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At least 1,099 records · Page 61Linked to original sources
PubMed1993

The ferredoxin:sulphite reductase gene from Synechococcus PCC7942.

The structural gene of the ferredoxin:sulphite reductase (EC 1.8.7.1) from the cyanobacterium Synechococcus PCC7942 (formerly 'Anacystis nidulans') was cloned and sequenced. The gene termed 'sir' was detected by heterologous Southern hybridisation with the structural gene cysI from Escherichia coli encoding the iron-sulphur haemoprotein of the NADPH:sulphite reductase. The open reading frame is comprised of 1875 bp encoding for a polypeptide of M(r) 70.028. The deduced amino acid sequence is 35.6% identical with the enterobacterial iron-sulphur haemoprotein. This putative fd-dependent sulphite reductase is only distantly related to the fd-dependent nitrite reductase (binary matching coefficient SAB: 0.23) or with the NADPH-sulphite reductase (SAB: 0.32). Highly conserved residues are found within the two Cys clusters forming the reactive Fe4S4-sirohaem centre of the enzyme. Expression of the sir gene using a fusion vector gave a single gene product which is immunologically related with the fd-sulphite reductase from the wild-type bacterium.

Amino Acid Sequence↗
PubMed1995

The genomic organization of the gene encoding a nitrate-inducible ferredoxin-NADP+ oxidoreductase from rice roots.

A genomic clone of the gene encoding a nitrate-inducible ferredoxin-NADP+ oxidoreductase (FNR) from rice (Oryza sativa L.) roots has been isolated and its nucleotide sequence determined. The clone contains 3897 nucleotides of the gene which consists of six exons interrupted by five introns. The transcription start site was determined by primer extension analysis which locates 64 bp upstream of the ATG translation initiation codon. The 5'-flanking region contains canonical TATA- and CAAT-boxes, and a potential Sp1-binding site. Four ATCAA(A/C) and two inverted TTTGAT sequences are localized in the promoter region and a TGTAA motif occurs three times in the 3'-untranslated region. No significant similarity was found when the 5' flanking region was compared with that of the photosynthetic FNR gene.

Base Sequence↗
PubMed1995

The oxidation-reduction properties of spinach thioredoxins f and m and of ferredoxin:thioredoxin reductase.

Oxidation-reduction midpoint potentials have been determined, using cyclic voltammetry, for the active-site disulfide/dithiol couples of spinach thioredoxins f and m and of spinach ferredoxin:thioredoxin reductase (FTR) and for a component likely to be the [4Fe-4S] cluster of FTR. Values for the midpoint potentials (n = 2) of -210 +/- 10 mV were determined for both thioredoxins f and m. Two redox centers were detected in FTR, with midpoint potential values of -230 +/- 10 mV (n = 2) and +340 +/- 30 mV, respectively. Alkylation of the active-site cysteines of FTR by treatment of the enzyme with N-ethylmaleimide (NEM) eliminates the component with the -230 mV midpoint potential, allowing one to assign this value to the active site disulfide/dithiol couple. Inasmuch as the only other electron-carrying center known to be present in FTR is the [4Fe-4S] cluster, it appears likely that the high-potential component can be attributed to this redox moiety. The midpoint potential value of the high-potential feature shifts slightly, to +380 +/- 20 mV, in the NEM-treated enzyme.

Chloroplast Thioredoxins↗
PubMed1977

Spectroscopic studies of the oxidation-reduction properties of three forms of ferredoxin from Desulphovibrio gigas.

Electron paramagnetic resonance spectra were recorded of three forms of Desulphovibrio gigas ferredoxin, FdI, FdI' and FdII. The g = 1.94 signal seen in dithionite-reduced samples is strong in FdI, weaker in FdI' and very small in FdII. The g = 2.02 signal in the oxidized proteins is weak in FdI and strongest in FdII. It is concluded that most of the 4Fe-4S centres in FdI change between states C- and C2-; FdI' contain both types of centre. There is no evidence that any particular centre can change reversibly between all three oxidation states. Circular dichroism spectra show differences between FdI and FdII even in the diamagnetic C2- state. The redox potentials of the iron-sulphur centres of the three oligomers (forms) are different. After formation of the apo-protein of FdII and reconstitution with iron and sulphide, the protein behaves more like FdI, showing a strong g = 1.94 signal in the reduced states.

Binding Sites↗
PubMed1980

Antiferromagnetic exchange interaction in the two-iron-two-sulphur ferredoxin from the blue-green alga Spirulina maxima studied with a highly sensitive magnetic balance.

1. A highly sensitive magnetic balance of the Faraday type is described. 2. The magnetic susceptibility of the oxidized and reduced forms of the two-iron-two-sulphur ferredoxin from the blue-green alga Spirulina maxima has been measured over a wide temperature range. 3. The results can be interpreted within a simple model involving antiferromagnetically coupled iron atoms at the active site. The coupling, expressed as --J, is estimated to be 182 +/- 20/cm and 98 +5/-10 /cm for the oxidized and reduced forms, respectively.

Binding Sites↗
PubMed1985

The lack of a solvent accessible hydroxide or water ligand to iron at the 3Fe center of Azotobacter vinelandii ferredoxin I.

The X-ray crystal structure of Azotobacter vinelandii ferredoxin I (FdI) describes a planar 3Fe-3S center in which one of the iron atoms is ligated to a solvent accessible oxo ligand, presumably from water or hydroxide (Ghosh et al., (1982) J. Mol. Biol. 158, 73-109). Efforts to displace the proposed oxo ligand with cyanide were unsuccessful, even in 80% dimethylsulfoxide. In addition, comparison of the electron spin echo envelopes for H2O- and D2O-equilibrated samples of FdI showed only a slight deuterium modulation, far less than would be expected were water to be bound as an iron ligand. These results do not support the presence of a solvent accessible oxo ligand to the 3Fe center as described in the X-ray crystal structure.

Azotobacter↗
PubMed1986

Ferredoxin-dependent reduction of nitroimidazole derivatives in drug-resistant and susceptible strains of Trichomonas vaginalis.

The inhibitory effect of a range of nitroimidazole-derivatives on H2 production by metronidazole resistant (CDC-85) and susceptible (C1-NIH) Trichomonas vaginalis strains was investigated. The 2-, 4-, and 5-nitro-derivatives used had one-electron reduction potentials within the range -250 to -525 mV. Nitroimidazole concentrations giving 50% inhibition of H2 production (kiH2) for compounds with one-electron reduction potentials in the range -250 to -425 mV were found to be similar for both strains tested. Compounds with one-electron reduction potentials below -425 mV give 10-fold higher KiH2 values for the metronidazole resistant isolate. Both strains showed increased KiH2 for compounds with potentials lower than -500 mV. The addition of 2.1 kPa (0.02 atm) O2 to the gas phase resulted in increasing the kiH2 values for all the compounds tested, but had the greater effect on results obtained with the resistant isolate using nitroimidazoles in the range -425 to -490 mV. The results enable the proposal that the resistant isolate CDC-85 has a ferredoxin with altered redox properties or reduced intracellular levels.

Animals↗
PubMed1985

Selective oxidative destruction of iron-sulfur clusters. Ferricyanide oxidation of Azotobacter vinelandii ferredoxin I.

The destructive oxidation of aerobically isolated 7Fe Azotobacter vinelandii ferredoxin I [(7Fe)FdI] by Fe(CN)3-6 is examined using low-temperature magnetic circular dichroism (MCD) and EPR. The results demonstrate that oxidation of the [3Fe-3S] cluster occurs only after essentially complete destruction of the [4Fe-4S] cluster. It is therefore feasible by controlled Fe(CN)3-6 oxidation to obtain a partially metallated form of FdI, (3Fe)FdI, containing only a [3Fe-3S] cluster. The MCD and EPR data demonstrate that the [3Fe-3S] cluster in (3Fe)FdI is essentially identical in structure to that in the native protein.

Azotobacter↗
PubMed1991

Does ferredoxin I (Azotobacter) represent a novel class of DNA-binding proteins that regulate gene expression in response to cellular iron(II)?

Azotobacter vinelandii (Av) and chroococcum (Ac) ferredoxin I contain [3Fe-4S]1 + 0 and [4Fe-4S]2+1+ clusters, when isolated aerobically, which undergo one-electron redox cycles at potentials of -460 +/- 10 mV (vs SHE) at pH 8.3 and -645 +/- 10 mV, respectively. The X-ray structure of Fd I (Av) reveals that the N-terminal half of the polypeptide folds as a sandwich of beta-strands which enclose the iron-sulphur clusters. The C-terminal sequence contains an amphiphilic alpha-helix of four turns which lies on the surface of the beta-barrel. Fd I (Av) controls expression of an unknown protein of Mr approximately 18,000. Fd I (Ac) will complex iron(II) avidly above pH approximately 8.0 only when the [3Fe-4S] cluster is reduced and provided that cellular nucleic acid is bound. Fd I (Ac) rigorously purified from nucleic acid does not undergo iron(II) uptake. These facts, together with recent evidence that the interconversion process [3Fe-4S]0 + Fe2+----[4Fe-4S]2+ in the iron-responsive element binding protein (IRE-BP) of eukaryotic cells is controlling protein expression at the level of mRNA [1991, Cell 64, 4771; 1991, Nucleic Acid Res. 19, 1739] leads to the following hypothesis. Fd I is a DNA-binding protein which interacts by single alpha-helix binding in the wide groove of DNA. The binding is regulated by iron(II) levels in the cell. The 7Fe form binds to DNA and represses gene expression. Only the DNA-bound form of the 7Fe Fd I will take up iron(II), not the form free in solution. Iron(II) becomes bound when the [3Fe-4S] cluster is reduced. The 8Fe Fd I thus generated no longer binds DNA and the gene is de-repressed. Sequence comparisons and the crystal structure suggests that the two central turns of the alpha-helix are important elements of the DNA-recognition process and that residues Gln69 and Glu73, which lie on the outer surface of the helix, hydrogen-bond with specific base pairs.

Amino Acid Sequence↗
PubMed1991

2D 1H NMR studies of oxidized 2(Fe4S4) ferredoxin from Clostridium pasteurianum.

Oxidized ferredoxin from Clostridium pasteurianum, containing two Fe4S4 clusters, has been investigated using 2D 1H NMR spectroscopy at 600 MHz. 2D NMR experiments allowed complete assignment of the sixteen isotropically shifted signals corresponding to the beta-CH2 protons of the eight metal coordinated cysteines. Geminal connectivities of Cys beta-CH2 protons were identified through magnitude COSY experiments and confirmed through 2D NOESY experiments. A few additional signals could be assigned to the corresponding alpha-CH protons. The importance of 2D experiments to achieve firm assignments of isotropically shifted signals in paramagnetic metalloproteins is stressed.

Clostridium↗
PubMed1992

Kinetic analysis of translocation into isolated chloroplasts of the purified ferredoxin precursor.

Time courses of the import into isolated chloroplasts of the purified ferredoxin precursor were measured at different protein concentrations. Analysis of the initial import rates indicates the presence of one saturable import system with an apparent Km value of approximately 100 nM and a Vmax corresponding to the uptake of approximately 2.5 x 10(4) precursor proteins per minute per chloroplast. We conclude that the in vitro observed activity of the chloroplast protein import machinery, functioning independently from cytosolic factors, would be enough to allow chloroplast development at physiological rate.

Biological Transport↗
PubMed1992

The haemoglobin-like protein (HMP) of Escherichia coli has ferrisiderophore reductase activity and its C-terminal domain shares homology with ferredoxin NADP+ reductases.

Three soluble ferrisiderophore reductases (FsrA, FsrB and FsrC) were detected in Escherichia coli. FsrB was purified and identified as the haemoglobin-like protein (HMP) by size and N-terminal sequence analyses. HMP was previously isolated as a dihydropteridine reductase and is now shown to have ferrisiderophore reductase activity. Database searches revealed that the C-terminal region of HMP (FsrB) is homologous to members of a family of flavoprotein oxidoreductases which includes ferredoxin NADP+ reductase (FNR). The combination of FNR-like and haemoglobin-like regions in HMP (FsrB) represents a novel pairing of functionally and structurally distinct domains. Structure-function properties of other FNR-like proteins, including LuxG and VanB, are also discussed.

Bacterial Proteins↗
PubMed1993

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.

Adenosine Triphosphate↗
PubMed1993

Selective interaction of ferricyanide with cluster I of Clostridium pasteurianum 2[Fe4S4] ferredoxin.

Treatment of Clostridium pasteurianum ferredoxin (CpFd) with stoichiometric amounts of potassium ferricyanide results in the specific conversion of cluster I into a Fe3S4+ species while leaving cluster II unaltered. Ferricyanide-treated CpFd derivative has been purified and characterized through biochemical and spectroscopical techniques. The cluster conversion process is reversible and reconstitution of native CpFd has been afforded under appropriate conditions.

Amino Acid Sequence↗
PubMed1994

The pH dependent spectral properties of Clostridium pasteurianum 2[Fe4S4] ferredoxin.

The recently assigned 1H NMR hyperfine signals of Clostridium pasteurianum ferredoxin were investigated over the pH range 8-12 to monitor possible pH-dependent conformational changes of the protein. For very high pH values minor perturbations were detected in the chemical shifts of three signals assigned to beta-CH2 cysteine protons of cluster II, while cluster I was not affected at all. These chemical shift variations, which can be fitted to a single pKa = 10.9, are interpreted as an effect of deprotonation of the phenolic group of Tyr-2, located reasonably close to cluster II. This hypothesis has been supported by means of other techniques such as CD and absorption spectroscopy that, on turn, are able to reveal minor pH-dependent spectral variations at high pH. Finally a UV difference experiment has provided further evidence for deprotonation of the phenolic group of Tyr-2. The possible influence of deprotonation of Tyr-2 on the redox properties of cluster II is discussed.

Bacterial Proteins↗
PubMed1994

NMR structures of ferredoxin chloroplastic transit peptide from Chlamydomonas reinhardtii promoted by trifluoroethanol in aqueous solution.

The 32-amino acid transit peptide of the unicellular green alga Chlamydomonas reinhardtii ferredoxin has been synthesized and analysed by NMR spectroscopy and circular dichroism. The results show that while the peptide is unstructured in water, it undergoes an alpha-helix formation from residue 3 to 13 in a 30:70 molar-ratio mixture of 2,2,2-trifluoroethanol. The remainder of the peptide is still unstructured in CF3CD2OD/H2O mixtures, but is distributed on a side opposite to a hydrophobic ridge formed by Met5, Phe9 and Val13 on the induced alpha-helix. The NMR structures driven by 2,2,2-trifluoroethanol in aqueous solution, are discussed in terms of potent interactions with the chloroplast envelope and its translocation molecular machinery.

Amino Acid Sequence↗
PubMed1995

Transit sequence-dependent binding of the chloroplast precursor protein ferredoxin to lipid vesicles and its implications for membrane stability.

The binding of the transit peptide (trfd) and precursor of the chloroplast protein ferredoxin (prefd) to large unilamellar lipid vesicles was investigated in relation to the lipid composition of the bilayer. Prefd binds with a dissociation constant of 0.27 microM to vesicles with a composition corresponding to the chloroplast envelope outer membrane. Binding is mediated by the transit sequence. From an analysis of binding to vesicles containing the individual lipid components it could be concluded that anionic lipids are mainly responsible for binding, emphasizing the importance of electrostatics for the transit sequence-lipid interaction. Binding is also mediated by the specific chloroplast glycolipid monogalactosyldiacylglycerol. Monolayer experiments revealed that in this case a more extended domain of the transit sequence inserts into the lipid layer. Precursor binding does not result in a loss of vesicle barrier function. However, high concentrations of trfd do cause release of vesicle-enclosed carboxyfluorescein. The results are discussed in the light of the chloroplast protein import process, with special emphasis on the role of monogalactosyldiacylglycerol.

Amino Acid Sequence↗
PubMed1996

Evidence for an association of ndh B, ndh J gene products and ferredoxin-NADP-reductase as components of a chloroplastic NAD(P)H dehydrogenase complex.

Using non-denaturing gel electrophoresis and staining with nitro-blue tetrazolium, we reveal the presence of two NAD(P)H oxidoreductase activity bands within thylakoids membranes of Solanum tuberosum L. Second dimension SDS-PAGE and Western analysis show that one of the activity bands contains several polypeptides, two of them being recognized by antibodies directed against peptides corresponding to conserved domains of chloroplastic genes products NDH B and NDH J (at 32 and 18 kDa, respectively). Both activity bands also contain a polypeptide (around 36 kDa) recognized by an antibody directed against ferredoxin-NADP(+)-reductase (FNR). We conclude from these results that both chloroplastic ndh B and ndh J gene products are components of a thylakoid NAD(P)H dehydrogenase complex. The association with FNR is suggested to allow the complex to use NADPH instead of NADH as a preferential substrate.

Amino Acid Sequence↗