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PubMed1979

[Effects of specific reagents and urea on the reactivity of non-heme iron and thiol groups of pea and corn ferredoxins].

The reactivities of the SH-groups of pea and corn ferredoxins were found to be different. One or two SH-groups in the molecule of pea ferredoxin and one SH-group in the molecule of corn ferredoxin are readily available for the thyol group specific reagents. Four SH-groups of both ferredoxins are completely masked, i. e. available for the thyol reagents only after protein denaturation in the presence of urea. The rates of SH-group interaction with the sulfhydryl reagents in corn ferredoxin are lower than those in pea ferredoxin. The non-haem iron of pea ferredoxin interacts with the complex formers far more rapidly as compared to corn ferredoxin. The ferredoxins tested differ in the amount of iron atoms. The latter require the presence of oxygen for their complete interaction with the complex formers.

Ferredoxins↗
PubMed1983

[Properties of two forms of ferredoxin from Rhodopseudomonas capsulata].

Electrophoretically homogenous preparations of two forms of ferredoxin were isolated from the nitrogen-fixing cells of the purple non-sulphur bacterium Rhodopseudomonas capsulata B10. The values of Mr for ferredoxins I and II are 12000 and 18000, respectively. Ferredoxin I contains 8 atoms of Fe+2 and 8 atoms of S2-; ferredoxin II--4 atoms of Fe2+ and 4 atoms of S2- per molecule. The ferredoxins differ also in their absorption spectra, stability and catalytic activity during electron transfer to nitrogenase of Rh. capsulata. The reduction of C2H2 and evolution of H2 in the presence of ferredoxin I occurs twice as fast as compared to that in the presence of ferredoxin II. Ferredoxin I synthesis takes place in the nitrogen-fixing cells of Rh. capsulata alone, whereas ferredoxin II formation does not depend on the growth conditions.

Electron Transport↗
PubMed1993

Overexpression in Escherichia coli and affinity purification of chick kidney ferredoxin.

Vertebrate ferredoxins are 12-14-kDa iron-sulfur proteins, some of which transfer electrons to mitochondrial cytochrome P450s. The function of many of these cytochrome P450s is to catalyze stereospecific hydroxylation of endogenous steroids. As part of our interest in the kidney mitochondrial 1 alpha-hydroxylation of 25-hydroxyvitamin D3, we have constructed an expression plasmid coding for a fusion protein containing the chick kidney ferredoxin. We subcloned chick kidney ferredoxin cDNA, obtained from our vitamin D-deficient chick kidney library by polymerase chain reaction (Brandt, M. E., Gabrik, A. H., and Vickery, L. E. (1991) Gene (Amst.) 97, 113-117) into Qiagen's pQE9, which contains an N-terminal 6xHis tag (peptide sequence for 6 adjacent histidines present in the recombinant proteins). The coding sequence was preceded by a factor Xa cleavage site. The resulting plasmid, pQTcFdx, was overexpressed in Escherichia coli, and the soluble fusion protein was purified from the cell lysate in one step by Ni(II)-nitrilotriacetic acid-agarose chromatography. We obtained 7-10 mg of greater than 99% homogeneous fusion protein from a 1-liter culture and 4-6 mg of mature ferredoxin cleaved by factor Xa. The fusion protein possessed an absorption spectrum and an electron paramagnetic resonance spectrum quantitatively indistinguishable from those published for ferredoxin purified from adrenal glands and placenta or expressed in E. coli with another vector. The fusion protein was active in supporting the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 in a reconstitution assay of a solubilized, partially purified preparation of cytochrome P450 from vitamin D-deficient chick kidney. We conclude that the procedure described here is an efficient way to produce and purify vertebrate ferredoxin; the [2Fe-2S] cofactor is assembled in vivo and effectively incorporated into the fusion protein in E. coli; slight alterations at the N terminus do not alter incorporation of the [2Fe-2S] cofactor or the biological activity of ferredoxin, and post-translational modifications, such as phosphorylation, are not an absolute requirement for ferredoxin electron transporting activity. The recombinant ferredoxin can be used for physical studies and other structure-function studies.

Amino Acid Sequence↗
PubMed2002

Browsing gene banks for Fe2S2 ferredoxins and structural modeling of 88 plant-type sequences: an analysis of fold and function.

One-hundred-and-seventy-nine sequences of Fe2S2 ferredoxins and ferredoxin precursors were identified in and retrieved from currently available protein and cDNA databases. On the basis of their cluster-binding patterns, these sequences were divided into three groups: those containing the CX4CX2CXnC pattern (plant-type ferredoxins), those with the CX5CX2CXnC pattern (adrenodoxins), and those with a different pattern. These three groups contain, respectively, 139, 36, and 4 sequences. After excluding ferredoxin precursors in the first group, two subgroups were identified, again based on their cluster-binding patterns: 88 sequences had the CX4CX2CX29C pattern, and 29 had the CX4CX2CXmC (m not equal 29) pattern. The structures of the 88 ferredoxins with the CX4CX2CX29C pattern were modeled based on the available experimental structures of nine proteins within this same group. The modeling procedure was tested by building structural models for the ferredoxins with known structures. The models resulted, on average, in being within 1 A of the backbone root-mean-square deviation from the corresponding experimental structures. In addition, these structural models were shown to be of high quality by using assessment procedures based on energetic and stereochemical parameters. Thus, these models formed a reliable structural database for this group of ferredoxins, which is meaningful within the framework of current structural genomics efforts. From the analysis of the structural database generated it was observed that the secondary structural elements and the overall three-dimensional structures are maintained throughout the superfamily. In particular, the residues in the hydrophobic core of the protein were found to be either absolutely conserved or conservatively substituted. In addition, certain solvent-accessible charged groups, as well as hydrophobic groups, were found to be conserved to the same degree as the core residues. The patterns of conservation of exposed residues identified the regions of the protein that are critical for its function in electron transfer. An extensive analysis of protein-protein interactions is now possible. Some conserved interactions between residues have been identified and related to structural and/or functional features. All this information could not be obtained from the analyses of the primary sequences alone. Finally, the analysis of the sequences of the related subgroup featuring the CX4CX2CXmC (m not equal 29) cluster-binding pattern in the light of the structural and functional insights provided by the inspection of the mentioned structural database affords some hints on the functional features of ferredoxins belonging to this subgroup.

Amino Acid Sequence↗
PubMed1981

The involvement of ferredoxin-NADP+ reductase in cyclic electron transport in chloroplasts.

The sites of action, in spinach thylakoid, of known inhibitors of electron transport at the reducing end of photosystem I have been more accurately located by parallel investigation of effects on three partial reactions: photo-reduction (from water) of added NADP+, photoreduction of added cytochrome c, and dark reduction of cyto-chrome c by added NADPH. Comparison with inhibitory effects on cyclic electron flow (registered by the slow phase of the electrochromic response during repetitive flash excitation) permitted assessment of the role of ferredoxin and ferredoxin-NADP+ reductase (ferredoxin: NADP+ oxidoreductase, EC 1.18.1.3) in the cyclic electron transport pathway around photosystem I. Disulfodisalicylidenepropane-1,1-diamine inhibited all the above partial reactions except the ferredoxin-dependent photoreduction of cytochrome C. thereby indicating its interference with the reductase or the complexation between reductase and ferredoxin. Studies with purified ferredoxin-NADP+ reductase established it as the sensitive component. Cyclic flow is also sensitive to the above inhibitor and thus presumably involves the reductase. Supporting evidence for this came from studies of inhibition by substituted maleimides, which are inhibitors of electron transfer through the isolated reductase; these also inhibited the slow phase of the electrochromic response and all partial reactions except the photoreduction of cytochrome c. In contrast, an antiserum against the reductase affected only reactions involving NADP. The conclusion is drawn that the pathway of cyclic electron transport includes both ferredoxin and ferredoxin-NADP+ reductase, but not the NADP-binding site on the reductase.

Chloroplasts↗
PubMed1988

Ferredoxin and cytochrome P-450scc concentrations in granulosa cells of porcine ovaries during follicular cell growth and luteinization.

The concentrations of cytochrome P-450scc and ferredoxin, two of the three proteins which comprise the mitochondrial steroidogenic electron transport chain, were measured in granulosa and luteal cells from porcine ovaries by an immunoblot procedure. During the follicular phase of the ovarian cycle the concentration of cytochrome P-450scc increased 5-fold and ferredoxin increased 3-fold. When the large follicles developed into corpora lutea the cytochrome P-450scc concentration increased a further 7-fold while ferredoxin increased only 3-fold. These changes were coincident with an overall 4-fold increase in the concentration of ferredoxin reductase during follicular cell development and luteinization. Analysis of the data revealed that the concentration of ferredoxin, which shuttles electrons from ferredoxin reductase to cytochrome P-450scc, was always adequate to saturate both the reductase and cytochrome P-450scc. This came about from a co-ordinate increase in the concentration of cytochrome P-450scc and the concentration of ferredoxin minus ferredoxin reductase.

Animals↗
PubMed1988

Isolation and purification of mature bovine adrenocortical ferredoxin with an elongated carboxyl end.

Mature bovine adrenocortical ferredoxin (adreno-ferredoxin) was extracted from fresh adrenal glands at pH 9.0. Extraction and purification at this alkaline pH protected the mature adreno-ferredoxin molecule from proteolytic degradation. The mature adreno-ferredoxin was extensively purified by a rapid procedure including two kinds of column chromatography, hydrophobic and ion exchange. The purified adreno-ferredoxin was homogeneous on the basis of two HPLC analyses, hydrophobic and ion exchange, and had the highest purity so far reported. Then it was digested by trypsin and the carboxyl-terminal peptide was isolated from the tryptic digest by a novel column chromatographic method using a cation-exchange HPLC column, TSK-gel SP-5PW. The carboxyl-terminal amino acid was isoleucine, so the adreno-ferredoxin had 127 amino acid residues, the longest polypeptide so far determined chemically for bovine adreno-ferredoxin. Only Glu-128 was lacking within the carboxyl-terminal elongated peptide that was found by nucleotide sequencing of the adreno-ferredoxin gene. There was no evidence obtained on whether the deletion of Glu-128 was due to so-called carboxyl-terminal processing or to proteolytic degradation during storage and purification.

Adrenal Cortex↗
PubMed1992

Expression of human ferredoxin in Saccharomyces cerevisiae: mitochondrial import of the protein and assembly of the [2Fe-2S] center.

Vertebrate ferredoxins function in the transfer of reducing equivalents from NADPH:ferredoxin oxidoreductase to cytochrome P450 enzymes involved in steroid metabolism. We report here the expression of human mitochondrial ferredoxin in the yeast Saccharomyces cerevisiae. The full-length ferredoxin protein containing the ferredoxin mitochondrial leader sequence could not be stably expressed in S. cerevisiae, but a fusion protein consisting of the mature portion of ferredoxin linked to the mitochondrial leader sequence of the S. cerevisiae cytochrome c oxidase subunit Va protein (COX5a) could be stably expressed. The COX5a:ferredoxin fusion protein was targeted to the mitochondria as a preprotein and was cleaved at the normal processing site of the COX5a presequence during import into the matrix. Absorption spectra and electron transfer activity of the isolated fusion protein established that the [2Fe-2S] center was correctly assembled and incorporated into the recombinant ferredoxin in this heterologous system.

Amino Acid Sequence↗
PubMed1990

Purification and characterization of a 7Fe ferredoxin from Streptomyces griseus.

A ferredoxin has been purified from Streptomyces griseus grown in soybean flour-containing medium. The homogeneous protein has a molecular weight near 14,000 as determined by both PAGE and size exclusion chromatography. The iron and labile sulfide content is 6-7 atoms/mole protein. EPR spectroscopy of native S. griseus ferredoxin shows an isotropic signal at g = 2.01 which is typical of [3Fe-4S]1+ clusters and which quantitates to 0.9 spin/mole. Reduction of the ferredoxin by excess dithionite at pH 8.0 produces an EPR silent state with a small amount of a g = 1.95 type signal. Photoreduction in the presence of deazaflavin generates a signal typical of [4Fe-4S]1+ clusters at much higher yields (0.4-0.5 spin/mole) with major features at g-values of 2.06, 1.94, 1.90 and 1.88. This latter EPR signal is most similar to that seen for reduced 7Fe ferredoxins, which contain both a [3Fe-4S] and [4Fe-4S] cluster. In vitro reconstitution experiments demonstrate the ability of the S. griseus ferredoxin to couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450soy for NADPH-dependent substrate oxidation. This represents a possible physiological function for the S. griseus ferredoxin, which if true, would be the first functional role demonstrated for a 7Fe ferredoxin.

Chemical Phenomena↗
PubMed1995

Regulation of ferredoxin gene in steroidogenic and nonsteroidogenic cells.

Ferredoxin is an electron transport intermediate for all the mitochondrial cytochromes P450. It is especially abundant in steroidogenic organs where it functions in steroid biosynthesis. The regulation of ferredoxin gene expression was studied in both steroidogenic and nonsteroidogenic cell lines. In steroidogenic cell line Y1, the expression of ferredoxin was stimulated by cAMP and repressed slightly by angiotensin II and phorbol ester PMA. These drugs exhibited the same effect on the basal promoter of the ferredoxin gene, which includes one TATA box and an SP1 site. In human adrenocortical cell line H295, the stimulation of the ferredoxin gene by cAMP was blocked by cycloheximide, as observed in bovine adrenocortical cell culture. In nonsteroidogenic cell lines such as HeLa and COS-1, the stimulation of ferredoxin gene expression by cAMP was not observed, although basal expression was strong. Transfection studies showed that the ferredoxin promoter could not be stimulated by cAMP in nonsteroidogenic cells. Therefore the steroidogenic cell-specific regulation and the general expression pattern appears to be a property unique to the ferredoxin gene.

Adrenal Cortex↗
PubMed1998

PsaC subunit of photosystem I is oriented with iron-sulfur cluster F(B) as the immediate electron donor to ferredoxin and flavodoxin.

The PsaC subunit of photosystem I (PS I) binds two [4Fe-4S] clusters, F(A) and F(B), functioning as electron carriers between F(X) and soluble ferredoxin. To resolve the issue whether F(A) or F(B) is proximal to F(X), we used single-turnover flashes to promote step-by-step electron transfer between electron carriers in control (both F(A) and F(B) present) and HgCl2-treated (F(B)-less) PS I complexes from Synechococcus sp. PCC 6301 and analyzed the kinetics of P700+ reduction by monitoring the absorbance changes at 832 nm in the presence of a fast electron donor (phenazine methosulfate (PMS)). In control PS I complexes exogenously added ferredoxin, or flavodoxin could be photoreduced on each flash, thus allowing P700+ to be reduced from PMS. In F(B)-less complexes, both in the presence and in the absence of ferredoxin or flavodoxin, P700+ was reduced from PMS only on the first flash and was reduced from F(X)- on the following flashes, indicating lack of electron transfer to ferredoxin or flavodoxin. In the F(B)-less complexes, a normal level of P700 photooxidation was detected accompanied by a high yield of charge recombination between P700+ and F(A)- in the presence of a slow donor, 2,6-dichlorophenol-indophenol. This recombination remained the only pathway of F(A)- reoxidation in the presence of added ferredoxin, consistent with the lack of forward electron transfer. F(A)- could be reoxidized by methyl viologen in F(B)-less PS I complexes, although at a concentration two orders of magnitude higher than is required in wild-type PS I complexes, thus implying the presence of a diffusion barrier. The inhibition of electron transfer to ferredoxin and flavodoxin was completely reversed after reconstituting the F(B) cluster. Using rate versus distance estimates for electron transfer rates from F(X) to ferredoxin for two possible orientations of PsaC, we conclude that the kinetic data are best compatible with PsaC being oriented with F(A) as the cluster proximal to F(X) and F(B) as the distal cluster that donates electrons to ferredoxin.

Biophysical Phenomena↗
PubMed1997

Transcriptional regulation of the CYP11A1 and ferredoxin genes.

The first step in the synthesis of all steroids is the cleavage of cholesterol side chain, catalyzed by an electron transport system located in mitochondria consisting of ferredoxin reductase, ferredoxin, and cytochrome P450scc. These proteins are present in adrenal, gonad, placenta, and some parts of the brain. In addition, ferredoxin and ferredoxin reductase are also found in the kidney and liver. Whereas ferredoxin reductase levels remain constant in the cell, ferredoxin and P450scc levels are stimulated by trophic hormones using cAMP as an intracellular messenger. The ferredoxin promoter is relatively simple, consisting of a TATA box and two Sp1-binding sites. This simple module is enough to direct cAMP-dependent transcription in a steroidogenic cell-specific fashion. The regulatory region for the P450scc gene is more complex, containing many protein binding sites for different regulation purposes. Its TATA box directs cAMP-dependent transcription in a cell-type-specific manner. A transcription factor, steroidogenic factor 1 (SF1), activates P450scc gene expression. The tissue-specific expression of the P450scc gene is probably accomplished through the interaction of SF1 with other protein factors located further upstream of the control region. SF1 may also be involved in the cAMP response. An upstream region binding to cAMP-Responsive Element Binding Protein CREB and AP1 can respond to cAMP for gene activation. These analyses of regulatory elements provide the structural architecture for transcriptional regulation of the ferredoxin and the CYP11A11 gene.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

NMR spectroscopic studies of the hydrogenosomal [2Fe-2S] ferredoxin from Trichomonas vaginalis: hyperfine-shifted 1H resonances.

The hyperfine-shifted 1H NMR resonances of oxidized and reduced Trichomonas vaginalis ferredoxin, a functionally unique [2Fe-2S] ferredoxin, have been studied. The oxidized protein spectrum displayed a pattern of six broad upfield-shifted resonances between 13 and 40 ppm with chemical shifts distinct from those of other [2Fe-2S] ferredoxins. All hyperfine 1H resonances of the oxidized ferredoxin displayed anti-Curie temperature dependences. Reduced T. vaginalis ferredoxin displayed hyperfine resonances both upfield and downfield of the diamagnetic region. These resonances showed Curie temperature dependences. Overall the hyperfine-shifted NMR spectrum of T. vaginalis ferredoxin, along with other spectroscopic properties, suggested different structural properties for the active center of oxidized hydrogenosomal ferredoxins from those of other [2Fe-2S] ferredoxins.

Animals↗
PubMed1983

Primary structure of the two (4 Fe-4 S) clusters ferredoxin from Desulfovibrio desulfuricans (strain Norway 4).

The primary structure of a ferredoxin isolated from D. desulfuricans Norway strain, which we called ferredoxin II (Fd II) has been elucidated. This ferredoxin is a dimer constituted of two identical subunits of molecular weight 6000. In ferredoxin II two (4 Fe-4 S) centers are present per subunit instead of one (Fe-S) center as is the case for the other ferredoxins isolated from Desulfovibrio and for Fd I from the same organism. The comparison of amino-acid sequences shows that ferredoxin II presents more homologies with clostridial type ferredoxin than with the ferredoxins from D. gigas and D. africanus.

Amino Acid Sequence↗
PubMed2003

NMR studies of a ferredoxin from Haloferax mediterranei and its physiological role in nitrate assimilatory pathway.

Haloferax mediterranei is a halophilic archaeon that can grow in aerobic conditions with nitrate as sole nitrogen source. The electron donor in the aerobic nitrate reduction to ammonium was a ferredoxin. This ferredoxin has been purified and characterised. Air-oxidized H. mediterranei ferredoxin has a UV-visible absorption spectra typical of 2Fe-type ferredoxins with an A420/A280 of 0.21. The nuclear magnetic resonance (NMR) spectra of the ferredoxin showed similarity to those of ferredoxins from plant and bacteria, containing a [2Fe-2S] cluster. The physiological function of ferredoxin might be to serve as an electron donor for nitrate reduction to ammonium by assimilatory nitrate (EC 1.6.6.2) and nitrite reductases (EC 1.7.7.1). The apparent molecular weight (Mr) of the ferredoxin was estimated to be 21 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Ferredoxins↗
PubMed1995

NMR of Chromatium vinosum ferredoxin: evidence for structural inequivalence and impeded electron transfer between the two [4Fe-4S] clusters.

The 2[4Fe-4S] ferredoxin from Chromatium vinosum has been investigated by 1H and 13C nuclear magnetic resonance. 1H NMR sequence-specific assignments have been obtained for a large majority of the residues. They indicate that the protein folds along a pattern similar to that previously evidenced for shorter 2[4Fe-4S] ferredoxins. However, C. vinosum ferredoxin differs from other ferredoxins by the occurrence of a turn in an eight amino acid region separating two successive cysteines, Cys-40 and Cys-49, liganding one cluster. Also, the unique C-terminal end of C. vinosum ferredoxin contains a 10 amino acid alpha-helix which interacts with one side of the above turn. The only cysteine of the sequence not involved in the ligation of the [4Fe-4S] clusters is Cys-57. Specific NMR experiments helped characterizing the signals arising from the ligands of these clusters: most of them display properties reminiscent of those of homologous ferredoxins, except for the signals associated with Cys-40. Despite the general similarity between C. vinosum ferredoxin and other 2[4Fe-4S] ferredoxins, the electron paramagnetic resonance and NMR spectra of the former reduced protein are significantly different from those previously observed for S = 1/2 [4Fe-4S]+ clusters. In addition, the intramolecular electron transfer rate in C. vinosum is far slower than in other similar cases. This is the first report of impeded electron exchange between two [4Fe-4S] clusters expected to be less than 12 A apart.

Amino Acid Sequence↗
PubMed1995

Probing the role of electrostatic forces in the interaction of Clostridium pasteurianum ferredoxin with its redox partners.

The ability of several low-potential redox proteins to mediate electron transfer between Clostridium pasteurianum pyruvate-ferredoxin oxidoreductase and hydrogenase has been evaluated in a coupled enzymatic assay. The active electron mediators, whatever their structure, must have a reduction potential compatible with the two enzymes, but for proteins of similar potentials, a marked specificity is displayed by 2[4Fe-4S] ferredoxins of the clostridial type. Such ferredoxins are small proteins exchanging electrons with many enzymes involved in the metabolism of anaerobic bacteria. The forces underlying the interactions of ferredoxin with hydrogenase and pyruvate-ferredoxin oxidoreductase have been examined with an emphasis on electrostatics: site-directed mutagenesis experiments have been used to individually convert all conserved glutamates and aspartates of C. pasteurianum ferredoxin into either neutral or positively charged amino acids. Also, up to four of these residues have been replaced simultaneously. The biological activities of the resulting variants depend very little on the number and the distribution of the anionic side chains on the surface of the ferredoxin. Only those molecular forms for which the immediate environment of the clusters is perturbed, independently of the charge distribution, display variations in their catalytic properties. It is concluded that electron transfer between C. pasteurianum 2[4Fe-4S] ferredoxin and its partners is far less dependent on electrostatic interactions than in many other well-documented electron transfer systems.

Amino Acid Sequence↗
PubMed1982

Complete amino acid sequence of the 4Fe-4S, thermostable ferredoxin from Clostridium thermoaceticum.

The complete amino acid sequence of the 4Fe-4S ferredoxin from the thermophilic bacterium Clostridium thermoaceticum has been determined. The protein is extremely thermostable and is the only known clostridial ferredoxin to contain a single [4Fe-4S] cluster. The sequence totals 63 residues and includes the first tryptophan (Trp-26) reported for a clostridial ferredoxin, and other amino acids not commonly found in clostridial or clostridial-like ferredoxins: methionine (Met-1), histidine (His-33), arginine (Arg-49), and leucine (Leu-9, -19, and -31). Sequence homology to clostridial and other 8Fe-8S ferredoxins is limited to eight to nine residues at the amino-terminal sulfhydryl grouping (Cys-10, -13, -16, and -20) and two to five residues in the carboxyterminal region. This ferredoxin is, thus, sequentially distinct from all known clostridial ferredoxins and from other bacterial ferredoxins in both the 8Fe-8S and 4Fe-4S classes.

Amino Acid Sequence↗