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Query: UMLS:C1832526 (PCC)
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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.
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PMID:Complex formation between ferredoxin and ferredoxin-NADP+ reductase from Anabaena PCC 7119: cross-linking studies. 131 39

Ferredoxin-NADP+ reductase from the cyanobacterium Anabaena sp. PCC 7119 was chemically modified by the alpha-dicarbonyl reagent phenylglyoxal. The studies of the inactivation by this compound, which is specific for arginyl residues, of both the diaphorase and NADPH-cytochrome c reductase activities, characteristic of the enzyme, are indicative of the involvement of at least one group of this kind in the binding site of NADP+ and a second one implicated in the interaction with ferredoxin. After specific cleavage of a FNR sample incubated with [7-14C]phenylglyoxal, two major labeled peptides were identified. The peptide which exhibited the higher degree of modification corresponded to residues 208-242. It contained four arginine residues but only two of them were the target of the modification: Arg224 and Arg233. Protection studies with protein substrates and sequence comparison with other reductases allow us to propose that these residues in Anabaena sp. PCC 7119 FNR must be involved in the interaction with the pyridine nucleotide. The second peptide corresponds to residues 75-103 and although it contains three arginine residues, Arg77 is the only one that exhibits the modification. This residue seems to be a key one in the interaction of this reductase with ferredoxin.
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PMID:Identification of arginyl residues involved in the binding of ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 to its substrates. 144 67

The iron-stress-induced genes isiA and isiB have been cloned and sequenced from the marine unicellular cyanobacterium Synechococcus sp. PCC 7002. These genes code for a photosystem II chlorophyll-binding protein and flavodoxin respectively. The genes form a dicistronic operon that is transcriptionally activated under iron-stress conditions to produce an abundant monocistronic message containing isiA and a much less abundant dicistronic message that also contains isiB. The arrangement of these genes, their transcriptional control and the relative abundance of the monocistronic and dicistronic messages produced under iron stress parallels the pattern shown by the freshwater cyanobacterium Synechococcus sp. PCC 7942. The genes for the corresponding proteins found under iron-replete conditions, CP-43 and ferredoxin, have also been cloned and sequenced. Northern blot analysis indicates that both of these genes are constitutively expressed under both iron-stress and iron-replete conditions.
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PMID:An iron stress operon involved in photosynthetic electron transport in the marine cyanobacterium Synechococcus sp. PCC 7002. 152 3

Ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 is chemically modified by pyridoxal 5'-phosphate. The incorporation of 2 +/- 0.3 mol pyridoxal 5'-phosphate/mol ferredoxin-NADP+ reductase inhibited NADPH-cytochrome c reductase activity by up to 95% while 55% of diaphorase activity still remained. Considerable protection against inactivation was afforded by ferredoxin. Chymotryptic cleavage of the modified enzyme was performed, the peptides were separated by high performance liquid chromatography, and the peptides containing pyridoxamine 5'-phosphate were identified by their fluorescence and by their absorbance at 325 nm. Three major labelled peptides were found. Their sequences were comprised of residues 46-54, 231-235 and 289-295. Lys-53 and -294 were the residues which presented the highest degree of modification and seem to be involved in the ferredoxin binding site of ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119.
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PMID:Lysine residues on ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 involved in substrate binding. 154 17

Ferredoxin-glutamate synthase from the unicellular cyanobacterium Synechococcus sp. PCC 6301 has been purified using, as main steps, ethanol fractionation in the presence of high ionic strength, ion-exchange chromatography and ferredoxin-Sepharose affinity chromatography. The overall process yielded an homogeneous enzyme with a specific activity of 30 U/mg protein, after a purification of 2800-fold with a recovery of 43%. The molecular mass of the native protein was 156 kDa, as calculated from its Stokes radius (rS, 4.32 nm) and sedimentation coefficient (S20,w, 8.46 S). The size was also estimated by SDS/PAGE as 160 kDa, indicating that the native protein was a monomer. The enzyme exhibited absorption maxima at 279, 370 and 438 nm and a A279/A438 absorbance ratio of 11. One molecule of FMN, but not FAD, was found/molecule native protein. The addition of dithionite resulted in the loss of the absorption peak at 438 nm, which was restored by the addition of 2-oxoglutarate, thus indicating that the prosthetic group is functional in catalysis. Classical hyperbolic kinetics with substrate inhibition was seen for 2-oxoglutarate. The Km values determined for glutamine and ferredoxin were 0.7 mM and 7 microM, respectively, and the apparent Km for 2-oxoglutarate was estimated to be 1.7 mM. Azaserine and 6-diazo-5-oxo-L-norleucine were potent inhibitors of the activity, while pyridoxal 5-phosphate, known to react with Lys residues, partially inactivated the enzyme. This ferredoxin-dependent glutamate synthase is, as far as we know, the first purified from prokaryotic organisms and resembles its counterpart from chloroplasts, suggesting that cyanobacterial glutamate synthase may have been the ancestor of ferredoxin-glutamate synthase in plants.
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PMID:Purification and characterization of the ferredoxin-glutamate synthase from the unicellular cyanobacterium Synechococcus sp. PCC 6301. 158 84

The unicellular cyanobacterium Synechocystis sp PCC 6803 is capable of synthesizing two different Photosystem-I electron acceptors, ferredoxin and flavodoxin. Under normal growth conditions a [2Fe-2S] ferredoxin was recovered and purified to homogeneity. The complete amino-acid sequence of this protein was established. The isoelectric point (pI = 3.48), midpoint redox potential (Em = -0.412 V) and stability under denaturing conditions were also determined. This ferredoxin exhibits an unusual electrophoretic behavior, resulting in a very low apparent molecular mass between 2 and 3.5 kDa, even in the presence of high concentrations of urea. However, a molecular mass of 10,232 Da (apo-ferredoxin) is calculated from the sequence. Free thiol assays indicate the presence of a disulfide bridge in this protein. A small amount of ferredoxin was also found in another fraction during the purification procedure. The amino-acid sequence and properties of this minor ferredoxin were similar to those of the major ferredoxin. However, its solubility in ammonium sulfate and its reactivity with antibodies directed against spinach ferredoxin were different. Traces of flavodoxin were also recovered from the same fraction. The amount of flavodoxin was dramatically increased under iron-deficient growth conditions. An acidic isoelectric point was measured (pI = 3.76), close to that of ferredoxin. The midpoint redox potentials of flavodoxin are Em1 = -0.433 V and Em2 = -0.238 V at pH 7.8. Sequence comparison based on the 42 N-terminal amino acids indicates that Synechocystis 6803 flavodoxin most likely belongs to the long-chain class, despite an apparent molecular mass of 15 kDa determined by SDS-PAGE.
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PMID:Ferredoxin and flavodoxin from the cyanobacterium Synechocystis sp PCC 6803. 163 77

Methods are described for the simultaneous isolation of ferredoxin-NADP+ reductase, ferredoxin and flavodoxin from large quantities of the cyanobacterium Anabaena PCC 7119 allowing the use of a single batch of cells. The ultraviolet-visible spectra and the extinction coefficients of ferredoxin-NADP+ reductase and ferredoxin were determined. The purification procedure also yields enriched fractions of phycobiliproteins and cytochrome c553.
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PMID:Purification of ferredoxin-NADP+ reductase, flavodoxin and ferredoxin from a single batch of the cyanobacterium Anabaena PCC 7119. 166 99

Flavodoxin from the nitrogen-fixing cyanobacteria Anabaena PCC 7119 forms an electron-transfer complex with ferredoxin--NADP+ reductase (FNR) from the same organism. The complex is mainly governed by electrostatic interactions between side-chain amino groups of the reductase and carboxyl residues of flavodoxin. In order to localize the binding site on flavodoxin, chemical modification of its carboxyl groups has been carried out. Treatment of flavodoxin with a water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), in the presence of a nucleophile, glycine ethyl ester, caused a time-dependent modification of the protein that is responsible for the loss of its ability to participate as electron carrier in the photoreduction of NADP+ by chloroplast membranes, and also in NADPH--cytochrome-c reductase activity, by about 85%. Nevertheless, the ability of flavodoxin to receive electrons from the reducing side of photosystem I was much less affected. The inhibition was enhanced at low pH, suggesting that carboxylic acid groups were the target of chemical modification. Treated flavodoxin failed to form covalent complexes with FNR and the dissociation constant for the non-covalent complex with FNR was fourfold higher. After tryptic digestion of a sample of flavodoxin modified by EDC in the presence of [1-14C]glycine ethyl ester, two major radioactive peptides were isolated. The first protein fragment contained three carboxylic residues (Asp123, Asp126 and Asp129), corresponding to the region where long-chain flavodoxins show an insert compared to short-chain flavodoxins. The second peptide corresponded to a similar region, either in the amino acid sequence or in the three-dimensional structure of the protein and also containing three carboxyl groups (Asp144, Glu145 and Asp146). Four of these carboxyl groups (Asp123, Asp126, Asp144 and Asp146) are highly conserved in all long-chain flavodoxins, suggesting that they could play an essential role in substrate recognition.
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PMID:Identification of specific carboxyl groups on Anabaena PCC 7119 flavodoxin which are involved in the interaction with ferredoxin-NADP+ reductase. 173 24

The molecular structure of the oxidized form of the [2Fe-2S] ferredoxin isolated from the cyanobacterium Anabaena species strain PCC 7120 has been determined by X-ray diffraction analysis to a nominal resolution of 2.5 A and refined to a crystallographic R factor of 18.7%. Crystals used in this investigation belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 37.42 A, b = 38.12 A, and c = 147.12 A and two molecules in the asymmetric unit. The three-dimensional structure of this ferredoxin was solved by a method that combined X-ray data from one isomorphous heavy-atom derivative with noncrystallographic symmetry averaging and solvent flattening. As in other plant-type [2Fe-2S] ferredoxins, the iron-sulfur cluster is located toward the outer edge of the molecule, and the irons are tetrahedrally coordinated by both inorganic sulfurs and sulfurs provided by protein cysteine residues. The main secondary structural elements include four strands of beta-pleated sheet and three alpha-helical regions.
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PMID:Crystallization and structure determination to 2.5-A resolution of the oxidized [2Fe-2S] ferredoxin isolated from Anabaena 7120. 190 76

Structural and chemical properties of a flavodoxin from Anabaena PCC 7119 are described. The first 36 residues of the amino-terminal amino acid sequence have been determined and show extensive homology with flavodoxins isolated from other sources. Anabaena flavodoxin exhibits a net negative change (-3) in the helix-1 segment as found with other cyanobacterial flavodoxins Synechococcus 6301 (Anacystis nidulans) and Nostoc MAC, but in contrast to the net positive charge found in this region in the case of flavodoxins isolated from nitrogen-fixing bacteria (Azotobacter and Klebsiella). The FMN cofactor can be reversibly resolved from the apoprotein by trichloroacetic acid treatment. Apoflavodoxin, thus prepared, binds FMN with a Kd value of 0.1 nM and binds riboflavin with a decreased affinity (Kd = 5 microM) at pH 7.2. The apoprotein is stable in dilute solutions at pH values around 7 but readily denatures at pH 8 as judged from loss in flavin-binding ability and by ultraviolet circular dichroism spectroscopy. Oxidation-reduction potential studies at pH values of 7 and 8 show OX/SQ couples of -195 mV and -255 mV, respectively, and show SQ/HQ couples of -390 mV and -418 mV, respectively. From these data, the binding constant for the FMN semiquinone is calculated to be approx. 5-fold tighter and the binding of the FMN hydroquinone is approx. 10(5)-fold weaker than that of the oxidized FMN to the apoprotein. Anabaena flavodoxin functions as an effective mediator of electron transfer from ferredoxin-NADP(+)-reductase to cytochrome c with a turnover number [4.5-5) x 10(3) min-1); a values similar to that determined for Anabaena ferredoxin. The flavodoxin binds tightly to the reductase with Kd values of 6.4 and 8.5 microM at pH values of 7.0 and 8.0, respectively.
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PMID:Structural and chemical properties of a flavodoxin from Anabaena PCC 7119. 211 31


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