Spirulina ferredoxin-NADP+ reductase. Further characterization with an improved preparation

The preparation procedure for Spirulina ferredoxin-NADP+ reductase (ferredoxin: NADP+ oxidoreductase, EC 1.18.1.2, FNR) was improved by adding protease inhibitors, phenylmethylsulfonylfluoride (PMSF) and EDTA, through the whole process of preparation and by introducing an affinity chromatography step on Blue Sepharose CL-6B. The addition of the inhibitors largely prevented the formation of the minor component (FNR I), and the affinity gel chromatography simplified the preparation process, shortening the exposure period of FNR to proteolysis. However, complete removal of the heterogeneity of FNR found at the amino (N)-terminal region was not achieved even by applying the new method. The affinity chromatography on the Blue Sepharose gel was also effective in purifying spinach FNR. The affinity of this gel for Spirulina FNR was compared with that for the enzyme derived from spinach leaves. The spinach enzyme had a higher affinity than the Spirulina one. Both enzymes showed the highest affinities to Blue Sepharose at 20--30 mM NaCl concentration. The N-terminal sequence analysis revealed that there was 4 forms, which were probably modifications produced by exopeptidase action during the preparation, or even in the living cells. The longest component gave the N-terminal sequence Ala-Lys-Thr-Asp-Ile-Pro-Val-Asn-Ile-Tyr-. The others lacked amino acids successively one by one from the N-terminus. In contrast, the carboxyl(C)-terminal residues of all 4 FNR forms were tyrosine. The probable C-terminal sequence was predicted to be -Trp-His-Val-Gln-Thr-Tyr based on a study of a cyanogen bromide peptide.     

Novel forms of ferredoxin and ferredoxin-NADP reductase from spinach roots

Ferredoxin and the enzyme catalyzing its reduction by NADPH, ferredoxin-NADP reductase (ferredoxin-NADP+ oxidoreductase or FNR), were found to be present in roots of spinach (Spinacia oleracea). Localization experiments with endosperm of germinating castor beans (Ricinus communis), a classical nonphotosynthetic tissue for cell fractionation studies, confirmed that ferredoxin and FNR are localized in the plastid fraction. Both proteins were purified from spinach roots and found to resemble their leaf counterparts in activity, spectral properties, and complex formation, but to differ in amino acid composition and amino terminal sequence. The results indicate that the primary structures of the FNR and ferredoxin of spinach roots differ from that of the corresponding leaf proteins. Together with earlier findings, the present results provide evidence that nonphotosynthetic plastids, including those of roots, are capable of reducing ferredoxin with heterotrophically generated NADPH.     

NADPH-cytochrome P-450 oxidoreductase: flavin mononucleotide and flavin adenine dinucleotide domains evolved from different flavoproteins

The FMN-binding domain of NADPH-cytochrome P-450 oxidoreductase, residues 77-228, is homologous with bacterial flavodoxins, while the FAD-binding domain, residues 267-678, shows a high degree of similarity to two FAD-containing proteins, ferredoxin-NADP+ reductase and NADH-cytochrome b5 reductase. Comparison of these proteins to glutathione reductase, a flavoprotein whose three-dimensional structure is known, has permitted tentative identification of FAD- and cofactor-binding residues in these proteins. The remarkable conservation of sequence between NADPH-cytochrome P-450 oxidoreductase and ferredoxin-NADP+ reductase, coupled with the homology of the FMN-binding domain of the oxidoreductase with the bacterial flavodoxins, implies that NADPH-cytochrome P-450 oxidoreductase arose as a result of fusion of the ancestral genes for these two functionally linked flavoproteins.     

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.     

Chromatographic resolution of chicken phosvitin. Multiple macromolecular species in a classic vitellogenin-derived phosphoprotein.

The 622-residue amino acid sequence of the hydrophilic domain in the porcine NADPH-cytochrome P-450 reductase (EC 1.6.2.4) is reported. The structural data required to complete the sequences published previously [Vogel, Kaiser, Witt & Lumper (1985) Biol. Chem. Hoppe-Seyler 366, 577-587] and to establish the primary structure of the porcine hydrophilic domain have been obtained by sequencing proteolytic subfragments derived from CNBr fragments and by characterizing the overlapping S-[14C]methylmethionine-containing peptides isolated from tryptic digests of the [14C]methyl-labelled hydrophilic domain. The hydrophilic domain displays 91.8% positional identity with that of the corresponding domain in the rat NADPH-cytochrome P-450 reductase. The region Val528-Ser678 in the NADPH-cytochrome P-450 reductase shows a significant homology to the sequence Ile165-Tyr314 in the spinach ferredoxin-NADP+ oxidoreductase. A model for the secondary structure of the hydrophilic domain has been derived by computer-assisted analysis of the amino acid sequence. Cys472 and Cys566 are protected against chemical modification in the NADP+ complex of the NADPH-cytochrome P-450 reductase.     

Comparative Studies on Ferredoxin-NADP+ Oxidoreductase Isoenzymes Derived from Different Organs by Antibodies Specific for the Radish Root- and Leaf-Enzymes

Determination of the prosthetic group and titration of sulfhydryl group of ferredoxin-NADP+ oxidoreductase (FNR) from roots of radish (Raphanus sativus var acanthiformis cv Miyashige) confirmed its similarity to leaf-FNR. Antisera directed against radish root-FNR and leaf-FNR distinguished the enzyme forms from roots and leaves of radish as well as other flowering plants. The FNR isoenzymes showed organ-specific distributions. In horsetail (Equisetum arvense L.) and cultured liverwort cells (Marchantia polymorpha), at least two FNR isoenzymes were distinguished by the antisera. FNR from Chlorella vulgaris reacted only with the anti-root-FNR antiserum. FNR from a cyanobacterium, Spirulina spp., failed to react with either antiserum.     

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].     

The sulfhydryl groups of ferredoxin-NADP+ oxidoreductases: is a disulfide bond really present?

The sulfhydryl groups of three ferredoxin-NADP+ oxidoreductases [EC 1.18.1.2] (FNR) two from blue-green algae, Spirulina sp. and Synechococcus sp., respectively, and one from spinach, were analyzed by carboxymethylation and titration with sulfhydryl reagents under different conditions. Five sulfhydryl groups in Spirulina and spinach enzymes and four in Synechococcus enzyme were found to be in reduced forms. There was no disulfide bond in any FNR tested. The results in the present experiment contradict those reported by Karplus et al. [Biochemistry 23, 6576-6583 (1984)].     

Isolation and characterization of two ferredoxin-NADP+ reductases from Spirulina platensis.

Two ferredoxin-NADP+ reductases (FNRs I and II) [EC 1.6.7.1] were purified from a blue-green alga, Spirulina platensis, by (NH4)2SO4 fractionation, gel filtration on Sephadex G-100 and DEAE-Sephadex A-50 chromatography. FNRs I and II were both FAD-containing enzymes with molecular weights of 33,000, and could photochemically reduce NADP+ to the same extent in the presence of S. platensis ferredoxin, using FNR-depleted membrane fragments of S. platensis. They had similar physical and enzymatic properties, except for chemical properties such as the amino (N)-terminal sequences and the patterns of their peptide maps. The significance of the presence of two FNRs in S. platensis as as of the multiple forms found in other organisms is discussed.     

The ferredoxin-NADP+ oxidoreductase-binding protein is not the 17-kDa component of the cytochrome b/f complex

The complex between ferredoxin-NADP+ oxidoreductase and its proposed membrane-binding protein (Vallejos, R. H., Ceccarelli, E., and Chan, R. (1984) J. Biol. Chem. 259, 8048-8051) was isolated from spinach thylakoids and compared with isolated cytochrome b/f complex containing associated ferredoxin NADP+ oxidoreductase (Clark, R. D., and Hind, G. (1983) J. Biol. Chem. 258, 10348-10354). There was no immunological cross-reactivity between the 17.5-kDa binding protein and an antiserum raised against the 17-kDa polypeptide of the cytochrome complex. Association of ferredoxin-NADP+ oxidoreductase with the binding protein or with the thylakoid membrane gave an allotopic shift in the pH profile of diaphorase activity, as compared to the free enzyme. This effect was not seen in enzyme associated with the cytochrome b/f complex. Identification of the 17.5-kDa binding protein as the 17-kDa component of the cytochrome b/f complex is ruled out by these results.     

Molecular heterogeneity of ferredoxin-NADP+ reductase from spinach leaves

Highly purified ferredoxin-NADP+ reductase from spinach leaves showed at least eight different protein bands in the electrofocused gel. All of them were catalytically active and were adsorbed on a ferredoxin-Sepharose 4B affinity column. The N-terminal amino acid sequence of the main component species was analyzed by the automatic Edman degradation method. It was found that when the reductase was stored at 4 degrees C, new protein bands appeared in isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoreses, but the appearance of the bands was suppressed by the addition of a protease inhibitor, diisopropyl fluorophosphate. This indicates that the molecular heterogeneity of the reductase may result from the digestion with a protease present in spinach leaves.     

Small changes in the activity of chloroplastic NADP(+)-dependent ferredoxin oxidoreductase lead to impaired plant growth and restrict photosynthetic activity of transgenic tobacco plants

A ferredoxin-NADP+ oxidoreductase (FNR) cDNA from tobacco (Nicotiana tabacum cv. Samsun) was cloned and sequenced. Comparison of the deduced amino acid sequence revealed high identity to FNR proteins from Capsicum annuum, Pisum sativum, Spinacia oleracea and Vicia faba. Transgenic tobacco plants were generated that constitutively express the FNR cDNA in reverse orientation between the CaMV 35S promoter and the polyadenylation signal of the octopine synthase gene. Plants expressing the FNR antisense gene showed lower levels of FNR mRNA and protein accumulation, which was paralleled by a decrease in FNR activity. As a consequence, NADPH levels declined whereas NADP+ levels increased, leading to an unaltered NADP(H) pool. Growth rates, chlorophyll content and net CO2 uptake rates at high and low irradiances were strongly reduced in FNR antisense tobacco plants. These changes were accompanied by an over-reduced state of P700 as estimated by absorption changes at 820 nm. FNR control coefficients determined for the photosynthetic rate at saturating (C(R) = 0.94) and limiting (C(R) = 0.70) light conditions revealed a prominent role of this reductase in the regulation of photosynthesis.     

Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase

NADPH-sulfite reductase flavoprotein (SiR-FP) was purified from a Salmonella typhimurium cysG strain that does not synthesize the hemoprotein component of the sulfite reductase holoenzyme. cysJ, which codes for SiR-FP, was cloned from S. typhimurium LT7 and Escherichia coli B, and both genes were sequenced. Physicochemical analyses and deduced amino acid sequences indicate that SiR-FP is an octamer of identical 66-kDa peptides and contains 4 FAD and 4 FMN per octamer. Potentiometric titrations of SiR holoenzyme, SiR-FP, and FMN-depleted SiR-FP yielded the following redox potentials for the prosthetic groups at pH 7.7: E'1 (FMNH./FMN) = -152 mV; E'2 (FMNH2/FMNH.) = -327 mV; E'3 (FADH./FAD) = -382 mV; E'4 (FADH2/FADH.) = -322 mV. Microcoulometric titration of SiR-FP at 25 degrees C yielded data which were in full agreement with these potentials. Spectroscopic and catalytic studies of native SiR-FP and of SiR-FP depleted of FMN support the following electron flow sequence: NADPH----FAD----FMN. FMN can then contribute electrons to the hemoprotein component of sulfite reductase, as well as to cytochrome c and various diaphorase acceptors. The FMN is postulated to cycle between the FMNH2 and FMNH. oxidation states during catalysis; in this sense SiR-FP shares a catalytic mechanism with NADPH-cytochrome P-450 oxidoreductase. SiR-FP domains involved in binding FMN, FAD, and NADPH are proposed from amino acid sequence homologies with Desulfovibrio vulgaris flavodoxin (Dubourdieu, M., and Fox, J.L. (1977) J. Biol. Chem. 252, 1453-1463) and spinach ferredoxin-NADP+ oxidoreductase (Karplus, P.A., Walsh, K.A., and Herriott, J. R. (1984) Biochemistry 23, 6576-6583). Comparison of the deduced amino acid sequences of SiR-FP and NADPH-cytochrome P-450 oxidoreductase (Porter, T. D., and Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U. S.A. 82, 973-977) also showed identities that suggest these two proteins are descended from a common precursor, which contained binding regions for both FMN and FAD.     

Ferredoxin:NADP oxidoreductase of Cyanophora paradoxa: purification, partial characterization, and N-terminal amino acid sequence.

The ferredoxin:NADP+ oxidoreductase of the protist Cyanophora paradoxa, as a descendant of a former symbiotic consortium, an important model organism in view of the Endosymbiosis Theory, is the first enzyme purified from a formerly original endocytobiont (cyanelle) that is found to be encoded in the nucleus of the host. This cyanoplast enzyme was isolated by FPLC (19% yield) and characterized with respect to the uv-vis spectrum, pH optimum (pH 9), molecular mass of 34 kDa, and an N-terminal amino acid sequence (24 residues). The enzyme shows, as known from other organisms, molecular heterogeneity. The N-terminus of a further ferredoxin:NADP+ oxidoreductase polypeptide represents a shorter sequence missing the first four amino acids of the mature enzyme.     

Regulation of the NADH and NADPH-ferredoxin oxidoreductases in clostridia of the butyric group.

NADH and NADPH-ferredoxin oxidoreductases have been studied in Clostridium acetobutylicum, Cl. tyrobutyricum and Cl. pasteurianum. The study of the distribution and regulation of these enzymatic activities in well-defined culture conditions, reveals that the essential function of NADPH-ferredoxin oxidoreductase is to produce NADPH, while NADH-ferredoxin oxidoreductase can, depending on cellular conditions, produce or oxidize NADH. When these Clostridia use glycolysis, regulation of the NADH-ferredoxin oxidoreductase by acetyl-CoA (obligatory activator of NADH-ferroxin reductase activity) and by NADH (competitive inhibitor of ferredoxin-NAD+ reductase activity) allow the enzymes to function correlatively with glyceraldehyde-3-phosphate dehydrogenase and thus control the levels of NAD+ and NADH in the cell. In Cl. tyrobutyricum and Cl. pasteurianum, the ferredoxin-NADP+ reductase activities are regulated by NAD+ and NADH in accordance with the intracellular concentrations of these coenzymes. In Cl. tyrobutyricum growing on pyruvate/acetate, NADH and NADPH-ferredoxin reductase activities cannot be detected; only the ferredoxin-NAD+ and ferredoxin-NADP+ reductase activities are found. In this Clostridium, regulation of the ferredoxin-NADP+ reductase activity is the same whether it is grown on glucose or pyruvate. Contrary to this, the ferredoxin-NAD+ reductase activity undergoes a drastic change, since NADH no longer controls the enzymatic activity. In this case regulation is no longer necessary, since glyceraldehyde-3-phosphate dehydrogenase does not function.     

Quantitative structure activity relationships for the electron transfer reactions of Anabaena PCC 7119 ferredoxin-NADP+ oxidoreductase with nitrobenzene and nitrobenzimidazolone derivatives: mechanistic implications.

The steady state single electron reduction of polynitroaromatics by ferredoxin-NADP+ oxidoreductase (EC 1.18.1.2) from cyanobacterium Anabaena PCC 7119 has been studied and quantitative structure activity relationships are described. The solubility of the polynitroaromatics as well as their reactivity towards ferredoxin-NADP+ oxidoreductase are markedly higher than those for previously studied mononitroaromatics and this enabled the independent measurement of the kinetic parameters-k(cat) and Km. Interestingly, the natural logarithm of the bimolecular rate constant, k(cat)/Km, and also the natural logarithm of k(cat) correlate with the calculated energy of the lowest unoccupied molecular orbital of the polynitroaromatic substrates. The minimal kinetic model in line with these quantitative structure activity relationships is a ping-pong mechanism which includes substrate binding equilibria in the second half reaction.     

Optimal conditions for post-translational uptake of proteins by isolated chloroplasts. In vitro synthesis and transport of plastocyanin, ferredoxin-NADP+ oxidoreductase, and fructose-1,6-bisphosphatase

Many polypeptides translated in the cytosol enter the chloroplast where they assemble into macromolecular complexes. The transport of these polypeptides into the plastid can be examined in vitro by mixing isolated chloroplasts with pea poly(A) RNA translation products. Following optimization of both translation in the wheat germ system and the conditions during in vitro uptake, we observe the post-translational transport of over 100 polypeptides; many remain in the soluble phase of the organelle while others integrate into the thylakoid membranes. Most products transported in vitro co-migrate with in vivo products on sodium dodecyl sulfate-polyacrylamide gels. Furthermore, with the improved conditions, we demonstrate the transport of plastocyanin, ferredoxin-NADP+ oxidoreductase, and fructose-1,6-bisphosphatase into isolated plastids. While we have not been able to detect any cell-free translation product that is immunologically related to fructose-1,6-bisphosphatase, both plastocyanin and ferredoxin-NADP+ oxidoreductase are synthesized as precursors in vitro. These precursors are imported into the organelle where they are processed to the size of their mature counterparts. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of the precursor to plastocyanin is 15,000 larger than the mature product and the precursor to ferredoxin-NADP+ oxidoreductase is 8,000 larger than the mature product.     

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.     

Chloroplast NADH dehydrogenase from Pisum sativum: characterization of its activity and cloning of ndhK gene

The pea chloroplast ndhK gene coding for a component of a NADH-plastoquinone oxidoreductase has been cloned and sequenced. This gene codes for a polypeptide of 227 amino acids and a predicted molecular mass of 25,495 Da which belongs to the family of the 20 kDa PSST subunit of the bovine mitochondrial complex I. A fragment of this gene has been overexpressed in Escherichia coli, and antibodies against the expressed polypeptide recognize a protein of the predicted molecular mass from pea thylakoid membranes. This polypeptide is a component of a protein complex with NADH dehydrogenase activity and is not associated with ferredoxin-NADP+ reductase.