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PubMed2002

Ferredoxin-mediated reactivation of the chlorocatechol 2,3-dioxygenase from Pseudomonas putida GJ31.

In the chlorobenzene degrader Pseudomonas putida GJ31, chlorocatechol is formed as an intermediate and cleaved by a meta-cleavage extradiol chlorocatechol dioxygenase, which has previously been shown to be exceptionally resistant to inactivation by substituted catechols. The gene encoding this dioxygenase ( cbzE) is preceded by a gene ( cbzT) potentially encoding a ferredoxin, the function of which was studied. The cbzT gene product was overproduced in Escherichia coli and purified in recombinant form. Two homologous proteins, CdoT and AtdS, encoded by genes identified in strains degrading nitrobenzene and aniline, respectively, were also purified and characterized. All three proteins showed spectroscopic properties typical for [2Fe-2S] ferredoxins. The chlorocatechol dioxygenase from strain GJ31 (CbzE) was fully inactivated when 4-methylcatechol was used as substrate. Inactivated CbzE could be rapidly reactivated in vitro in the presence of purified CbzT and a source of reductant. It is inferred that the ability of strain GJ31 to metabolize both chlorobenzene and toluene might depend on the regeneration of the chlorocatechol dioxygenase activity mediated by CbzT. Three CbzT-like ferredoxins, including AtdS, were found to be competent in the reactivation of CbzE, whereas XylT, a protein known to mediate reactivation of the catechol dioxygenase from P. putida mt2 (XylE), was ineffective. Accordingly, CbzT formed a covalent complex with CbzE when cross-linked with a carbodiimide, whereas XylT did not. In the reverse situation, CbzT was found to reactivate XylE as efficiently as XylT and formed an heterologous covalent complex with this enzyme upon cross-linking. We conclude that CbzT, CdoT and AtdS are isofunctional ferredoxins that appear to be involved in the reactivation of their cognate catechol dioxygenases. Based on primary structure comparisons, residues of the ferredoxins possibly involved in the molecular interaction with catechol dioxygenases were identified and their significance is discussed.

Amino Acid Sequence↗

PubMed2006

Characterization of a novel ferredoxin with N-terminal extension from Clostridium acetobutylicum ATCC 824.

A gene (CAC2657) encoding a ferredoxin (EFR1) from the strictly anaerobic soil bacterium Clostridium acetobutylicum was cloned and expressed in Escherichia coli. The ferredoxin gene encodes a polypeptide of 27 kDa that incorporates 2[4Fe-4S] clusters. An extended N-terminal region of 187 amino acid (aa) residues precedes ferredoxin domain. The EFR1 expressed in E. coli is a trimeric protein. The iron and sulfur content of the reconstituted protein agrees with that expected of a trimeric form of the protein. The ferredoxin domain of EFR1 is closely related to ferredoxin of C. pasteurianum; and can be fitted to the X-ray crystal structure with a root mean square deviation of 0.62 As for the Calpha atoms of the generated 3D simulation model. In cultures of C. acetobutylicum the efr1 gene shows higher relative expression on induction with Trinitrotoluene (TNT) compared to that from uninduced control cultures.

Amino Acid Sequence↗

PubMed2005

Characterization of [3Fe-4S] ferredoxin DbfA3, which functions in the angular dioxygenase system of Terrabacter sp. strain DBF63.

Dibenzofuran 4,4a-dioxygenase (DFDO) from Terrabacter sp. strain DBF63 is comprised of three components, i.e., terminal oxygenase (DbfA1, DbfA2), putative [3Fe-4S] ferredoxin (ORF16b product), and unidentified ferredoxin reductase. We produced DbfA1 and DbfA2 using recombinant Escherichia coli BL21(DE3) cells as a native form and purified the complex to apparent homogeneity. We also produced and purified a putative [3Fe-4S] ferredoxin encoded by ORF16b, which is located 2.5 kb downstream of the dbfA1A2 genes, with E. coli as a histidine (His)-tagged form. The reconstructed DFDO system with three purified components, i.e., DbfA1A2, His-tagged ORF16b product, and His-tagged PhtA4 (which is a tentative reductase derived from the phthalate dioxygenase system of strain DBF63) could convert fluorene to 9-fluorenol (specific activity: 14.4 nmol min(-1) mg(-1)) and convert dibenzofuran to 2,2',3-trihydroxybiphenyl. This indicates that the ORF16b product can transport electrons to the DbfA1A2 complex; and therefore it was designated DbfA3. Based on spectroscopic UV-visible absorption characteristics and electron paramagnetic resonance spectra, DbfA3 was elucidated to contain a [3Fe-4S] cluster. Ferredoxin interchangeability analysis using several types of ferredoxins suggested that the redox partner of the DbfA1A2 complex may be rather specific to DbfA3.

Actinomycetales↗

PubMed2006

Effect of two intermediate electron donors, NADPH and FADH(2), on Spirulina Delta (6)-desaturase co-expressed with two different immediate electron donors, cytochrome b (5) and ferredoxin, in Escherichia coli.

When the gene desD encoding Spirulina Delta(6)-desaturase was heterologously expressed in E. coli, the enzyme was expressed without the ability to function. However, when this enzyme was co-expressed with an immediate electron donor, i.e. the cytochrome b (5) domain from Mucor rouxii, the results showed the production of GLA (gamma-linolenic acid), the product of the reaction catalyzed by Delta(6)-desaturase. The results revealed that in E. coli cells, where cytochrome b (5) is absent and ferredoxin, a natural electron donor of Delta(6)-desaturase, is present at a very low level, the cytochrome b (5) domain can complement for the function of ferredoxin in the host cells. In the present study, the Spirulina-ferredoxin gene was cloned and co-expressed with the Delta(6)-desaturase in E. coli. In comparison to the co-expression of cytochrome b ( 5 ) with the Delta(6)-desaturase, the co-expression with ferredoxin did not cause any differences in the GLA level. Moreover, the cultures containing the Delta(6)-desaturase co-expressed with cytochrome b (5) and ferredoxin were exogenously supplied with the intermediate electron donors, NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) and FADH(2) (flavin adenine dinucleotide, reduced form), respectively. The GLA level in these host cells increased drastically, by approximately 50%, compared to the cells without the intermediate electron donors. The data indicated that besides the level of immediate electron donors, the level of intermediate electron donors is also critical for GLA production. Therefore, if the pools of the immediate and intermediate electron donors in the cells are manipulated, the GLA production in the heterologous host will be affected.

Base Sequence↗

PubMed1987

Evidence from high-pressure liquid chromatography for the existence of two ferredoxins in plants.

Crude ferredoxin preparations were obtained from blue-green algae, green algae, ferns, and higher plants. We analyzed the preparations by high-pressure liquid chromatography using two different types of columns, a hydrophobic phenyl-5PW column and an ion-exchange DEAE-5PW column. Two ferredoxins were detected in all plants analyzed. The ferredoxins from some plants were separated by use of both columns and those from others were separated by one of the two columns. Thus, there were three possible ways in which pairs of ferredoxins from a single species of plant could be separated. We suspect that there are two different ferredoxins in most if not all species of plants.

Chlorella↗

PubMed1991

Monoclonal antibody studies of ferredoxin:NADP+ oxidoreductase.

Eleven independent monoclonal antibodies, all IgG's, have been raised against the ferredoxin:NADP+ oxidoreductase of spinach leaves. All 11 monoclonal antibodies were able to produce substantial inhibition of the NADPH to 2,6-dichlorophenol indophenol (DCPIP) diaphorase activity of the enzyme, but none of the antibodies produced any significant inhibition of electron flow from NADPH to ferredoxin catalyzed by the enzyme. Spectral perturbation assays were used to demonstrate that antibody interaction with NADP+ reductase did not interfere significantly with the binding of either ferredoxin or NADP+ to the enzyme. Ultrafiltration binding assays were used to confirm that the monoclonal antibodies did not interfere with complex formation between ferredoxin and the enzyme. These results have been interpreted in terms of the likely presence of one or more highly antigenic epitopes at the site where the nonphysiological electron acceptor, DCPIP, binds to the enzyme. Furthermore, the results suggest that the site where DCPIP is reduced differs from both of the two separate sites at which the two physiological substrates, ferredoxin and NADP+/NADPH, are bound.

Antibodies, Monoclonal↗

PubMed1992

pH-induced conformational changes in spinach ferredoxin: steady-state and time-resolved fluorescence studies.

Steady-state and time-resolved fluorescence techniques were used to monitor pH-induced conformational changes in spinach ferredoxin. An increase was seen in the wave-length maximum of tryptophan-73 (Trp-73) emission, from 325 nm below pH 6.0 to 342 nm above pH 7.0, indicating significantly diminished hydrophobicity, at pH 7.0, in the environment of the indole ring. Raising the solution pH from 6.0 to 7.6 also decreased the binding of the detergent Brij-96, showing that the ferredoxin molecule as a whole became more hydrophilic at higher pH. Nonionic (acrylamide) and ionic (I- and Cs+) quenchers were used to probe the tryptophan environment. Trp-73 is partially shielded from I-, presumably by negatively charged residues, as predicted from the amino acid sequence and three-dimensional structure of plant-type ferredoxins. Ionic strength and pH effects on tryptophan fluorescence lifetimes follow a pattern common to single-tryptophan proteins: the emission decays can be fit to a biexponential model in which the lifetime of the excited state increases with increasing pH. The indication of a pH-induced conformational change in the range pH 6.0 to 7.6 is discussed with reference to the physiological association of ferredoxin with ferredoxin:NADP+ oxidoreductase and the rise in chloroplast stromal pH in the light.

Detergents↗

PubMed1980

Biological activity of synthetic molybdenum-iron-sulphur, iron-sulphur and iron-selenium analogues of ferredoxin-type centres.

The molybdenum-iron-sulphur cluster [Fe6Mo2S8(SCH2CH2OH)9]3-, which contains two Fe3MoS4 cubane-like centres, is the best plausible analogue available to date for the molybdenum site of the nitrogenase enzymes. The iron-sulphur cluster [Fe4S4(S . CH2CH2OH)4]2- and the iron-selenium cluster [Fe4Se4(S . CH2CH2OH)4]2- are structural analogues of the ferredoxin Fe4S4 active centre. All three clusters would replace ferredoxin and mediate electron transfer to Clostridium pasteurianum hydrogenase in a H2-evolving system with sodium dithionite as the electron donor. The clusters would not replace hydrogenases which themselves are unable to evolve H2 from reduced ferredoxins. The molybdenum-iron-sulphur cluster would also replace ferredoxin in a chloroplast-ferredoxin-hydrogenase H2 evolving system.

Binding Sites↗

PubMed1980

Cyclic photophosphorylation reactions catalyzed by ferredoxin, methyl viologen and anthraquinone sulfonate. Use of photochemical reactions to optimize redox poising.

The flavin analogue 5-deazariboflavin is a convenient catalyst for the photoreduction of low-potential redox compounds. In an anaerobic medium with Tricine buffer as the electron donor, 5-deazariboflavin is capable of photo-reducing both ferredoxin and methyl viologen. We have used this method to conduct a comparative study of the Photosystem I photophosphorylation activities supported by the reduced forms of ferredoxin, methyl viologen and anthraquinone sulfonate. All of these catalysts are capable of generating high rates (200-500 mumol ATP/h per mg chlorophyll) of cyclic photophosphorylation, but only the activity dependent on ferredoxin exhibits sensitivity to antimycin A. This finding suggests that the size of the catalyst and its ability to approach the thylakoid membrane, rather than low-redox potential, governs antimycin A sensitivity. Ferredoxin-catalyzed activity is, however, less sensitive to inhibition by dibromothymoquinone than are the activities supported by methyl viologen and anthraquinone sulfonate. This discrepancy is due to binding of the inhibitor by ferredoxin.

Anthraquinones↗

PubMed1990

Soluble cytochromes and ferredoxins from the marine purple phototrophic bacterium, Rhodopseudomonas marina.

Four soluble c-type cytochromes, the high redox potential 4-Fe-S ferredoxin known as HiPIP, a large molecular weight 2-Fe-S ferredoxin and a 4-Fe-S 'bacterial' ferredoxin, were isolated from extracts of two strains of Rps. marina. Cytochrome c-550, cytochrome c' and cytochrome c-549 were previously described, and we have extended their characterization. Cytochrome c-558, which has not previously been observed in Rps. marina, appears to be a low-spin isozyme of the more commonly observed high-spin cytochrome c'. HiPIP, which was not observed in previous work, was found to be abundant in Rps. marina. The 2-Fe-S ferredoxin, which has previously been observed only in Rps. palustris, has a native size greater than 100 kDa and a subunit size of 17 kDa. The 'bacterial' ferredoxin appears to have only a single four-iron-sulfur cluster. We examined photosynthetic membranes by difference spectroscopy and found abundant c-type cytochromes. Approximately one-quarter of the heme can be reduced by ascorbate and the remainder by dithionite. There is 2 nm difference between the high-potential heme (554 nm) and the low (552 nm). These characteristics resemble those of the tetraheme reaction center cytochrome of Rps. viridis. In addition to the electron transfer components, we found small amounts of a fluorescent yellow protein which has spectral resemblance to a photoactive yellow protein from Ec. halophila.

Bacterial Proteins↗

PubMed1993

Small subunits of Photosystem I reaction center complexes from Synechococcus elongatus. II. The psaE gene product has a role to promote interaction between the terminal electron acceptor and ferredoxin.

Function of a subunit polypeptide (the psaE gene product) of Photosystem I (PS I) reaction center complexes was investigated by comparing the reactivity of the reduced iron-sulfur centers (FA/FB)- with ferredoxin among Synechococcus PS I complexes which had been variously depleted of this polypeptide. Ferredoxin at or below 1 microM can accept electrons from (FA/FB)- effectively competing with the back reaction between P-700+ and (FA/FB)- in the thylakoid membranes and PS I complexes that contained all the eight small subunits. The high reactivity of (FA/FB)- with low concentrations of ferredoxin was observed in PS I complexes which contain only the products of psaC, psaD and psaE genes but not in complexes which carry the psaC, psaD, psaL and psaK gene products but no psaE gene product. Varied amounts of the psaE gene product were extracted by treatment with different concentrations of a cationic detergent, dodecyltrimethylammonium bromide, and 2.5 M NaCl. The solubilized polypeptide was then reconstituted to the depleted complexes. The magnitudes of the back reaction that could be suppressed by addition of ferredoxin at or below 1 microM were well correlated to the amounts of the psaE polypeptide remained bound or rebound to the complexes. It is concluded that the product of the psaE gene has a role to promote the interaction between the terminal bound electron acceptor and ferredoxin. A high autooxidizability of (FA/FB)- and contrasting effects of lipophilic cations and anions on the rate of the back reaction from (FA/FB)- to P-700+ were also reported.

Cetrimonium↗

PubMed1979

Modification of arginyl residues in ferredoxin-NADP+ reductase from spinach leaves.

Reaction of spinach leaves ferredoxin-NADP+ reductase (NADPH:ferredoxin oxidoreductase, EC 1.6.7.1) with alpha-dicarbonyl compounds results in a biphasic loss of activity. The rapid phase yields modified enzyme with about 30% of the original activity, but no change in the Km for NADPH. Only partial protection against inactivation is provided by NADP+, NADPH and their analogs, whereas ferredoxin affords complete protection. The reductase inactivated to 30% of original activity shows a loss of about two arginyl residues, whereas only one residue is lost in the NADP+-protected enzymes. The data suggest that the integrity of at least two arginyl residues are requested for maximal activity of ferredoxin-NADP+ reductase: one residue being located near the NADP+-binding site, the other presumably situated in the ferredoxin-binding domain.

Aldehydes↗

PubMed1976

Ferredoxin biosynthesis in Euglena gracilis.

Analysis of ferredoxin content in cultures of Euglena gracilis grown in the presence of selective antibiotic inhibitors of protein synthesis resulted in the following conclusions: 1. Ferredoxin is synthesized from cytoplasmic (80s-type) ribosomes; cycloheximide, a potent inhibitor of 80s translation completely abolished the synthesis, while the inhibitors of 70s translation chloramphenicol and erythromycin were not effective. In addition, ferredoxin was detected in a streptomycin-bleached mutant that lacks the chloroplast structure and chloroplast DNA. 2. Ferredoxin's transcript is presumably of nuclear origin; rifampicin, an inhibitor of chloroplast DNA-dependent RNA polymerase did not inhibit synthesis, while the streptomycin-bleached mutant continued to synthesize ferredoxin without chloroplast DNA.

Animals↗

PubMed1979

A theoretical interpretation of the variations of some physical parameters within the [2Fe-2S] ferredoxin group.

A model is proposed to explain the variation of some physical parameters within the reduced [2Fe-2S] ferredoxin group. According to this model, the main effects result from a variable mixing of some d orbitals of the Fe2+ ion owing to rhombic distortion of the active site having the same geometrical character, but different in intensity, for each protein. Some peculiar experimental results such as the axial electron paramagnetic resonance spectra of adrenal ferredoxin and Pseudomonas putida ferredoxin and the electric field gradient tensor of P. putida ferredoxin are explained without assuming properties drastically different from those of the other ferredoxins, as had been suggested in the literature.

Ferredoxins↗

PubMed1983

Chemical modification of spinach ferredoxin with diethylpyrocarbonate: evidence for the essential nature of the histidyl residue.

Spinach ferredoxin contains a single ferredoxin which can be chemically modified with diethylpyrocarbonate. By varying the concentration of diethylpyrocarbonate modified ferredoxins could be prepared which had only one or both of the imidazole nitrogens of the histidine modified. A small amount of tyrosine was also modified. Ferredoxin with only one of the imidazole nitrogens modified was fully active in NADP photoreduction by chloroplast membranes. This activity was lost as the second imidazole nitrogen was modified. The results suggest an essential role for the single histidine of ferredoxin.

Diethyl Pyrocarbonate↗

PubMed1986

Heterogeneity of adrenocortical ferredoxin.

Bovine adrenocortical ferredoxin (adreno-ferredoxin) was purified from adrenocortical mitochondria by an improved method that included hydrophobic chromatography on Toyopearl gels. The purified ferredoxin was electrophoretically homogeneous. It was further separated into five fractions by hydrophobic chromatography on a TSK-gel phenyl-5PW column with a high-pressure liquid chromatography system. The properties of the three main fractions were examined. The fractions had identical absorption spectra and almost the same activity in an NADPH-cytochrome c reducing system. Their amino-terminal sequences all corresponded to the reported sequence, but the carboxyl-terminal residues were glycine or serine, not alanine as reported. These results indicate that these adreno-ferredoxins had additional amino acid residues at the carboxyl end. It seems that adreno-ferredoxin extracted from mitochondria undergoes proteolytic attack during purification to become heterogeneous.

Adrenal Cortex↗

PubMed1992

Lysine residues on ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 involved in substrate binding.

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.

Amino Acid Sequence↗

PubMed1994

Cloning, sequencing and overexpression of the Desulfovibrio gigas ferredoxin gene in E. coli.

We have cloned the gene encoding Desulfovibrio gigas ferredoxin using a photodigoxigenin-labelled probe synthesized with the polymerase chain reaction. The DNA sequence of the gene predicts a polypeptide of 58 residues after removal of the initial formyl methionine (polypeptide M(r) = 6,276). The ferredoxin gene was expressed in aerobically grown E. coli behind the lac promoter of pUC18 resulting in a high level of ferredoxin expression which comprises about 10% of the total cell protein. EPR analysis of recombinant ferredoxin revealed the presence of a [3Fe-4S] cluster which is characteristic of native D. gigas ferredoxin II.

Amino Acid Sequence↗