The root form of ferredoxin-NADP oxidoreductase is expressed in rice coleoptiles for the assimilation of nitrate

Two ferredoxin-dependent proteins, nitrite reductase and glutamate synthase, play a role in nitrate assimilation during the anaerobic germination of rice (Oryza sativa L.). This paper reports the expression of the root form of ferredoxin-NADP⁺ oxidoreductase (FNR), the protein responsible for providing reduced ferredoxin in rice coleoptiles. Using an antibody against FNR, a protein with the expected molecular mass for root FNR (35 kDa) was recognized by Western blot analysis in extracts from aerobic and anaerobic coleoptiles. The enzyme is synthesized de novo, as shown by immunoprecipitation of the radiolabeled 35-kDa protein from anaerobic seedlings grown in the presence of [³⁵S]methionine. Northern blot analysis with specific probes for root and leaf FNR showed the presence of mRNA for the root form but not for the leaf form, in both aerobic and anaerobic rice coleoptiles. The inductive effect of exogenous nitrate on the expression of FNR is further evidence for the presence of the root type of FNR in rice coleoptiles. The importance of the expression of root FNR during the anaerobic development of rice seedlings is discussed. Received: 7 October 1996 / Accepted: 22 January 1997     

Characterization of cyanobacterial ferredoxin-NADP+ oxidoreductase molecular heterogeneity using chromatofocusing

Chromatofocusing has been used as an analytical tool to check preparations of the enzyme ferredoxin-NADP+ oxidoreductase (EC 1.18.1.2) purified in either the presence or absence of the serine protease inhibitor phenylmethylsulfonyl fluoride from the cyanobacterium Anabaena sp. strain 7119. Only one isoelectric species was found when the crude extract was processed in the presence of the protease inhibitor. Nevertheless, when the inhibitor was omitted, four ionic forms of the enzyme--showing apparent pI's in the range 4.3-4.6--were separated after chromatofocusing of the purified preparation. These forms were found to differ in their specific activities, exhibiting, on the other hand, lower values than the single one obtained in the presence of the protease inhibitor. Analysis by acrylamide gel electrophoresis revealed virtually a single main protein band except for the ionic form of pI 4.39, which was clearly resolved into two active components. Except for the more basic form, which seems to be an homodimer of Mr 80,000, all the protein components were found to be monomeric species in the range Mr 33,000-38,000. These results indicate that the molecular heterogeneity of the ferredoxin-NADP+ oxidoreductase purified from the cyanobacterium Anabaena sp. strain 7119 may result from the activity of a protease present in the whole cell homogenates. On the other hand, these data also point out that chromatofocusing should be considered as an effective technique in the isolation and characterization of the different molecular forms of this enzyme.     

Interaction of NADPH and triazine dyes with ferredoxin-NADP+ oxidoreductase

The ability of NADPH to compete for binding with other ligands of known affinity has been used to provide values for the Kd of NADPH with ferredoxin-NADP+ oxidoreductase (EC 1.18.1.2) (FNR). When the competing ligand is procion red, which binds with a red-shift in spectrum, or Woodwards reagent K(N-ethyl-5-phenylisoxazolium 3'-sulfonate), which covalently modifies an active site carboxyl residue, the calculated Kd for the NADPH-FNR complex is greater than 8 or 0.08 mM, respectively. Because of the feeble (or non-existent) ability of NADPH to dislodge procion red, we propose that this dye and NADPH are not binding at the same site. Procion red must, however, bind additionally at the active site (presumably without spectral perturbation) as it is a competitive inhibitor of NADPH in ferricyanide reduction assays and more crucially proves to be a novel substrate itself, being reduced to a leuco form which can be reoxidised by oxygen. Although a Kd for the NADPH-FNR complex of 0.08 mM is reasonable, we point out the difficulty of interpreting this value and question its physiological significance.     

Subcellular localization of ferredoxin-NADP+ oxidoreductase in phycobilisome retaining oxygenic photosysnthetic organisms

Ferredoxin-NADP⁺ oxidoreductase (FNR) catalyzing the terminal step of the linear photosynthetic electron transport was purified from the cyanobacterium Spirulina platensis and the red alga Cyanidium caldarium. FNR of Spirulina consisted of three domains (CpcD-like domain, FAD-binding domain, and NADP⁺-binding domain) with a molecular mass of 46 kDa and was localized in either phycobilisomes or thylakoid membranes. The membrane-bound FNR with 46 kDa was solublized by NaCl and the solublized FNR had an apparent molecular mass of 90 kDa. FNR of Cyanidium consisted of two domains (FAD-binding domain and NADP⁺-binding domain) with a molecular mass of 33 kDa. In Cyanidium, FNR was found on thylakoid membranes, but there was no FNR on phycobilisomes. The membrane-bound FNR of Cyanidium was not solublized by NaCl, suggesting the enzyme is tightly bound in the membrane. Although both cyanobacteria and red algae are photoautotrophic organisms bearing phycobilisomes as light harvesting complexes, FNR localization and membrane-binding characteristics were different. These results suggest that FNR binding to phycobilisomes is not characteristic for all phycobilisome retaining oxygenic photosynthetic organisms, and that the rhodoplast of red algae had possibly originated from a cyanobacterium ancestor, whose FNR lacked the CpcD-like domain.     

Purification and properties of ferredoxin-NADP+ oxidoreductase from the nitrogen-fixing cyanobacteria Anabaena variabilis

The isolation and characterization of ferredoxin-NADP+ -oxidoreductase from Anabaena variabilis, a nitrogen-fixing, filamentous cyanobacterium, is described. Purified enzyme was obtained in four steps with a 55% yield and 300-fold purification utilizing chromatographic separations on DEAE-cellulose and Cibacron Blue-Sepharose columns. The enzyme is quite similar but not identical to the spinach enzyme as judged by isoelectric focusing, molecular weight determination, and amino acid composition. N-terminal sequence analysis allowed identification of 28 of the first 33 residues. Alignment with the corresponding sequences from spinach and Spirulina FNR preparations was possible. A higher degree of homology was found with the Spirulina enzyme than with the spinach enzyme. Small differences with the spinach enzyme were also shown by absorption and circular dichroism spectral measurements. Oxidation-reduction potential measurements of the bound FAD coenzyme show an Em = -320 mV at pH 7 for the two-electron process. Complex formation between the reductase and ferredoxin from the same organism was observed by difference absorption spectroscopy with a Kd = 4 microM. Similar Kd and difference absorption properties were observed on complex formation with spinach ferredoxin.     

Essential histidyl residues of ferredoxin-NADP+ oxidoreductase revealed by diethyl pyrocarbonate inactivation

Diethyl pyrocarbonate inhibited diaphorase activity of ferredoxin-NADP+ oxidoreductase with a second-order rate constant of 2 mM-1 X min-1 at pH 7.0 and 20 degrees C, showing a concomitant increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives. Activity could be restored by hydroxylamine, and the pH curve of inactivation indicated the involvement of a residue having a pKa of 6.8. Derivatization of tyrosyl residues was also evident, although with no effect on the diaphorase activity. Both NADP+ and NADPH protected the enzyme against inactivation, suggesting that the modification occurred at or near the nucleotide binding domain. The reductase lost all of its diaphorase activity after about two histidine residues had been blocked by the reagent. In differential-labeling experiments with NADP+ as protective agent, it was shown that diaphorase inactivation resulted from blocking of only one histidyl residue per mole of enzyme. Modified reductase did not bind pyridine nucleotides. Modification of the flavoprotein in the presence of NADP+, i.e., with full preservation of diaphorase activity, resulted in a significant impairment of cytochrome c reductase activity, with a second-order rate constant for inactivation of about 0.5 mM-1 X min-1. Reversal by hydroxylamine and spectroscopic data indicated that this second residue was also a histidine. Ferredoxin afforded only slight protection against this inhibition. Conversely, carbethoxylation of the enzyme did not affect complex formation with the ferrosulfoprotein. Redox titration of the modified reductase with NADPH and with reduced ferredoxin suggested that the second histidine might be located in the electron pathway between FAD and ferredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of ferredoxin-NADP+ oxidoreductase. Evidence for the formation of a functional preholoenzyme in the cytoplasmic compartment

Biosynthesis of ferredoxin-NADP+ reductase in higher plants was investigated in relation with the mechanism of formation of the holoenzyme. The putative precursor of the flavoprotein, obtained after cell-free translation on a wheat germ extract primed with poly(A)-rich mRNA, was able to spontaneously bind free FAD, rendering a functional prereductase. The newly synthesized preholoenzyme showed diaphorase and cytochrome c reductase activities, an apparent molecular mass of 45 kDa, and contained FAD as the only flavin cofactor. It gave a positive reaction towards antisera against mature ferredoxin-NADP+ reductase. On the other hand, intracellular distribution of flavin-synthesizing enzymes indicates that FAD formation occurs in the cytoplasm; that is, in the same compartment as the site of reductase synthesis. On the basis of the preceding data a model is presented for the biosynthesis of the enzyme in vivo, involving conjugation of the apoprotein with FAD in the cytoplasm, followed by transport of the preholoreductase across the chloroplast envelope to reach its final destiny in the thylakoid membrane.     

Assembly of plant ferredoxin-NADP+ oxidoreductase in Escherichia coli requires GroE molecular chaperones.

We have recently reported the expression in Escherichia coli of an enzymatically competent ferredoxin-NADP+ oxidoreductase from cloned pea genes encoding either the mature enzyme or its precursor protein (Ceccarelli, E. A., Viale, A. M., Krapp, A. R., and Carrillo, N. (1991) J. Biol. Chem. 266, 14283-14287). Processing to the mature form by bacterial protease(s) and FAD assembly occurred in the bacterial cytosol. Expression of ferredoxin-NADP+ reductase in chaperonin-deficient (groE-) mutants of E. coli resulted in partial reductase assembly at permissive growth temperatures (i.e. 30 degrees C), and in total breakdown of holoenzyme assembly, and accumulation as aggregated inclusion bodies at non-permissive temperatures (i.e. 42 degrees C). Coexpression in these mutants of a cloned groESL operon from the phototrophic bacterium Chromatium vinosum resulted in partial or total recoveries of ferredoxin-NADP+ reductase assembly. The overall results indicate that bacterial chaperonins are required for the productive folding/assembly of eucaryotic ferredoxin-NADP+ reductase expressed in E. coli.     

Induction of ferredoxin-NADP+ oxidoreductase and ferredoxin synthesis in pea root plastids during nitrate assimilation

A 14.5 kDa protein with antigenic components in common with pea leaf ferredoxin was detected on transblots of the soluble proteins of pea root plastids. The amount of this protein was found to increase during the induction of nitrate assimilation in pea roots, reaching a maximal level at 8–12 h. Concurrent with this, a fourfold increase in NADPH-dependent ferredoxin-NADP⁺ oxidoreductase (FNR) activity was observed corresponding to an increase in the amount of this protein detected immunologically on transblots using a leaf FNR antibody. These changes were not observed in plastids from roots of plants grown on ammonia or depleted of nitrogen. It is suggested that in addition to the already well reported induction by nitrate of nitrate reductase and nitrite reductase, there is a co-induction of a plastid located ferredoxin and FNR. Both these proteins are necessary for the transfer of reductant generated by the oxidative pentose phosphate pathway to nitrite reductase.     

Rapid procedure for preparation of macrophage plasma membrane

This report describes a simple and efficient procedure for the isolation of plasma membrane from guinea pig peritoneal macrophages. The use of polycationic beads (Affi-gel 731 beads) facilitates rapid and high-clear separation of plasma membrane within 30 min. The final plasma membrane coated beads fraction has high specific activities of marker enzymes with little contamination with mitochondrial, lysosomal or cytoplasmal markers.     

The function of chloroplast ferredoxin-NADP+ oxidoreductase positively regulates the accumulation of bamboo mosaic virus in Nicotiana benthamiana

A gene down-regulated in Nicotiana benthamiana after bamboo mosaic virus (BaMV) infection had high identity to the nuclear-encoded chloroplast ferredoxin NADP⁺ oxidoreductase gene (NbFNR). NbFNR is a flavoenzyme involved in the photosynthesis electron transport chain, catalysing the conversion of NADP⁺ into NADPH. To investigate whether NbFNR is involved in BaMV infection, we used virus-induced gene silencing to reduce the expression of NbFNR in leaves and protoplasts. After BaMV inoculation, the accumulation of BaMV coat protein and RNA was significantly reduced. The transient expression of NbFNR fused with orange fluorescent protein (OFP) localized in the chloroplasts and elevated the level of BaMV coat protein. These results suggest that NbFNR could play a positive role in regulating BaMV accumulation. Expressing a mutant that failed to translocate to the chloroplast did not assist in BaMV accumulation. Another mutant with a catalytic site mutation could support BaMV accumulation to some extent, but accumulation was significantly lower than that of the wild type. In an in vitro replication assay, the replicase complex with FNR inhibitor, heparin, the RdRp activity was reduced. Furthermore, BaMV replicase was revealed to interact with NbFNR in yeast two-hybrid and co-immunoprecipitation experiments. Overall, these results suggest that NbFNR localized in the chloroplast with functional activity could efficiently assist BaMV accumulation.     

Interaction of ferredoxin-NADP+ oxidoreductase with triazine dyes a rapid purification method by affinity chromatography

The triazine dyes, Cibacron blue F3GA and Procion red HE3B inhibited diaphorase activity of ferredoxin-NADP⁺ reductase, in a competitive manner with respect to NADPH. The K_i values were 1.5 and 0.2 μM, respectively. Binding of the dyes to the flavoprotein, as measured by difference spectroscopy, indicated an apparent stoichiometry of 1 mol dye/mol reductase and was prevented by NADP⁺ or high ionic strength. Chemical modification of a lysine residue and a carboxyl group at the NADP(H) binding site of the enzyme prevented complex formation with Procion red. Procion red showed a higher affinity for ferredoxin-NADP⁺ reductase than Cibacron blue. The K_d values were 1.9 and 5 μM, respectively. Once covalently linked to a Sepharose matrix, the triazine compounds specifically bind the flavoprotein. The interaction is partially electrostatic and partially hydrophobic. The enzyme can be eluted by high concentrations of salt or low concentrations of the corresponding coenzyme. The use of this affinity column allows the rapid purification of ferredoxin-NADP⁺ oxidoreductase from spinach leaves with good yields.     

Isolation and sequencing of an active-site peptide from spinach ferredoxin-NADP+ oxidoreductase after affinity labeling with periodate-oxidized NADP+

Spinach ferredoxin-NADP+ oxidoreductase was inactivated by treatment with 2',3'-dialdehyde NADP+ (periodate-oxidized NADP+), which selectively modifies a lysine residue at the nucleotide-binding domain of the enzyme. The identity of the derivatized residue was ascertained by thin-layer chromatography of the protein hydrolysate. Reductase that had been labeled with periodate-oxidized NADP+ and NaB3H4 was treated with trypsin, and samples of the tryptic digest were subjected to reverse-phase high-performance liquid chromatography. The radioactivity profiles showed modification of one specific peptide. The primary structure of this peptide was found to be Gly-Glu-Lys*-Met-Tyr-Ile-Gln-Thr-Arg, where Lys* represents the derivatized lysine. The sequence obtained corresponds to residues 242-250 in the primary structure of spinach ferredoxin-NADP+ reductase recently reported [Karplus et al. (1984) Biochemistry 23, 6576-6583].     

Studies of the multiple forms of ferredoxin-NADP oxidoreductase from spinach.

Six different forms of ferredoxin-NADP Oxidoreductase from spinach were separated by isoelectric focusing and were found to differ in their specific activities in various assay systems and in their affinity for NADPH. The forms of the enzyme were found to be interconvertible when the enzyme was stored in the cold.     

Interaction of ferredoxin-NADP+ oxidoreductase with triazine dyes. A rapid purification method by affinity chromatography

The triazine dyes, Cibacron blue F3GA and Procion red HE3B inhibited diaphorase activity of ferredoxin-NADP+ reductase, in a competitive manner with respect to NADPH. The Ki values were 1.5 and 0.2 microM, respectively. Binding of the dyes to the flavoprotein, as measured by difference spectroscopy, indicated an apparent stoichiometry of 1 mol dye/mol reductase and was prevented by NADP+ or high ionic strength. Chemical modification of a lysine residue and a carboxyl group at the NADP(H) binding site of the enzyme prevented complex formation with Procion red. Procion red showed a higher affinity for ferredoxin-NADP+ reductase than Cibacron blue. The Kd values were 1.9 and 5 microM, respectively. Once covalently linked to a Sepharose matrix, the triazine compounds specifically bind the flavoprotein. The interaction is partially electrostatic and partially hydrophobic. The enzyme can be eluted by high concentrations of salt or low concentrations of the corresponding coenzyme. The use of this affinity column allows the rapid purification of ferredoxin-NADP+ oxidoreductase from spinach leaves with good yields.     

Expression, assembly, and processing of an active plant ferredoxin-NADP+ oxidoreductase and its precursor protein in Escherichia coli

The flavoprotein ferredoxin-NADP+ reductase (FNR) catalyzes the final step of the photosynthetic electron transport chain, i.e. the reduction of NADP+ by ferredoxin. A cloned FNR cDNA from a pea library (Newman, B., and Gray, J. (1988) Plant Mol. Biol. 10, 511-520) was used to construct plasmids which express the apoenzyme in Escherichia coli. Two recombinant vectors were prepared, one containing the sequence corresponding to the mature enzyme and another including, in addition, the sequence of the transit peptide that directs FNR to the chloroplast. These proteins were expressed as fusion products to the NH2-terminal portion of beta-galactosidase. In both cases, a 35-kDa immunoreactive polypeptide was the major product, suggesting that the proteins were processed in vivo. NH2-terminal sequence determination of the purified recombinant proteins indicate cleavage at positions -1/-2 with respect to the normal processing site in chloroplasts. The processed enzymes showed enzymatic activities and spectral properties that were similar or identical to those of native plant FNR. When a La protease-deficient E. coli strain was used as a host, the expressed FNR precursor was found to be poorly processed, associated to bacterial pellets, and showed no detectable FNR activity. The overall results indicate that acquisition of the native enzyme conformation and assembly of the prosthetic group takes place in the bacterial host, generating an enzyme that is, as far as studied, indistinguishable from plant FNR.     

Rapid preparation in high yield of pure formate dehydrogenase from Pichia angusta

Biomimetic dye ligand chromatography and reversible ionic strength-dependent protein precipitation enabled isolation of formate dehydrogenase from the yeast Pichia angusta in purity of >95%. The enzyme has a specific NAD-linked activity of 5.7 units/mg and was obtained in stable form, yield of 88% and concentration of about 20 mg/ml. © Rapid Science Ltd. 1998     

Immunological studies of the binding protein for chloroplast ferredoxin-NADP+ reductase

Monospecific rabbit antibodies against the ferredoxin-NADP+ reductase binding protein of spinach thylakoids were obtained and characterized. The immunoglobulin G (IgG) fraction gave single precipitation arcs with the purified antigen or with Triton X-100 extracts of thylakoids or the reductase binding protein complex. Antibodies against the flavoprotein behave similarly. Both antibodies agglutinated thylakoids and precipitated the diaphorase activity of a Triton X-100 extract of these membranes. Isolated Fab fragments of the IgG anti-binding protein inhibited NADP+ photoreduction in a time- and Fab concentration-dependent manner. The presence of ferredoxin diminished the rate of inhibition. In the light, the inactivation rate was higher than in dark and this effect was abolished in the presence of uncouplers. These results suggest that the binding protein is protruding from the thylakoids and could be sensing the proton gradient.     

Crystal structure analysis of Bacillus subtilis ferredoxin-NADP(+) oxidoreductase and the structural basis for its substrate selectivity

Bacillus subtilis yumC encodes a novel type of ferredoxin‐NADP⁺ oxidoreductase (FNR) with a primary sequence and oligomeric conformation distinct from those of previously known FNRs. In this study, the crystal structure of B. subtilis FNR (BsFNR) complexed with NADP⁺ has been determined. BsFNR features two distinct binding domains for FAD and NADPH in accordance with its structural similarity to Escherichia coli NADPH‐thioredoxin reductase (TdR) and TdR‐like protein from Thermus thermophilus HB8 (PDB code: 2ZBW). The deduced mode of NADP⁺ binding to the BsFNR molecule is nonproductive in that the nicotinamide and isoalloxazine rings are over 15 Å apart. A unique C‐terminal extension, not found in E. coli TdR but in TdR‐like protein from T. thermophilus HB8, covers the re‐face of the isoalloxazine moiety of FAD. In particular, Tyr50 in the FAD‐binding region and His324 in the C‐terminal extension stack on the si‐ and re‐faces of the isoalloxazine ring of FAD, respectively. Aromatic residues corresponding to Tyr50 and His324 are also found in the plastid‐type FNR superfamily of enzymes, and the residue corresponding to His324 has been reported to be responsible for nucleotide specificity. In contrast to the plastid‐type FNRs, replacement of His324 with Phe or Ser had little effect on the specificity or reactivity of BsFNR with NAD(P)H, whereas replacement of Arg190, which interacts with the 2′‐phosphate of NADP⁺, drastically decreased its affinity toward NADPH. This implies that BsFNR adopts the same nucleotide binding mode as the TdR enzyme family and that aromatic residue on the re‐face of FAD is hardly relevant to the nucleotide selectivity.