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PubMed2004

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.

Anabaena↗
PubMed1990

Reciprocal post-translational regulation of renal 1 alpha- and 24-hydroxylases of 25-hydroxyvitamin D3 by phosphorylation of ferredoxin. mRNA-directed cell-free synthesis and immunoisolation of ferredoxin.

We have used a cell-free rabbit reticulocyte translational system programmed with polyadenylated [poly(A)+] RNA prepared from chick kidney tissue to study the synthesis of nascent ferredoxin, a class of iron-sulphur-containing proteins functional in the renal mitochondrial 1 alpha- and 24-hydroxylases of 25-hydroxyvitamin D3. The synthesis of ferredoxin was monitored by determining [35S]methionine incorporation into ferredoxin and quantified by SDS/PAGE and autoradiography after immunoprecipitation from the total translation products. Compared with normal controls, vitamin D deprivation caused a significant increase in the net synthesis of nascent ferredoxin with an Mr of 12,000-13,000. [3H]Orotate incorporation as uridine into kidney poly(A)+ RNA was stimulated by aminophylline, a potent inducer of 25-hydroxyvitamin D3 24-hydroxylase; however, the amount of nascent ferredoxin synthesis was the same as in normal controls. Also, partially purified chick kidney mitochondrial cyclic AMP-stimulated protein kinase catalysed the phosphorylation of ferredoxin in vitro. The catalytic activity of the ferredoxin in 1 alpha- and 24-hydroxylations of 25-hydroxyvitamin D3 in reconstituted systems consisting of cytochrome P-450 and ferredoxin reductase was altered with ferredoxin phosphorylation. The phosphorylation caused inhibition of the 1 alpha-hydroxylase activity while at the same time it stimulated the 24-hydroxylase. Authentic 1 alpha,25- and 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 were used as standards to monitor the separation of the enzymic products by h.p.l.c. using methanol/water (4:1, v/v) as solvent. These results indicate that, in the absence of vitamin D or its metabolites in the deficient state, the synthesis of ferredoxin necessary for the 1 alpha-hydroxylase is accentuated, whereas the stimulation of the 24-hydroxylase requires the phosphorylation of existing ferredoxin without a net gain in its synthesis. This would suggest a post-translational regulation of the 1 alpha- and 24-hydroxylases. A model delineating the various aspects of this study is presented.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

Inactivation of clostridial ferredoxin and pyruvate-ferredoxin oxidoreductase by sodium nitrite.

Clostridial ferredoxin and pyruvate-ferredoxin oxidoreductase activity was investigated after in vitro or in vivo treatment with sodium nitrite. In vitro treatment of commercially available Clostridium pasteurianum ferredoxin with sodium nitrite inhibited ferredoxin activity. Inhibition of ferredoxin activity increased with increasing levels of sodium nitrite. Ferredoxin was isolated from normal C. pasteurianum and Clostridium botulinum cultures and from cultures incubated with 1,000 micrograms of sodium nitrite per ml for 45 min. The activity of in vivo nitrite-treated ferredoxin was decreased compared with that of control ferredoxin. Pyruvate-ferredoxin oxidoreductase isolated from C. botulinum cultures incubated with 1,000 micrograms of sodium nitrite per ml showed less activity than did control oxidoreductase. It is concluded that the antibotulinal activity of nitrite is due at least in part to inactivation of ferredoxin and pyruvate-ferredoxin oxidoreductase.

Clostridium↗
PubMed1992

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.

Anabaena↗
PubMed1988

On the specificity of pig adrenal ferredoxin (adrenodoxin) and spinach ferredoxin in electron-transfer reactions.

Spinach leaf ferredoxin and ferredoxin:NADP oxidoreductase as well as pig adrenodoxin and adrenodoxin reductase have been purified to homogeneity. Ferredoxin-NADP reductase and adrenodoxin-NADP reductase can perform the same diaphorase reactions (dichloroindophenol, ferricyanide and cytochrome c reduction) albeit not with the same efficiency. Despite the differences in their redox potentials, animal and plant ferredoxins can be used as heterologous substrates by the ferredoxin-NADP reductases from both sources. In heterologous systems, however, the ferredoxin/adrenodoxin concentrations must be increased approximately 100-fold in order to reach rates similar to those obtained in homologous systems. Ferredoxin and adrenodoxin can form complexes with the heterologous reductases as demonstrated by binding experiments on ferredoxin-Sepharose or ferredoxin-NADP-reductase-Sepharose and by the realization of difference spectra. Adrenodoxin also weakly substitutes for ferredoxin in NADP photoreduction, and can be used as an electron carrier in the light activation of the chloroplastic enzyme NADP-dependent malate dehydrogenase. In addition adrenodoxin is a good catalyst of pseudocyclic photophosphorylation, but not of cyclic phosphorylation and can serve as a substrate of glutamate synthase. These results are discussed with respect to the known structures of plant and animals ferredoxins and their respective reductases.

Adrenal Glands↗
PubMed1993

Charge pair interactions stabilizing ferredoxin-ferredoxin reductase complexes. Identification by complementary site-specific mutations.

Ferredoxin reductase (Fd-reductase) supplies electrons to mitochondrial steroid hydroxylase cytochrome P450 enzymes via a [2Fe-2S] ferredoxin. Chemical labeling studies with bovine Fd-reductase have implicated Lys-243 as important in binding to bovine ferredoxin (Hamamoto, I., Kazutaka, K., Tanaka, S., and Ichikawa, Y. (1988) Biochim. Biophys. Acta 953, 207-213). We have used site-directed mutagenesis to examine the role of charged residues in this region of human Fd-reductase in ferredoxin binding. Mutant proteins were expressed in Escherichia coli and were assayed for activity by ferredoxin-mediated electron transfer to cytochrome c. Replacement of Lys-242 (homologous to Lys-243 in bovine Fd-reductase) with Gln and replacement of Arg-241 with Ser had little effect (2.7- and 3.6-fold increased Km, respectively). In contrast, mutants at positions 239 and 243 (R239S and R243Q) exhibited markedly lower affinity for ferredoxin (17.5- and 1,600-fold increased Km, respectively). Studies were also carried out with two ferredoxin charge mutants shown previously to have lowered affinity for Fd-reductase (Coghlan, V. M., and Vickery, L. E. (1991) J. Biol. Chem. 266, 18606-18612). Comparisons of the binding of ferredoxin mutants D76N and D79N to Fd-reductase mutants R239S and R243Q suggest that Arg-239 and Arg-243 of Fd-reductase each interact directly with both Asp-76 and Asp-79 of ferredoxin during formation of the complex between the two proteins. These results support the hypothesis that specific electrostatic interactions involving this region are important in stabilizing the ferredoxin-Fd-reductase complex.

Amino Acid Sequence↗
PubMed1986

Chemical modification and cross-linking as probes of regions on ferredoxin involved in its interaction with ferredoxin: NADP reductase.

Ferredoxin which had been modified with glycine ethylester in the presence of a water-soluble carbodiimide to the extent of one carboxyl-group modified per ferredoxin was subjected to peptide mapping in an attempt to locate the site(s) of modification. The peptide mapping was done by HPLC and analysis of the resulting chromatogram allowed assignment of peaks to various segments in the amino acid sequences of the two isozymes of ferredoxin. The modified ferredoxin appeared to be a mixture of ferredoxin derivatives in which modification had occurred in three areas of the molecule. Although unable to identify the specific residues modified, it has been shown that modification is localized in the regions of residues 26-30, 65-70, and 92-94. The possibility that these regions of ferredoxin may define its binding site for ferredoxin: NADP reductase is discussed. Peptide mapping studies on a covalently linked adduct between ferredoxin and ferredoxin: NADP reductase also support these regions of ferredoxin as being important in the interaction between the two proteins.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and nucleotide sequences of bovine hepato-ferredoxin cDNA; identical primary structures of hepato- and adreno-ferredoxins.

1. The ferredoxin from bovine liver mitochondria, so-called hepatoredoxin, was purified and characterized as to its molecular weight, optical absorption spectrum and amino acid composition. 2. These properties were found to be very similar to those of adreno-ferredoxin. 3. To clarify the molecular basis of tissue specificity, the ferredoxin clones were obtained from a bovine liver library and the cDNA sequence of hepato-ferredoxin was determined. 4. The nucleotide sequence of hepato-ferredoxin clone was found to be identical to that of adreno-ferredoxin clone except for a single nucleotide in the 3' non-translated region. 5. Identical amino acid sequence of the two ferredoxins was confirmed by determining the partial amino acid sequence of the purified hepato-ferredoxin. 6. The results indicated that the organ specific activity of purified ferredoxin could not be explained by the different primary structure nor different RNA processing. 7. Other factors may be involved in the tissue specific properties of ferredoxins.

Adrenal Glands↗
PubMed1988

Tertiary structure of Bacillus thermoproteolyticus [4Fe-4S] ferredoxin. Evolutionary implications for bacterial ferredoxins.

The structure of a low-potential [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus has been solved using anomalous scattering data from iron atoms in the diffraction data of native crystals and refined partially to a crystallographic R-factor of 0.33, with 2.3 A (1 A = 0.1 nm) resolution data. The least-squares refinement based on the Bijvoet differences has determined that the four iron atoms in the cluster are an equal distance, approximately 2.8 A, apart. The NH ... S hydrogen bonds between polypeptide nitrogen atoms, and both cysteine and inorganic sulfur atoms, are present, as in ferrodoxin from Peptococcus aerogenes. The polypeptide chain of the B. thermoproteolyticus ferredoxin has a fold closely similar to that of 2[4Fe-4S] ferredoxin from P. aerogenes. The structural correspondence indicates strongly that both types of ferredoxin evolved from a common ancestor. The second cluster-binding region in P. aerogenes ferredoxin corresponds to the alpha-helix in B. thermoproteolyticus ferredoxin. The secondary-structure predictions strongly suggest that the alpha-helix is generally present in the monocluster-type ferredoxins. The conformational change to alpha-helix, insertions of a loop and a protrusion, as well as the absence of the second cluster in B. thermoproteolyticus ferredoxin, result in the lack of 2-fold symmetry present in P. aerogenes ferredoxin. So, the track of gene duplication is no longer detectable in the tertiary structure alone. The evolutionary events that may have occurred in the ferredoxins with the [4Fe-4S] cluster are discussed.

Amino Acid Sequence↗
PubMed1985

Chemical modification of carboxyl groups on spinach ferredoxin alters its ability to interact with ferredoxin:NADP reductase.

Treatment of spinach ferredoxin with glycine ethyl ester in the presence of a water soluble carbodiimide resulted in the modification of 3-4 carboxyl groups and decreased the ability of ferredoxin to participate in NADP photoreduction by chloroplast membranes by about 80%. The ability of the modified ferredoxin to receive electrons from the reducing side of Photosystem I was relatively unaffected. These findings suggest that the modified ferredoxin is unable to interact with ferredoxin:NADP reductase. This has been verified by demonstration that the modified ferredoxin fails to produce difference spectra typical of a ferredoxin-ferredoxin:NADP reductase complex when added to ferredoxin:NADP reductase.

Ferredoxin-NADP Reductase↗
PubMed1984

Properties of ferredoxin reductase and ferredoxin from the bovine corpus luteum.

Ferredoxin reductase and ferredoxin were purified from the bovine corpus luteum and their properties compared to the corresponding adrenal proteins. The luteal and adrenal proteins had similar absorbance spectra and molecular weights. Evidence was obtained from spectrophotometric titrations for formation of 1:1 complexes between luteal ferredoxin reductase and ferredoxin and between ferredoxin and cytochrome P-450scc. Adrenal ferredoxin reductase and ferredoxin were equally as effective as luteal ferredoxin reductase and ferredoxin in supporting cholesterol side-chain cleavage by luteal cytochrome P-450scc.

Adrenal Glands↗
PubMed1995

Tertiary structure of [2Fe-2S] ferredoxin from Spirulina platensis refined at 2.5 A resolution: structural comparisons of plant-type ferredoxins and an electrostatic potential analysis.

The structure of plant-type [2Fe-2S] ferredoxin isolated from Spirulina platensis has been refined using diffraction data to 2.5 A resolution by alternate cycles of simulated annealing and manual revision of the model. The final R factor is 19.9% for 2,912 reflections with F > 2 sigma F between 8.0 and 2.5 A resolution. S. platensis ferredoxin, like other plant-type [2Fe-2S] ferredoxins, has a major alpha-helix flanking a sheet consisting of four beta strands. The present refinement revises the conformation of residues 56-71, in which a one-turn helix was identified. Superposition of the Spirulina ferredoxin structure on the structures of other ferredoxins that have been well refined showed structural perturbation at a few residues on the amino and carboxyl termini and the turn between the first and second beta-strands. The root-mean-square deviations of the corresponding C alpha atoms of the pairs of ferredoxins range from 0.90 to 1.17 A for all the residues, but from 0.64 to 0.70 A if the few perturbed residues are excluded. Therefore, it may be concluded that the main-chain foldings of all the plant-type [2Fe-2S] ferredoxins are essentially the same. Electrostatic potential analysis showed that the molecular surface around the cluster is negatively charged, whereas that of the beta-sheet of the other side is positively charged. The interaction between ferredoxin and ferredoxin-NADP+ reductase is discussed on the basis of the charge distributions of these molecules and biochemical data.

Amino Acid Sequence↗
PubMed1996

Glutamate 94 of [2Fe-2S]-ferredoxins is important for efficient electron transfer in the 1:1 complex formed with ferredoxin-glutamate synthase (GltS) from Synechocystis sp. PCC 6803.

We have analyzed the role of critical amino acid residues involved in the interaction between ferredoxin and ferredoxin-glutamate synthase (GOGAT) encoded by the gltS gene from the cyanobacterium Synechocystis sp. PCC 6803. Our results indicated that the glutamate 94 residue of Anabaena 7120 ferredoxin (= E92 of the Synechocystis 6803 ferredoxin) was necessary for an efficient electron transfer to GOGAT comparable to ferredoxin:NADP-reductase, nitrite reductase and nitrate reductase [Schmitz and Böhme (1995) Biochim. Biophys. Acta 1231, 335-341]. The K(m) value determined for wt-ferredoxins and mutant E94Q (and E92Q) was 1 muM, respectively, and activity loss of E94Q was due to a lowered Vmax. Exchange of residue F65 for aliphatic substitutions, which was crucial to electron transfer to ferredoxin:NADP-reductase and nitrite reductase, exhibited only small effects on glutamate synthase-dependent activity while heterocyst ferredoxin and flavodoxin were almost inactive as electron donors. In contrast to data reported for the spinach system, the stoichiometry of the cross-linked complex between ferredoxin and glutamate synthase was 1:1.

Amino Acid Oxidoreductases↗
PubMed1995

Ferredoxin-dependent redox system of a thermoacidophilic archaeon, Sulfolobus sp. strain 7. Purification and characterization of a novel reduced ferredoxin-reoxidizing iron-sulfur flavoprotein.

To elucidate the ferredoxin-dependent redox system of the thermoacidophilic, aerobic archaeon Sulfolobus sp. strain 7, a novel FeS flavoprotein, which can reoxidize the reduced 7Fe ferredoxin in vitro, has been purified and characterized (designated as IFP) using the cognate 7Fe ferredoxin and 2-oxoacid:ferredoxin oxidoreductase, a key enzyme of the archaeal tricarboxylic acid cycle. IFP consists of three non-identical subunits with apparent molecular masses of 87, 32, and 22 kDa, respectively, and contains at least two FMN (Em, 6.8 = -57 mV) and two plant-ferredoxin-type [2Fe-2S]2+,1+ clusters (Em, 6.8 = -260 mV)/alpha 2 beta 2 gamma 2 structure. Both FeS and flavin centers of IFP are slowly but fully reduced by the enzymatically reduced cognate ferredoxin under anaerobic conditions at 50 degrees C, but not by NAD(P)H. Thus, the ferredoxin-dependent redox system of Sulfolobus sp. strain 7 is tentatively proposed as follows: 2-oxoacid:ferredoxin oxidoreductase (thiamine pyrophosphate and [4Fe-4S] cluster)-->ferredoxin-->IFP ([2Fe-2S] cluster-->FMN).

Circular Dichroism↗
PubMed1991

1H NMR spectra of vertebrate [2Fe-2S] ferredoxins. Hyperfine resonances suggest different electron delocalization patterns from plant ferredoxins.

We report the observation of paramagnetically shifted (hyperfine) proton resonances from vertebrate mitochondrial [2Fe-2S] ferredoxins. The hyperfine signals of human, bovine, and chick [2Fe-2S] ferredoxins are described and compared with those of Anabaena 7120 vegetative ferredoxin, a plant-type [2Fe-2S] ferredoxin studied previously [Skjeldal, L., Westler, W. M., & Markley, J. L. (1990) Arch. Biochem. Biophys. 278, 482-485]. The hyperfine resonances of the three vertebrate ferredoxins were very similar to one another both in the oxidized state and in the reduced state, and slow (on the NMR scale) electron self-exchange was observed in partially reduced samples. For the oxidized vertebrate ferredoxins, hyperfine signals were observed downfield of the diamagnetic envelope from +13 to +50 ppm, and the general pattern of peaks and their anti-Curie temperature dependence are similar to those observed for the oxidized plant-type ferredoxins. For the reduced vertebrate ferredoxins, hyperfine signals were observed both upfield (-2 to -18 ppm) and downfield (+15 to +45 ppm), and all were found to exhibit Curie-type temperature dependence. This pattern and temperature dependence are distinctly different from those found with reduced plant-type ferredoxins which have signal centered around +120 ppm with Curie-type temperature dependence, assigned to cysteines which interact with Fe(III), and signals centered around +20 ppm with anti-Curie temperature dependence, assigned to cysteines which interact with Fe(II) [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Immobilized ferredoxins for affinity chromatography of ferredoxin-dependent enzymes.

An immobilized ferredoxin more stable than the conventional immobilized spinach ferrodoxin was prepared by reacting CNBr-Sepharose with ferredoxins isolated from barley and Synechococcus vulcanus, a thermophilic blue-green alga. The dissociation constants of immobilized ferredoxin from spinach, barley and S. vulcanus for spinach ferredoxin-NADP reductase were 0.922, 2.505 and 5.209 microM, respectively, whereas those for barley ferredoxin-NADP reductase were 1.159, 0.579 and 2.851 microM, respectively. The order of stability was S. vulcanus greater than barley greater than spinach. The immobilized ferredoxin was applied to the simultaneous detection of ferredoxin-dependent enzymes in spinach chloroplasts. Over 20 polypeptides were detected. Synechococcus ferredoxin could also be immobilized on a Toyopearl gel and repeatedly used in an automated high-performance liquid chromatographic system.

Chemical Phenomena↗
PubMed1997

A three-domain iron-sulfur flavoprotein obtained through gene fusion of ferredoxin and ferredoxin-NADP+ reductase from spinach leaves.

Ferredoxin and ferredoxin-NADP+ reductase are the two last partners of the photosynthetic electron-transfer chain, responsible for the final reduction of NADP+ to NADPH. Herein, we report the engineering and characterization of a novel protein molecule in which the electron-carrier protein (ferredoxin I) and the reductase (a flavoprotein) were covalently linked in a single polypeptide chain by gene fusion. The gene was obtained by joining the cDNAs encoding the respective proteins and subsequently by deleting the intervening sequence between them by site-directed mutagenesis. No extra amino acid residues were introduced between the C-terminus of ferredoxin I and the N-terminus of the flavoenzyme. The chimera was purified to homogeneity and characterized. The M(r) of the chimera apoprotein was 45,800 as determined by mass spectrometry, in agreement with the expected value of 45,846. Both flavin and iron-sulfur cluster were in 1:1 ratio with respect to the apoprotein. The chimera was found active as a diaphorase and, more interestingly, highly efficient as a cytochrome c reductase, without need for free ferredoxin addition in the assay medium. Several lines of evidence indicate that the ferredoxin and the reductase in the chimera assume a configuration quite similar to that in the dissociable physiological complex. Thus, the fusion protein could be a useful tool for studying the mechanism of protein-protein recognition and electron transfer in the ferredoxin-ferredoxin-NADP+ reductase system.

Amino Acid Sequence↗