.     

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.     

Monoclonal antibody studies of ferredoxin:NADP+ oxidoreductase.

Eleven independent monoclonal antibodies, all IgG's, have been raised against the ferredoxin:NADP+ oxidoreductase of spinach leaves. All 11 monoclonal antibodies were able to produce substantial inhibition of the NADPH to 2,6-dichlorophenol indophenol (DCPIP) diaphorase activity of the enzyme, but none of the antibodies produced any significant inhibition of electron flow from NADPH to ferredoxin catalyzed by the enzyme. Spectral perturbation assays were used to demonstrate that antibody interaction with NADP+ reductase did not interfere significantly with the binding of either ferredoxin or NADP+ to the enzyme. Ultrafiltration binding assays were used to confirm that the monoclonal antibodies did not interfere with complex formation between ferredoxin and the enzyme. These results have been interpreted in terms of the likely presence of one or more highly antigenic epitopes at the site where the nonphysiological electron acceptor, DCPIP, binds to the enzyme. Furthermore, the results suggest that the site where DCPIP is reduced differs from both of the two separate sites at which the two physiological substrates, ferredoxin and NADP+/NADPH, are bound.     

Evidence for the presence of a [2Fe-2S] ferredoxin in bean sprouts

An iron-sulfur protein with properties similar to those of ferredoxins found in the leaves of higher plants has been isolated from bean sprouts--a non-photosynthetic plant tissue. The bean sprout protein has a molecular mass of 12.5 kDa and appears to contain a single [2Fe-2S] cluster. The absorbance and circular dichroism spectra of the bean sprout protein resemble those of spinach leaf ferredoxin and the bean sprout protein can replace spinach ferredoxin as an electron donor for NADP+ reduction, nitrite reduction and thioredoxin reduction by spinach leaf enzymes. Although the reduced bean sprout protein (Em = -440 mV) is a slightly stronger reductant than spinach ferredoxin and appears to be less acidic than spinach ferredoxin, the two proteins are similar enough so that the bean sprout protein is recognized by an antibody raised against spinach ferredoxin.     

The location of the mobile electron carrier ferredoxin in vascular plant photosystem I

In this study, we present the location of the ferredoxin-binding site in photosystem I from spinach. Image analysis of negatively stained two-dimensional crystals indicates that the addition of ferredoxin and chemical cross-linkers do not significantly alter the unit cell parameters (for untreated photosystem I, a = 26.4 nm, b = 27.6 nm, and gamma = 90 degrees, space group p22(1)2(1) and for ferredoxin cross-linked photosystem I, a = 26.2 nm, b = 27.2 nm, and gamma = 90 degrees, space group p22(1)2(1)). Fourier difference analysis reveals that ferredoxin is bound on top of the stromal ridge principally interacting with the extrinsic subunits PsaC and PsaE. This location would be accessible to the stroma, thereby promoting efficient electron transfer away from photosystem I. This observation is significantly different from that of the ferredoxin binding site proposed for cyanobacteria. A model for the binding of ferredoxin in vascular plants is proposed and is discussed relative to observations in cyanobacteria.     

Antigenic similarities between ferredoxin-dependent nitrite reductase and glutamate synthase fromChlamydomonas reinhardtii

Polyclonal antisera were prepared against ferredoxin-nitrite reductase (EC 1.7.7.1) and ferredoxin-glutamate synthase (glutamate synthase (ferredoxin); EC 1.4.7.1) from the green algaChlamydomonas reinhardtii. The anti-glutamate synthase antibodies recognized both glutamate synthase and nitrite reductase, but inhibited only the ferredoxin-linked activity of the latter enzyme and not the activity dependent on methyl viologen. Analogously, the anti-nitrite reductase antibodies recognized glutamate synthase and nitrite reductase but the first enzyme was only poorly inhibited. Free ferredoxin protected the nitrite reductase against its inactivation by anti-glutamate synthase antibodies. These results indicate that the ferredoxin-dependent glutamate synthase and nitrite reductase from this alga share common antigenic determinants, and that these are located at the ferrodoxin-binding domains.     

Modification of arginyl residues in ferredoxin-NADP+ reductase from spinach leaves.

Reaction of spinach leaves ferredoxin-NADP+ reductase (NADPH:ferredoxin oxidoreductase, EC 1.6.7.1) with alpha-dicarbonyl compounds results in a biphasic loss of activity. The rapid phase yields modified enzyme with about 30% of the original activity, but no change in the Km for NADPH. Only partial protection against inactivation is provided by NADP+, NADPH and their analogs, whereas ferredoxin affords complete protection. The reductase inactivated to 30% of original activity shows a loss of about two arginyl residues, whereas only one residue is lost in the NADP+-protected enzymes. The data suggest that the integrity of at least two arginyl residues are requested for maximal activity of ferredoxin-NADP+ reductase: one residue being located near the NADP+-binding site, the other presumably situated in the ferredoxin-binding domain.     

Structural and mechanistic insight into the ferredoxin-mediated two-electron reduction of bilins

PEB (phycoerythrobilin) is one of the major open-chain tetrapyrrole molecules found in cyanobacterial light-harvesting phycobiliproteins. In these organisms, two enzymes of the ferredoxin-dependent bilin reductase family work in tandem to reduce BV (biliverdin IXα) to PEB. In contrast, a single cyanophage-encoded enzyme of the same family has been identified to catalyse the identical reaction. Using UV-visible and EPR spectroscopy we investigated the two individual cyanobacterial enzymes PebA [15,16-DHBV (dihydrobiliverdin):ferredoxin oxidoreductase] and PebB (PEB:ferredoxin oxidoreductase) showing that the two subsequent reactions catalysed by the phage enzyme PebS (PEB synthase) are clearly dissected in the cyanobacterial versions. Although a highly conserved aspartate residue is critical for both reductions, a second conserved aspartate residue is only involved in the A-ring reduction of the tetrapyrrole in PebB and PebS. The crystal structure of PebA from Synechococcus sp. WH8020 in complex with its substrate BV at a 1.55 ? (1 ?=0.1 nm) resolution revealed further insight into the understanding of enzyme evolution and function. Based on the structure it becomes obvious that in addition to the importance of certain catalytic residues, the shape of the active site and consequently the binding of the substrate highly determines the catalytic properties.     

Ferredoxin Cross-Links to a 22 kD Subunit of Photosystem I

We have used a cross-linking approach to study the interaction of ferredoxin (Fd) with photosystem I (PSI). The cross-linking reagent N-ethyl-3-(3-dimethylaminopropyl) carbodiimide was found to cross-link spinach Fd to a 22 kilodalton subunit of PSI in both isolated spinach (Spinacia oleracea) PSI complexes and spinach thylakoid membranes. The product had an apparent molecular weight of 38 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was identified as a cross-linked product using specific antibodies to Fd and the 22 kilodalton subunit. In both a native PSI complex (200 Chl/P700) and a PSI core complex (100 Chl/P700), a second cross-linked product at 36 kilodaltons was seen. The latter cross-reacted with an antibody to Fd but did not cross-react with antibodies directed against the 24.3, 22, 19, 17.3 or 8.5 kilodalton, or psaC subunits of PSI. Its composition remains to be determined. In thylakoids only the 38 kilodalton product was observed along with a cross-linked complex of Fd and Fd:NADP⁺ reductase.     

Molecular cloning and characterization of complementary DNA encoding for ferredoxin-dependent glutamate synthase in maize leaf

The sequence of ferredoxin-dependent glutamate synthase (EC 1.4.7.1) mRNA from maize has been determined. Complementary DNAs were isolated from a cDNA library of light-induced leaf poly(A)+ RNA constructed in an expression vector. An open reading frame beginning at an ATG codon at nucleotide 328 of the longest cDNA (5617-bases long) encoded 1616 amino acid residues. The amino terminus of the purified mature enzyme coincided with the cysteine residue at position 98 of the predicted sequence. This enzyme is homologous with the large subunit of Escherichia coli NADPH-dependent glutamate synthase having about 42% identical residues between the two proteins. The enzyme also contains a short region similar to a potential FMN-binding region of yeast flavocytochrome b2. The cDNA hybridizes to an RNA band about 5.5 kilobases whose steady-state level is markedly increased upon illumination of etiolated maize seedlings. Analysis of genomic DNA indicates the presence of a single-copy gene for ferredoxin glutamate synthase in maize.     

Complex formation between ferredoxin and ferredoxin-NADP+ reductase from Anabaena PCC 7119: cross-linking studies.

Ferredoxin-NADP+ reductase and ferredoxin from the cyanobacterium Anabaena PCC 7119 have been covalently cross-linked by incubation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The covalent adduct, which shows a molecular mass consistent with a 1:1 stoichiometry of the two proteins, maintains nearly 60% of the NADPH-cytochrome c reductase activity of the enzyme saturated with ferredoxin and this value is considerably higher than when equimolar amounts of both proteins are assayed. No ternary complexes with Anabaena flavodoxin or horse heart cytochrome c were formed, suggesting that the binding site on the enzyme is the same for ferredoxin and flavodoxin and that ferredoxin-NADP+ reductase and cytochrome c bind at a common site on ferredoxin. In the noncovalent complex, titrated at pH 7, the oxidation-reduction potential of ferredoxin becomes 15 mV more negative and that of ferredoxin-NADP+ reductase 27 mV more positive compared to the proteins alone. When covalently linked, the midpoint potential of the enzyme has a value similar to that in the noncovalent complex, while the ferredoxin potential is 20 mV more positive compared to ferredoxin alone. The changes in redox potentials have been used to estimate the dissociation constants for the interaction of the different redox forms of the proteins, based on the value of 1.21 microM calculated for the oxidized noncovalent complex.     

A new assay for ferredoxin-dependent glutamine oxoglutarate amino transferase (glutamate synthase) using electrolytically reduced methyl viologen

A new, quick assay is described for ferredoxin-dependent glutamate synthase from leaves, using electrolytically reduced methyl viologen. Activity, which is stimulated fivefold by ferredoxin, is measured spectrophotometrically as oxidation of methyl viologen. The assay does not require routine estimation of glutamate. Two molecules of glutamate were shown to be formed during the oxidation of two molecules of methyl viologen. Activities correspond to those previously reported for leaf homogenates.