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At least 1,063 records · Page 59Linked to original sources
PubMed1994

Some structural features of cluster-coordinating cysteines of Clostridium pasteurianum ferredoxin are revealed by 2D TOCSY 1H NMR on the oxidized protein.

Different sets of geminal J coupling constants for the eight beta-CH2 protons in the iron-coordinating cysteines in Clostridium pasteurianum ferredoxin were detected by 2D TOCSY 1H NMR experiments on the oxidized protein. Four resonances were characterized by quite similar high values of J, two more resonances had a J value about half of the former ones, while the last two had extremely low J values. These findings suggest that the cysteines required for cubane symmetry around the iron atoms are constrained into different geometries. The simplified model used for fine tuning of tau m in these TOCSY experiments is also presented and discussed.

Clostridium↗
PubMed1996

Elimination of nitrite from the explosive 2,4,6-trinitrophenylmethylnitramine (tetryl) catalyzed by ferredoxin NADP oxidoreductase from spinach.

Nitroreductase enzymes generally catalyze the reduction of nitroaromatic compounds to the corresponding amines. In contrast, ferredoxin NADP oxidoreductase (FNR), glutathione reductase, xanthine oxidase, and cytochrome c reductase catalyze the NADPH dependent elimination of the nitramine nitro group from 2,4,6-trinitrophenylmethylnitramine to form N-methylpicramide (NMP). Nitrite elimination was inhibited under aerobic conditions. Our results suggest that under aerobic conditions, tetryl is enzymatically reduced to the nitroanion radical which is then involved in the reduction of molecular oxygen. Under anaerobic conditions, the radical is reduced to NMP and nitrite is eliminated.

Anaerobiosis↗
PubMed2001

Characterization of recombinant Desulfovibrio gigas ferredoxin.

Dg ferredoxin gene was cloned using the polymerase chain reaction (PCR), inserted into vector pT7-7, and overexpressed in Escherichia coli (E. coli) grown in aerobic media. The recombinant protein is a dimer and contains a [3Fe-4S] cluster per monomer. EPR and (1)H NMR data of recombinant and wild-type protein are compared.

Cloning, Molecular↗
PubMed1995

Refined crystal structure of spinach ferredoxin reductase at 1.7 A resolution: oxidized, reduced and 2'-phospho-5'-AMP bound states.

The crystal structure of spinach ferredoxin-NADP(+)-oxidoreductase (FNR), determined by multiple isomorphous replacement at 2.6 A resolution, has been refined at 1.7 A resolution to an R-factor of 17.9%. The structure of FNR bound to the competitive inhibitor 2'-phospho-5'-AMP (P-AMP) has also been refined at 1.7 A to an R-factor of 17.4% and dithionite-reduced/P-AMP-bound FNR has been refined at 2.0 A to an R-factor of 14.9%. The P-AMP-bound structure was used to construct a model for the binding of NADP+. Over 200 solvation sites were included in each structure, and many of the best defined solvation sites stabilize buried turns. A bulk solvent correction obviated the need for a low-resolution data cutoff. An acidic side-chain likely to be responsible for the low pH requirement for crystallization has been identified. Three large networks of the hydrophobic side-chains help define the FNR structure. One of these contains a large cavity far from the active site, which coincides with the lone site of sequence heterogeneity in FNR, and may provide a site for membrane attachment. The reduced structure shows that Ser96 moves toward atom N-5 of FAD and a water molecule moves toward atom N-1 of FAD, while the flavin moiety remains planar. Possible sources of a proton that must be picked up upon reduction are discussed.

Binding Sites↗
PubMed1998

Determination of the structure of oxidised Desulfovibrio africanus ferredoxin I by 1H NMR spectroscopy and comparison of its solution structure with its crystal structure.

The solution structure of the 64 amino acid Fe4S4 ferredoxin I from Desulfovibrio africanus has been determined using two-dimensional 1H NMR spectroscopy. Sequence-specific assignments were obtained for 59 amino acid residues and the structure determined with the program DIANA on the basis of 549 nuclear Overhauser enhancement (NOE) upper distance limits, and four dihedral angle and 52 distance constraints for the Fe4S4 cluster. The NMR structure was refined using the simulated annealing and energy minimisation protocols of the program X-PLOR to yield a final family of 19 structures selected on the basis of good covalent geometry and minimal restraint violations. The r.m.s.d. values to the average structure for this family are 0.49(+/-0.07) A and 0.94(+/-0.09) A for the backbone and heavy-atoms of residues 3 to 62, respectively. The NMR structure has been compared to the previously reported X-ray structures for the two molecules within the asymmetric unit of the crystal, which have a network of seven hydrogen bonds between them. This intermolecular interface, involving residues 38, 40 to 43 and 46, has the same conformation in the solution structures showing that the crystal packing does not perturb the structure. There are three regions in which the NMR and X-ray structures differ: around the cluster, a turn involving residues 8 to 10, and a loop involving residues 29 to 32. In the family of solution structures the backbone of the loop region incorporating residues 29 to 32 is well-defined whilst in both of the X-ray molecules it is ill-defined. The small differences between the X-ray and NMR structures for the cluster environment and the turn between residues 8 to 10 probably reflects a lack of NMR constraints. The observation of relatively rapid amide NH hydrogen exchange of NH groups close to the cluster, together with rapid flipping for Phe25, which is also close to the cluster, indicates that the cluster environment is more dynamic than the corresponding regions of related Fe/S proteins.

Amino Acid Sequence↗
PubMed1994

Overexpression of ferredoxin I in Azotobacter vinelandii.

Azotobacter vinelandii has recently been used for a variety of genetic experiments which take advantage of its facile transformation system and its high-frequency homologous recombination. One gene that has been cloned and sequenced is the fdxA gene that encodes a small Fe-S protein called A. vinelandii ferredoxin I (AvFdI). Because this protein has been extensively characterized by X-ray crystallography and spectroscopic methods, we are using it as a model to address some general questions in Fe-S biochemistry. AvFdI is not a very abundant protein in wild-type cells, so to facilitate our biochemical studies we have developed the overexpression system described herein. The results show that AvFdI can be easily overproduced ca. 50-fold in its native background, by introducing multiple copies of the fdxA gene into A. vinelandii, on the broad-host-range multicopy plasmid, pKT230. The protein can be expressed from its own constitutive promoter or from the controlled nifH promoter. The overproduced protein has no deleterious effects on the organism and is identical to the protein produced by wild-type cells. This A. vinelandii-based system should be generally useful for the overproduction of other A vinelandii proteins or for the expression of genes from thermophilic or other organisms with similarly high G-C contents, or for the expression of O2-sensitive metalloproteins that are unstable in other systems.

Azotobacter vinelandii↗
PubMed1996

A cyanobacterial narB gene encodes a ferredoxin-dependent nitrate reductase.

The narB gene from the cyanobacterium Synechococcus sp. PCC 7942 was cloned downstream from the LacI-regulated promoter Ptrc in the Escherichia coli vector pTrc99A, rendering plasmid pCSLM1. Addition of isopropyl-beta-D-thiogalactoside to E. coli (pCSLM1) resulted in the parallel expression of a 76 kDa polypeptide and a nitrate reductase activity with properties identical to those known for nitrate reductase isolated from Synechococcus cells. As is the case for nitrate reductase from Synechococcus cells, either reduced methyl viologen or reduced ferredoxin could be used as an electron donor for the reduction of nitrate catalyzed by E. coli (pCSLM1) extracts. This data shows that narB is a cyanobacterial structural gene for nitrate reductase.

Base Sequence↗
PubMed1995

Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings.

Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163-168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a lambda gt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.

Amino Acid Oxidoreductases↗
PubMed1993

Sequence analysis of pre-ferredoxin-NADP(+)-reductase cDNA from Cyanophora paradoxa specifying a precursor for a nucleus-encoded cyanelle polypeptide.

A cDNA clone for pre-ferredoxin-NADP+ reductase (FNR) was obtained by screening a Cyanophora paradoxa expression library with antibodies specific for cyanelle FNR. The 1.4 kb transcript was derived from a single-copy gene. The precursor (41 kDa) and mature forms (34 kDa) of FNR were identified by western blotting of in vitro translation products and cyanelle extracts, respectively. The derived amino acid sequence of the mature form was corroborated by data from N-terminal protein sequencing and yielded identity scores from 58% to 62% upon comparison with cyanobacterial FNRs. Sequence conservation seemed to be even more pronounced in comparison with enzymes from higher plants, but using the neighbor joining method the C. paradoxa sequence was clearly positioned between the prokaryotic and eukaryotic sequences. The transit peptide of 65 or 66 amino acids appeared to be totally unrelated to those from spinach, pea and ice plant but showed overall characteristics of stroma-targeting peptides.

Amino Acid Sequence↗
PubMed1994

Characteristic features of the heterologous functional synthesis in Escherichia coli of a 2[4Fe-4S] ferredoxin.

Different strategies have been used to express synthetic genes all encoding Clostridium pasteurianum 2[4Fe-4S] ferredoxin (Fd) in Escherichia coli. The polypeptide can be produced as the C-terminal addition to a hybrid Cro::Protein A fusion protein lacking the metallic centers. The incorporation of the [4Fe-4S] clusters into the cleaved apoFd cannot be carried out in the same conditions as those affording holoFd from purified C. pasteurianum apoFd. In contrast, fully functional Fds can be produced from non-fused synthetic genes under the dependence of strong promoters. The yields of recombinant Fd, although sufficient to purify significant quantities of protein, are limited by the very short half-life of the 2[4Fe-4S] Fd in E. coli, irrespective of the expression system used. These features are characteristic of 2[4Fe-4S] Fds when compared with the far more stable recombinant rubredoxin, and probably other small iron-sulfur proteins which have already been produced in high yields. The reasons for the high turnover of 2[4Fe-4S] Fds are discussed.

Amino Acid Sequence↗
PubMed1993

The ORF encoding a putative ferredoxin-like protein downstream of the vnfH gene in Azotobacter vinelandii is involved in the vanadium-dependent alternative pathway of nitrogen fixation.

An open reading frame (ORF) in the same operon as, but downstream of, vnfH in Azotobacter vinelandii can code for a ferredoxin-like protein. The role this ORF may play in the vnf (vanadium-dependent alternative) pathway of nitrogen fixation was investigated. Site-directed mutagenesis was used to alter one base in each of the codons specifying amino acids 18 and 19 generating a unique Bg/II site. A kanamycin resistance cartridge was cloned into the Bg/II site. This construct was mobilized into A. vinelandii CA12 (delta nifHDK) strain by conjugation and the mutation was introduced into the genome by marker exchange. The resulting mutant was unable to fix nitrogen under conditions in which the vnf pathway of nitrogen fixation operates. This suggests that this ORF is functional and is essential for the vanadium-dependent alternative pathway of nitrogen fixation in A. vinelandii.

Amino Acid Sequence↗
PubMed1992

Transcriptional analysis and promoter mapping of the fdxA gene which encodes the 7Fe ferredoxin (FdII) of Rhodobacter capsulatus.

The structural gene (fdxA) coding for ferredoxin II (FdII) of the photosynthetic bacterium Rhodobacter capsulatus has been previously cloned and sequenced. Transcription of the fdxA gene was studied by mRNA analyses and by use of plasmid-borne fdxA::lacZ translational fusions. The transcription start site was mapped 23 bp upstream of the initiation codon, as deduced from analysis of mRNA by mung bean nuclease protection and primer extension experiments. A motif resembling the canonical sequence observed in sigma 70-dependent Escherichia coli promoters is present at the expected distance (-10/-35) from the proposed transcription start site. mRNA analysis by Northern hybridization revealed a fdxA-specific transcript of approximately 0.4 kb, indicating that fdxA is transcribed as a single gene.fdxA expression, as measured by the activity of the fdxA::lacZ fusion, was found to be constant during growth and reached a similar level under all growth conditions tested. These results suggest that FdII is constitutively synthesized in R. capsulatus.

Base Sequence↗
PubMed1993

Characterization of the promoter from the single-copy gene encoding ferredoxin-NADP(+)-oxidoreductase from spinach.

We describe a genomic DNA segment from spinach that bears part of the single-copy gene for ferredoxin-NADP(+)-oxidoreductase (FNR) including a 3.4 kb promoter sequence. Dissection of this DNA segment and its analysis in GUS (beta-glucuronidase) gene fusions in transgenic tobacco demonstrated that the promoter differs in structure from all other promoters for thylakoid protein genes studied to date. Two regions with light-responsive elements were identified. One is located within the first 118 bp upstream of the transcription initiation site. A second fragment covering nucleotide positions -220 to -119 is capable of conferring light-dependent GUS gene expression on two different minimal promoters. The latter fragment binds a transacting factor in gel-shift assays. None of the fragments carries cis elements known from other genes to be involved in light-controlled expression. Comparison of the light responsiveness of GUS gene fusions controlled by the -753/+231 and -118/+231 regions indicates that they respond differentially to phytochrome-dependent signals and that their expression in tobacco is not restricted to tissue with functional chloroplasts.

Amino Acid Sequence↗
PubMed1994

Structure-function relations for ferredoxin reductase.

Ferredoxin: NADP+ reductase is representative of a large family of flavoenzymes which catalyze the interchange of reducing equivalents between one-electron carriers and the two-electron-carrying nicotinamide dinucleotides. The structure of the enzyme from spinach is known at 1.7 A resolution and this structure, together with results of chemical modification and site-directed mutagenesis studies, gives insights into features of the structure that are important for function.

Amino Acid Sequence↗
PubMed1976

Ferredoxin-dependent photosynthetic reduction of nitrate and nitrite by particles of Anacystis nidulans.

The dark and light reduction of nitrate and nitrite by cell-free preparations of the blue-green alga Anacystis nidulans has been investigated. The three following methods have been successfully applied to the preparation of active particulate fractions from the alga cells: (a) shaking with glass beads, (b) lysozyme treatment and lysis of the resulting protoplasts, and (c) sonication. The two enzymes of the nitrate-reducing system-namely, nitrate reductase and nitrite reductase-are firmly bound to the isolated pigment-containing particles, and can be easily solubilized by prolonging the vibration or sonication time. Both enzymes-whether solubilized or bound to the particles-depend on reduced ferredoxin as the immediate electron donor. In its presence, the alga particles catalyze the gradual photoreduction of nitrate to nitrite and ammonia, a process that can thus be considered as one of the most simple and relevant examples of Photosynthesis. Some of the properties of nitrate reductase have been studied. Nitrate reductase as well as nitrite reductase are adaptive enzymes repressed by ammonia.

Cyanobacteria↗
PubMedDate not supplied

Sp1-like proteins function in the transcription of human ferredoxin genes.

We characterized a regulatory element located in the -76 to -62 region of the human ferredoxin gene. This region bound to Sp1-like proteins with low affinity, as shown using electrophoretic mobility shift, competition, antibody binding, and Southwestern experiments. The similarity of the regulatory element to Sp1 extends beyond its DNA-binding domain, as cloned Sp1 functioned equally well when fused to a peptide that bound to an irrelevant site. The function of these Sp1-binding sites is mediated through the cAMP-dependent protein kinase (PKA) signaling pathway, because reporter genes downstream of the Sp1-binding sites were not activated in a PKA-deficient cell line. Transfection of the catalytic subunit of PKA restored activated transcription. Similar Sp1-binding sites identified in the CYP11A1 and CYP21 genes also controlled cAMP-dependent transcription of the reporter gene. Our finding of the function of Sp1-like proteins in steroidogenic gene transcription adds one more role Sp1 plays in controlling physiological events.

Adrenal Cortex↗