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     

How is Ferredoxin-NADP Reductase Involved in the NADP Photoreduction of Chloroplasts?

NADP photoreduction of chloroplasts was discovered in 1951, and subsequent research was conducted to elucidate the enzymatic mechanisms involved in this reaction. In 1963, ferredoxin-NADP reductase (FNR; EC 1.18.1.2, ferredoxin-NADP oxidoreductase) was isolated and purified to a crystalline form. Because the reaction mechanism of ferredoxin-NADP reducing system was clarified in the isolated enzyme system, it was generally thought that the role of FNR in the NADP photoreduction of chloroplasts had been fully elucidated. However, the results of a reconstitution study using the crystallized FNR and the depleted grana, from which 'built-in' FNR had been eliminated, showed that the NADP photoreducing activity of reconstituted FNR was much lower than the original physiological activity, and as a result, more studies had to be continued. In 1985, a protein factor called 'connectein' was discovered, and it was shown that this new protein binds with two FNR molecules to form an FNR-connectein complex. Then in 1991, the FNR-connectein complex was formed using purified connectein and FNR, and after eliminating 'built-in' FNR, the reconstituted complex was bound to the depleted grana having reduced NADP photoreducing activity. The results showed that NADP photoreducing activity of the reconstituted system was comparable to the original physiological activity. This proved that the FNR-connectein complex, which binds to a specific site on the surface of thylakoid membrane, is functionally responsible for NADP photoreduction in chloroplasts.     

Purification of membrane-bound ferredoxin: NADP+ oxidoreductase and of plastocyanin from a detergent extract of washed thylakoids

A method is described for the isolation and purification of ferredoxin-NADP⁺ oxidoreductase (FNR, E.C. 1.18.1.2) and plastocyanin from spinach thylakoids. FNR is recovered from pools which are loosely and tightly bound to the membrane, with minimal disruption of pigment-protein complexes; yields can thus be higher than from procedures which extract only the loosely bound enzyme.Washed thylakoid membranes were incubated with the dipolar ionic detergent CHAPS (3-(3-cholamidopropyl-dimethylammonio)-1-propane-sulfonate). This provided an extract containing FNR and PC as its principal protein components, which could be rapidly separated from one another by chromatography on an anion-exchange column. FNR was purified to homogeneity (as judged from sodium dodecyl sulfate gel electrophoresis and the ratio between protein and flavin absorption maxima), using chromatography on phosphocellulose followed by batchwise adsorption to, and elution from hydroxylapatite. Plastocyanin was further purified on a Sephadex G-75 molecular sieve column.A typical yield, obtained in 3–4 days from 1 kg of deveined spinach leaves, was 7 mg of pure FNR (a single protein of M_r=37,000) and 3.5 mg of plastocyanin.Abbreviations CHAPS- 3-(3-cholamidopropyl-dimethylammonio)-1-propanesulfonate) - Chl- chlorophyll - FNR- ferredoxin-NADP⁺ oxidoreductase - Mops- 3-(N-morpholino) propanesulfonic acid - PC- plastocyanin - PMSF- phenylmethanesulfonylfluoride - SDS- sodium dodecyl sulfate - SDS-PAGE- sodium dodecyl sulfate polyacrylamide gel electrophoresis - Tricine- N-tris (hydroxymethyl) methylglycine     

Rate-determining steps in penicillopepsin-catalysed reactions

FNR regulates the expression of target genes in response to anaerobiosis. It resembles the catabolite gene activator or cAMP-receptor protein (CRP) except for the presence of an N-terminal cysteine cluster, which may form a redox-sensing iron-binding site. Site-directed mutagenesis has shown that 3 of the 4 cysteine residues in the N-terminal cluster (Cys-20, -23 and -29, but not Cys-16) and the only other cysteine residue (Cys-122), are essential for the normal activation and repression of PNR-dependent promoters. Deletion of residues Pro-3-Arg-9 (inclusive) had no effect, but FNR was inactivated by a frameshift extending through the C-terminal DNA-binding domain. Four independent in vivo mutants contained identical Gly-96→Asp substitutions, which may inactivate FNR by distorting a sharp turn between β-strands in the predicted structure.     

Effects of chemical modification of Anabaena flavodoxin and ferredoxin-NADP+ reductase on the kinetics of interprotein electron transfer reactions.

The influence of chemical modification of arginine residues (using phenylglyoxal) in ferredoxin-NADP+ reductase (FNR), and of carboxyl groups (using glycine ethyl ester) in flavodoxin (Fld), on the kinetics of electron transfer between FNR and Fld, and between ferredoxin (Fd) and FNR, was examined using laser flash photolysis methods. All proteins were obtained from the cyanobacterium Anabaena PCC7119. Reduction by laser-generated 5-deazariboflavin semiquinone of the FAD moiety of phenylglyoxal-modified FNR occurred with a second-order rate constant 2.5-fold smaller than that obtained for reduction of native FNR, indicating either a small degree of steric hindrance of the cofactor, or a decrease in its redox potential, upon chemical modification. In contrast, no changes were found in the kinetics of reduction of the FMN cofactor of Fld modified by glycine ethyl ester as compared with the native protein. The observed rate constants for reoxidation of Fdred (reduced Fd) by FNRox (oxidized FNR) were dramatically decreased (approximately 100-fold) when phenylglyoxal-modified FNR was used. In contrast to the reaction involving the native proteins, no ionic strength effects on kobs values were found. These results, and those obtained upon varying the protein concentration, indicate that the rate constant for complex formation and the attractive electrostatic interaction between the two proteins were greatly diminished by chemical modification of arginine residues of FNR. When phenylglyoxal-modified FNRsq (FNR semiquinone) was used to reduce Fldox (oxidized Fld), similar inhibitory effects were observed. In this case, the limiting first-order rate constant for Fldsq (Fld semiquinone) formation via intracomplex electron transfer from FNRsq was approximately 12-fold smaller than that obtained for the native FNR (600 s-1 vs 7000 s-1). Again, ionic strength effects were diminished. The glycine-ethyl-ester-modified Fld yielded a limiting first-order rate constant for intracomplex electron transfer from FNRsq to Fldox which was approximately 7-fold smaller (1000 s-1) than that obtained with native Fld, and ionic strength effects were again diminished. These results indicate that complex formation can still occur between modified FNR and native Fld, and between native FNR and modified Fld, but that the geometry of these complexes is altered so as to decrease the effectiveness of interprotein electron transfer. The results are discussed in terms of the specific structural features of the proteins involved.     

Mature ferredoxin-NADP reductase with a glutaminyl residue at N-terminus from spinach chloroplasts

When 35%-acetone extract of spinach chloroplasts was separated by SDS-PAGE, ferredoxin-NADP reductase (FNR) appeared as a single band at a molecular mass of 35 kDa. After the polypeptides on the SDS-PAGE plate were electroblotted onto PVDF membrane, the FNR band was cut out and analyzed for N-terminal structure in a gas-phase protein sequencer. Two different FNR peptides were identified: one with glutamine at its N-terminus (Gln-FNR) and the other with -pyroglutamic acid (tFNR) fraction was extracted from chloroplasts with their loosely bound FNR (lFNR) fraction removed in advance. The tFNR fraction contained Gln-FNR only. The Gln-FNR could be highly purified by affinity chromatography using a ferredoxin column. The purified Gln-FNR was digested with arginyl endopeptidase for peptide mapping and partial sequence analysis. Primary structure of Gln-FNR differed from that of lFNR loosely bound FNR - tFNR tightly bound FNR - -pyroglutamic acid at N-terminus     

The complete purification and characterization of three forms of ferredoxin-NADP(+) oxidoreductase from a thermophilic cyanobacterium Synechococcus elongatus

The petH gene, encoding ferredoxin-NADP(+) oxidoreductase (FNR), was isolated from a thermophilic cyanobacterium, Synechococcus elongatus (the same strain as Thermosynechococcus elongatus). The petH gene of S. elongatus was a single copy gene, and the N-terminal region of PetH showed a sequence similarity to the CpcD-phycobilisome linker polypeptide. The amino acid sequence of the catalytic domains of PetH was markedly similar to those from mesophilic cyanobacterial PetH and higher plant FNR. The enzymatically active FNR protein was purified to homogeneity from S. elongatus as three forms corresponding to the 45-kDa form retaining the CpcD-like domain, the 34-kDa form lacking the CpcD-like domain, and the 78-kDa complex with phycocyanin. The FNR in the 78-kDa complex was tolerant to proteolytic cleavage. However, the dissociation of phycocyanin from the 78-kDa complex induced to specific proteolysis between the CpcD-like domain and the FAD-binding domain to give rise to the 34-kDa form of FNR. The enzymatic activity of the 45-kDa form was thermotolerant, but the 45-kDa form readily aggregated under the storage at -30 degrees C. These results suggest that the association with phycocyanin via CpcD-like domain gives remarkable stability to S. elongatus FNR.     

Highly nonproductive complexes with Anabaena ferredoxin at low ionic strength are induced by nonconservative amino acid substitutions at Glu139 in Anabaena ferredoxin:NADP+ reductase

Ferredoxin (Fd) and ferredoxin:NADP(+) reductase (FNR) from Anabaena function in photosynthetic electron transfer (et). The et interaction between the FNR charge-reversal mutant E139K and Fd at 12 mM ionic strength (mu) is extremely impaired relative to the reaction with wt FNR, and the dependency of k(obs) on E139K concentration shows strong upward curvature at protein concentrations > or = 10 microM. However, at values of mu > or = 200 mM, reaction rates approach those of wild-type FNR, and normal saturation kinetics are observed. For the E139Q mutant, which is also significantly impaired in its et interaction with Fd at low FNR concentrations and low mu values, the dependency of k(obs) on E139Q concentration shows a smaller degree of upward curvature at mu = 12 and 100 mM and shows saturation kinetics at higher values of mu. wt FNR and the E139D mutant both show a slight amount of upward curvature at FNR concentrations >30 microM at mu = 12 mM but show the expected saturation kinetics at higher values of mu. These results are explained by a mechanism in which the mutual orientation of the proteins in the complex formed at low ionic strength with the E139K mutant is so far from optimal that it is almost unreactive. At increased E139K concentrations, the added mutant FNR reacts via a collisional interaction with the reduced Fd present in the unreactive complex. The et reactivity of the low ionic strength complexes depends on the particular amino acid substitution, which via electrostatic interactions alters the specific geometry of the interface between the two proteins. The presence of a negative charge at position 139 of FNR allows the most optimal orientations for et at ionic strengths below 200 mM.     

Domain exchange between isoforms of ferredoxin-NADP+ reductase produces a functional enzyme

Two isoforms of ferredoxin-NADP(+) reductase (FNR) exist in higher plants, the leaf (or photosynthetic) and the root (or non-photosynthetic) isoform, which have 48% amino acid sequence identity and display specific structural and functional features. With the aim to gain further insight into the structure-function relationship of this enzyme, we designed two novel chimeric flavoenzymes by swapping the structural domains between the leaf and the root isoforms. Characterization of the chimeras would allow dissection of the contribution of the individual domains to catalysis. The chimera obtained by grafting together the FAD-binding domain of the root-isoform and the NADP-binding domain of the leaf-isoform was inactive when expressed in Escherichia coli. On the other hand, the chimera assembled in the opposite way (leaf FAD-binding domain and root NADP-binding domain) was functional and was produced in the bacterial host to a level threefold higher than that of the parent enzymes. The protein was purified and found to be as stable as the natural isoforms. Limited proteolysis excluded the presence in the chimera of misfolded regions. The affinity of the chimera for ferredoxin I (Fd I) was similar to that of the leaf isoform, although interprotein electron-transfer was partially impaired. As occurs with the root isoform, the chimera bound NADP(+) with high affinity, while spectroscopic evidence suggested that the conformation adopted by the nicotinamide moiety bound to the chimera was similar to that observed in the leaf enzyme. Interestingly, the chimera, by combining favorable features from both parent isoforms, acquired a catalytic efficiency (k(cat)/K(m)), as an NADPH-dependent diaphorase, higher than those of both the root ( approximately 2-fold) and the leaf enzyme ( approximately 5-fold). Thus, molecular breeding between isozymes has improved the catalytic properties of FNR.     

Interaction of homologues of Hsp70 and Cpn60 with ferredoxin-NADP reductase upon its import into chloroplasts

A homologue of the 70-kDa heat-shock protein (Hsp70) was purified from pumpkin chloroplasts. The molecular mass of the purified protein was approximately 75 kDa and its N-terminal amino acid sequence was very similar to those of homologues of Hsp70 from bacterial cells and from the mitochondrial matrix and stroma of pea chloroplasts. The purified homologue of Hsp70 was found in the stroma of chloroplasts. To investigate the role(s) of the homologue of Hsp70 in the chloroplast stroma, we examined the possibility that the homologue of Hsp70 might interact with newly imported proteins to assist in their maturation (for example, in their folding and assembly). Ferredoxin NADP⁺ reductase (FNR) imported into chloroplasts in vitro could be immunoprecipitated with antisera raised against the homologue of Hsp70 from pumpkin chloroplasts and against GroEL from Escherichia coli, which is a bacterial homologue of chaperonin 60 (Cpn60), in an ATP-dependent manner, an indication that newly imported FNR interacts physically with homologues of Hsp70 and Cpn60 in chloroplasts. Time-course analysis of the import of FNR showed that imported FNR interacts transiently with the homologue of Hsp70 and that the association of FNR with the homologue of Hsp70 precedes that with the homologue of Cpn60. These results suggest that homologues of Hsp70 and Cpn60 in chloroplasts might sequentially assist in the maturation of newly imported FNR in an ATP-dependent manner.