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PubMed2004

Inhibition of pea ferredoxin-NADP(H) reductase by Zn-ferrocyanide.

Ferredoxin-NADP(H) reductases (FNRs) represent a prototype of enzymes involved in numerous metabolic pathways. We found that pea FNR ferricyanide diaphorase activity was inhibited by Zn2+ (Ki 1.57 microM). Dichlorophenolindophenol diaphorase activity was also inhibited by Zn2+ (Ki 1.80 microM), but the addition of ferrocyanide was required, indicating that the inhibitor is an arrangement of both ions. Escherichia coli FNR was also inhibited by Zn-ferrocyanide, suggesting that inhibition is a consequence of common structural features of these flavoenzymes. The inhibitor behaves in a noncompetitive manner for NADPH and for artificial electron acceptors. Analysis of the oxidation state of the flavin during catalysis in the presence of the inhibitor suggests that the electron-transfer process between NADPH and the flavin is not significantly altered, and that the transfer between the flavin and the second substrate is mainly affected. Zn-ferrocyanide interacts with the reductase, probably increasing the accessibility of the prosthetic group to the solvent. Ferredoxin reduction was also inhibited by Zn-ferrocyanide in a noncompetitive manner, but the observed Ki was about nine times higher than those for the diaphorase reactions. The electron transfer to Anabaena flavodoxin was not affected by Zn-ferrocyanide. Binding of the apoflavodoxin to the reductase was sufficient to overcome the inhibition by Zn-ferrocyanide, suggesting that the interaction of FNRs with their proteinaceous electron partners may induce a conformational change in the reductase that alters or completely prevents the inhibitory effect.

2,6-Dichloroindophenol↗
PubMed1998

Control of ferredoxin and Gal/GalNAc lectin gene expression in Entamoeba histolytica by a cis-acting DNA sequence.

The ferredoxin (fdx) and lectin (hgl5) promoters of Entamoeba histolytica contain the DNA sequence motif TATTCTATT (URE3). Previously we showed that mutation of the URE3 motif in the hgl5 lectin promoter results in an increase in promoter reporter activity. Mutation of this motif in the fdx promoter led to a 40-to-50% decrease in fdx promoter activity as measured by reporter gene activity and abundance of mRNA. E. histolytica nuclear proteins exhibited sequence-specific binding to the URE3 motif in electrophoretic mobility shift assays. These results support the regulation of both ferredoxin and lectin promoters by a nuclear protein(s) which recognizes the URE3 motif.

Acetylgalactosamine↗
PubMed1972

Four-iron (sulfide) ferredoxin from Bacillus polymyxa.

Ferredoxin from Bacillus polymyxa contains (per mole) four non-heme iron residues, four acid-labile sulfide residues, and four cysteine residues. Its molecular weight is approximately 8,800, and it has an oxidation-reduction potential (E(m)) of -390 mv. It is active as an electron carrier in several ferredoxin-linked enzyme systems.

Amino Acids↗
PubMed1986

Isolation and sequence of the gene for ferredoxin I from the cyanobacterium Anabaena sp. strain PCC 7120.

The structural gene for ferredoxin I, petF, from the cyanobacterium Anabaena sp. strain PCC 7120 has been isolated from a recombinant lambda library. Mixtures of tetradecanucleotides and heptadecanucleotides, each containing all possible DNA sequences corresponding to two separate regions of the ferredoxin amino acid sequence, were synthesized and used as hybridization probes to identify a genomic clone containing the coding sequence for the petF gene. The sequence of the entire petF coding region and portions of the 3'- and 5'-flanking regions was determined. The DNA sequence of petF suggests that, in contrast to the nucleus-encoded plant protein, cyanobacterial apoferredoxin is not synthesized as a higher-molecular-weight precursor. The Anabaena petF gene is a single-copy gene. During growth on complete medium it was transcribed into a monocistronic mRNA species of approximately 500 bases that initiated 100 base pairs upstream from the petF coding region.

Amino Acid Sequence↗
PubMed1993

Escherichia coli ferredoxin NADP+ reductase: activation of E. coli anaerobic ribonucleotide reduction, cloning of the gene (fpr), and overexpression of the protein.

A specific ribonucleoside triphosphate reductase is induced in anaerobic Escherichia coli. This enzyme, as isolated, lacks activity in the test tube and can be activated anaerobically with S-adenosylmethionine, NADPH, and two previously uncharacterized E. coli fractions. The gene for one of these, previously named dA1, was cloned and sequenced. We found an open reading frame coding for a polypeptide of 248 amino acid residues, with a molecular weight of 27,645 and with an N-terminal segment identical to that determined by direct Edman degradation. In a Kohara library, the gene hybridized between positions 3590 and 3600 on the physical map of E. coli. The deduced amino acid sequence shows a high extent of sequence identity with that of various ferredoxin (flavodoxin) NADP+ reductases. We therefore conclude that dA1 is identical with E. coli ferredoxin (flavodoxin) NADP+ reductase. Biochemical evidence from a bacterial strain, now constructed and overproducing dA1 activity up to 100-fold, strongly supports this conclusion. The sequence of the gene shows an apparent overlap with the reported sequence of mvrA, previously suggested to be involved in the protection against superoxide (M. Morimyo, J. Bacteriol. 170:2136-2142, 1988). We suggest that a frameshift introduced during isolation or sequencing of mvrA caused an error in the determination of its sequence.

Amino Acid Sequence↗
PubMed1994

Cloning, expression, and molecular characterization of the gene encoding an extremely thermostable [4Fe-4S] ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus.

The gene for ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, sequenced, and expressed in Escherichia coli. The coding region confirmed the determined amino acid sequence. Putative archaeon-type transcriptional regulatory elements were identified. The fdxA gene appears to be an independent transcriptional unit. Recombinant ferredoxin was indistinguishable from the protein purified from P. furiosus in its thermal stability and in the potentiometric and spectroscopic properties of its [4Fe-4S] cluster.

Amino Acid Sequence↗
PubMed1994

Transcriptional and translational analysis of ferredoxin and flavodoxin under iron and nitrogen stress in Anabaena sp. strain PCC 7120.

In Anabaena sp. strain PCC 7120, vegetative cell ferredoxin synthesis under iron starvation was repressed 25-fold, whereas heterocyst ferredoxin synthesis decreased only 2.8-fold. Induction of flavodoxin under iron depletion was independent of the availability of combined nitrogen. Under iron stress but in the presence of combined nitrogen, fdxH and nifH genes were transcriptionally active; although excision of the 11-kb element seemed to be completed, nitrogenase activity and the fdxH gene product were not detectable.

Adaptation, Biological↗
PubMed2001

Role of a ferredoxin gene cotranscribed with the nifHDK operon in N(2) fixation and nitrogenase "switch-off" of Azoarcus sp. strain BH72.

The endophytic diazotroph Azoarcus sp. strain BH72 is capable of infecting rice roots and of expressing the nitrogenase (nif) genes there. In order to study the genetic background for nitrogen fixation in strain BH72, the structural genes of nitrogenase (nifHDK) were cloned and sequenced. The sequence analysis revealed an unusual gene organization: downstream of nifHDK, a ferredoxin gene (fdxN; 59% amino acid sequence identity to R. capsulatus FdxN) and open reading frames showing 52 and 36% amino acid sequence identity to nifY of Pseudomonas stutzeri A15 and ORF1 of Azotobacter vinelandii were located. Northern blot analysis, reverse transcriptase PCR and primer extension analysis revealed that these six genes are located on one transcript transcribed from a sigma(54)-type promoter. Shorter transcripts sequentially missing genes of the 3' part of the full-length mRNA were more abundantly detected. Mutational analyses suggested that FdxN is an important but not the essential electron donor for dinitrogenase reductase. An in-frame deletion of fdxN resulted in reduced growth rates (59% +/- 9%) and nitrogenase activities (81%) in nitrogen-fixing pure cultures in comparison to the wild type. Nitrogenase activity was fully complemented in an fdxN mutant which carried a nifH promoter-driven fdxN gene in trans. Also, in coculture with the ascomycete Acremonium alternatum, where strain BH72 develops intracytoplasmic membrane stacks, the nitrogenase activity in the fdxN deletion mutant was decreased to 56% of the wild-type level. Surprisingly, the fdxN deletion also had an effect on the rapid "switch-off" of nitrogenase activity in response to ammonium. Wild-type strain BH72 and the deletion mutant complemented with fdxN in trans showed a rapid reversible inactivation of acetylene reduction, while the deletion mutant did not cease to reduce acetylene. In concordance with the hypothesis that changes in the redox state of NifH or electron flux towards nitrogenase may be involved in the mechanism of physiological nitrogenase switch-off, our results suggest that the ferredoxin may be a component involved in this process.

Azoarcus↗
PubMed1992

Transcription of the human ferredoxin gene through a single promoter which contains the 3',5'-cyclic adenosine monophosphate-responsive sequence and Sp 1-binding site.

We have investigated the functional elements involved in cAMP-stimulated transcription of the human ferredoxin gene. Unlike the bovine gene, the human gene lacked a second upstream RNA initiation site as demonstrated by sequence analysis of the exon boundary, lack of upstream RNA, and analysis of the promoter. The presence of a single promoter was determined by testing the ability of various gene segments to drive the expression of the chloramphenicol acetyltransferase gene after transfection into a mouse adrenal cell line Y1. Full promoter activity was conferred by a DNA fragment spanning -209 to +55, although the -94 to +55 fragment already provided some promoter activity. Transcription from the -94 to +55 segment was stimulated by 2-fold when 8-bromo-cAMP was added to the cell. Footprinting analyses showed two GC boxes at -50 to -70 and -87 to -108 were protected by proteins from both Y1 and HeLa cells. Competition experiments showed that a protein with a recognition sequence indistinguishable from Sp1 bound to these sites. When connected to a heterologous TATA box, the sequence at -76 to -42, which contained the proximal GC box, was able to confer a high level of basal transcription and cAMP stimulation. This sequence does not show sequence homology with the known cAMP-responsive element. Mutations or deletion of the Sp 1-binding site showed diminished basal transcription and defined the cAMP responsive sequence to be from -76 to -62. Therefore the cAMP-responsive sequence of the human ferredoxin gene was located at -76 to -62, which was adjacent to the Sp 1-binding site.

Amino Acid Sequence↗
PubMed2004

Comparison of the nucleotide sequences of ferredoxin-cDNAs among some Datura plants.

The nucleotide sequences of partial ferredoxin (Fd)-cDNAs (corresponding to the amino acid sequence of 22-87 in the total 97 amino acids of ferredoxin) were determined for Datura arborea, D. stramonium, D. metel, and related Datura plants. Non-synonymous substitutions were noted at 4 positions and a synonymous substitution was seen at position 82 (Gln [CAA] (arborea) vs. Gln [CAG] (stramonium and metel)). The nucleotide sequence of Fd-cDNA may provide more detailed information regarding the relative taxonomic positions of plants than the amino acid sequence. However, Datura plants in the same section (metel, fastuosa, and innoxia) and of different varieties (stramonium var. stramonium and stramonium var. tatula) showed identical Fd-cDNA nucleotide sequences. This result suggests that there are very close relationships among the plants in each group.

Amino Acid Sequence↗
PubMed1995

Cloning and nucleotide sequence of a leaf ferredoxin-nitrite reductase cDNA of rice.

A ferredoxin-nitrite reductase (EC 1.7.7.1) cDNA was isolated and sequenced from a lambda gt 11 cDNA library constructed from nitrate-induced greening shoots of rice (Oryza sativa L.) seedlings. The nucleotide sequence of the cDNA clone contains an open reading frame of 1788 nucleotides. There exists a strong bias for the third codon usage of G/C (95.5%) as in the case of the maize enzyme. The deduced amino acid sequence shows an overall homology to the maize (81%) and the dicot enzymes (70-74%), suggesting that the primary structure of ferredoxin-nitrite reductase is highly conserved in higher plants.

Amino Acid Sequence↗
PubMed2005

The plant-type ferredoxin-NADP+ reductase/ferredoxin redox system as a possible drug target against apicomplexan human parasites.

Apicomplexa are unicellular, obligate intracellular parasites of great medical importance. They include human pathogens like Plasmodium spp., the causative agent of malaria, and Toxoplasma gondii, an opportunistic parasite of immunosuppressed individuals and a common cause of congenital disease (toxoplasmosis). They alone affect several hundred million people worldwide so that new drugs, especially for plasmodial infections, are urgently needed. This review will focus on a recently emerged, potential drug target, a plant-type redox system consisting of ferredoxin-NADP(+) reductase (FNR) and its redox partner, ferredoxin (Fd). Both reside in an unique organelle of these parasites, named apicoplast, which is of algal origin. The apicoplast has been shown to be required for pathogen survival. In addition to other pathways already identified in this compartment, the FNR/Fd redox system represents a promising drug target because homologous proteins are not present in host organisms. Furthermore, a wealth of structural information exists on the closely related plant proteins, which can be exploited for structure-function studies of the apicomplexan protein pair. T. gondii and P. falciparum FNRs have been cloned, and the T. gondii enzyme was shown to be a flavoprotein active as a NADPH-dependent oxidoreductase. Both phylogenetic and biochemical analyses indicate that T. gondii FNR is similar in function to the isoform present in non-photosynthetic plastids whereby electron flow is from NADPH to oxidized Fd. The resulting reduced Fd is then presumably used as a reductant for various target enzymes whose nature is just starting to emerge. Among the likely candidates is the iron-sulfur cluster biosynthesis pathway, which is also located in the apicoplast and dependent on reducing power. Furthermore, lipoic acid synthase and enzymes of the isoprenoid biosynthetic pathway may be other conceivable targets. Since all these metabolic steps are vital for the parasite, blocking electron flow from FNR to Fd by inhibition of either FNR activity or its molecular interaction with Fd should also interfere with these pathways, ultimately killing the parasite. Although the three-dimensional structure of FNR from T. gondii is not yet known, experimental and computational evidence shows that apicomplexan and plant enzymes are very similar in structure. Furthermore, single amino acid changes can have profound effects on the enzyme activity and affinity for Fd. This knowledge may be exploited for the design of inhibitors of protein-protein interaction. On the other hand, specifically tailored NAD(P) analogues or mimetics based on previously described substances might be useful lead compounds for apicomplexan FNR inhibitors.

Animals↗
PubMed1988

7-Iron ferredoxin revisited.

The crystal structure of the 7Fe ferredoxin from Azotobacter vinelandii has been redetermined using area detector data to 2.7-A resolution and a new derivative. Tetragonal crystals of the protein were maintained at pH 8.0. The results show that the structure previously reported was in error and confirms a recent independent report of the structure (Stout, G.H., Turley, S., Sieker, L. C., and Jensen, L. H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, in press). The protein fold is similar to the homologous 8Fe ferredoxin structure for the N-terminal half of the protein; the C-terminal residues wrap around this structure. The structure contains a 3Fe cluster coordinated by cysteines 8, 16, and 49 and a 4Fe cluster coordinated by cysteines 20, 39, 42, and 45. However, there are two free sulfhydryls, cysteines 11 and 24, in the new model. Cysteine 24 is in contact with the [4Fe-4S] cluster. Cysteine 11 is shielded from solvent by residues 86-90.

Azotobacter↗
PubMed2000

Structural basis of the catalytic role of Glu301 in Anabaena PCC 7119 ferredoxin-NADP+ reductase revealed by x-ray crystallography.

The three-dimensional crystal structure of the Glu301Ala site-directed mutant of ferredoxin-NADP+ reductase from Anabaena PCC 7119 has been determined at 1.8A resolution by x-ray diffraction. The overall folding of the Glu301Ala FNR mutant shows no significant differences with respect to that of the wild-type enzyme. However, interesting conformational changes are detected in the side chain of another glutamate residue, Glu139, which now points towards the FAD cofactor in the active center cavity. The new conformation of the Glu139 side chain is stabilized by a network of five hydrogen bonds to several water molecules, which seem to hold the carboxylate side chain in a rather fixed position. This interacting network connects the Glu139 side chain to the Ser80 side chain through a series of three water molecules. These observations are discussed in terms of the reactivity of Glu301Ala ferredoxin-NADP+ reductase towards its substrates, and the role of Glu301 in the catalysis is re-examined. Moreover, a structural explanation of the different reoxidation properties of this mutant is given on the basis of the reported structure by modeling the hypothetical flavin C(4a)-hydroperoxide intermediate. The model shows that the distal oxygen of the peroxide anion could be in an appropriate situation to act as the proton donor in the reoxidation process.

Anabaena↗
PubMed2000

Ferredoxin III of Desulfovibrio africanus: sequencing of the native gene and characterization of a histidine-tagged form.

Desulfovibrio africanus ferredoxin III (Da FdIII) contains one [4Fe-4S](2+/1+) cluster and one [3Fe-4S](1+/0) cluster, bound by seven Cys residues, in which the [3Fe-4S] cluster is co-ordinated by the unusual sequence, Cys(11)-Xaa-Xaa-Asp(14)-Xaa-Xaa-Cys(17)-Xaa(n)-Cys(51)-Glu. The [3Fe-4S] core of this ferredoxin is so far unique in showing rapid bi-directional [3Fe-4S]<-->[4Fe-4S] cluster interconversion with a wide range of metal ions. In order to obtain protein for mutagenesis studies Da FdIII has been cloned, sequenced, and expressed as a hexa-histidine tagged (ht) polypeptide in Escherichia coli strain BL21(DE3) pLysS. Expression of ht Da FdIII, whether translated from a synthetic gene (pJB10) or from the native nucleotide sequence (pJB11), occurred at similar levels (approx. 6 mg.l(-1)), but without incorporation of metal clusters. The nucleotide sequence confirms the protein sequence reported previously [Bovier-Lapierre, Bruschi, Bonicel and Hatchikian (1987) Biochim. Biophys. Acta 913, 20-26]. Cluster incorporation was achieved using FeCl(3) together with cysteine sulphur transferase, NifS, plus cysteine to generate low levels of sulphide ions. Absorption and EPR spectroscopy show that both [3Fe-4S] and [4Fe-4S] clusters are correctly inserted. Thin-film electrochemistry provides evidence that the [3Fe-4S] cluster undergoes reversible cluster transformation in the presence of Fe(II) and Zn(II) ions with properties identical to the native protein. Nevertheless the protein has lower stability than native Da FdIII during chromatography. The one-dimensional 600 MHz NMR spectrum of the apoprotein indicates an unstructured protein with random coil chemical shifts whereas spectra of the reconstituted ht protein show secondary structural elements and 18 peaks shifted downfield of 9.6 p.p.m. The spectra are unique but have similarities with the shift patterns seen with 7Fe Desulfurolobus ambivalens Fd. The ht does not affect iron-sulphur cluster incorporation, but NMR evidence suggests that excess Fe binds to the tag. This may account for the lower stability of the ht compared with the native protein.

Amino Acid Sequence↗
PubMedDate not supplied

[Ferredoxin reduction by polarographic methods].

The reduction of iron-sulphur protein of the higher plant ferrodoxin has been studied by polarographical methods. Ferredoxin initiates a reversible wave with E1/2=--0,61 v (N. C. E.) at pH 7. Protein absorption greatly influences the electrochemical reduction. The protons have been shown to take part in the electrode reaction. The potentiometrically obtained data about the difference between E1/2 and E0=--0.70 v and its causative factors are discussed. As a result of the experiments with modification of ferredoxin active centre it has been concluded that the active centre participates in the polarographical reduction.

Ferredoxins↗
PubMed1976

[Pterin-protein complex and ferredoxin as possible components of a molecular complex in photosystem I].

Properties are studied of pterin-protein complex (PPC), possible precursor of ferredoxin in electrone transport chain PS-I. PPC is shown to form chelate complex with Fe ions which are capable to activate the process of light reduction of NADP+ by chloroplasts in the absence of ferrodoxin. A possibility of dark reduction of NADP+ with the participation of light pre-activated PPC is found. Two-step scheme of electrone transport from P700 to ferredoxin is supposed. The data obtained indicate that one of sites of electrone transport into interior part of tilakoid is located on the reduction side of PS-I. An additional possibility of NADP+ reduction with the participation of PPC-Fe in vivo is suggested.

Chloroplasts↗
PubMed1991

Potentiometric and electron nuclear double resonance properties of the two spin forms of the [4Fe-4S]+ cluster in the novel ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus.

Pyrococcus furiosus ferredoxin contains a single [4Fe-4S] that exists in both S = 1/2 (20%) and S = 3/2 (80%) ground states in the reduced protein. We report here on the temperature-dependent potentiometric properties of the two spin forms, their stability, and on the structural features that differentiate them. The midpoint potential (Em) of the cluster in either spin state was determined at -365 mV (30 degrees C, pH 8.0). By rapidly freezing samples for EPR analyses, it was shown that the Em values of both spin states appear to change by -1.7 mV/degrees C over the range 20 degrees-80 degrees C, and by -6 mV/degrees C between 80 and 89 degrees C. The Em values and the relative amounts of the S = 1/2 and S = 3/2 forms of the cluster were unaffected by pH (6.8-10.5), even at 85 degrees C, and were unchanged by the presence of NaCl (1.0 M), sodium dodecyl sulfate (10%, w/v) or ethylene glycol (50%, v/v), even at 80 degrees C. The S = 1/2 form of the [4Fe-4S]+ cluster was found to exhibit a strongly coupled 1H ENDOR resonance (A = 22 MHz) that was exchangeable with the solvent. Such a large coupling has not been observed in any other iron-sulfur protein. Since a unique feature of this 4Fe-ferredoxin is that only 3 cysteinyl residues appear to be coordinated to the [4Fe-4S] cluster, the ENDOR data are consistent with an H2O molecule being a ligand to the unique Fe site. The S = 3/2 form of the [4Fe-4S]+ cluster exhibited a similar, strongly coupled 1H ENDOR resonance, but in this spin state it was not exchangeable with the solvent. This suggests that the [4Fe-4S]+ cluster exhibiting the S = 3/2, but not the S = 1/2 ground state, is "shielded" from the solvent, presumably by neighboring amino acid residues. In view of the pH dependence of the midpoint potential of the two spin states, the fourth ligand to the cluster and the source of the strongly coupled 1H ENDOR resonance is probably an OH- rather than H2O molecule.

Archaea↗