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PubMed1997

Y13C Azotobacter vinelandii ferredoxin I. A designed [Fe-S] ligand motif contains a cysteine persulfide.

Ferredoxins that contain [4Fe-4S]2+/+ clusters often obtain three of their four cysteine ligands from a highly conserved CysXXCysXXCys sequence motif. Little is known about the in vivo assembly of these clusters and the role that this sequence motif plays in that process. In this study, we have used structure as a guide in attempts to direct the formation of a [4Fe-4S]2+/+ in the [3Fe-4S]+/0 location of native (7Fe) Azotobacter vinelandii ferredoxin I (AvFdI) by providing the correct three-dimensional orientation of cysteine ligands without introducing a CysXXCysXXCys motif. Tyr13 of AvFdI occupies the position of the fourth ligating cysteine in the homologous and structurally characterized 8Fe ferredoxin from Peptococcus aerogenes and a Y13C variant of AvFdI could be easily modeled as an 8Fe protein. However, characterization of purified Y13C FdI by UV-visible spectra, circular dichroism, electron paramagnetic resonance spectroscopies, and by x-ray crystallography revealed that the protein failed to use the introduced cysteine as a ligand and retained its [3Fe-4S]+/0 cluster. Further, electrochemical characterization showed that the redox potential and pH behavior of the cluster were unaffected by the substitution of Tyr by Cys. Although Y13C FdI is functional in vivo it does differ significantly from native FdI in that it is extremely unstable in the reduced state possibly due to increased solvent exposure of the [3Fe-4S]0 cluster. Surprisingly, the x-ray structure showed that the introduced cysteine was modified to become a persulfide. This modification may have occurred in vivo via the action of NifS, which is known to be expressed under the growth conditions used. It is interesting to note that neither of the two free cysteines present in FdI was modified. Thus, if NifS is involved in modifying the introduced cysteine there must be specificity to the reaction.

Amino Acid Sequence↗
PubMed1999

Metronidazole resistance in the protozoan parasite Entamoeba histolytica is associated with increased expression of iron-containing superoxide dismutase and peroxiredoxin and decreased expression of ferredoxin 1 and flavin reductase.

To obtain insight into the mechanism of metronidazole resistance in the protozoan parasite Entamoeba histolytica, amoeba trophozoites were selected in vitro by stepwise exposures to increasing amounts of metronidazole, starting with sublethal doses of 4 microM. Subsequently, amoebae made resistant were able to continuously multiply in the presence of a 40 microM concentration of the drug. In contrast to mechanisms of metronidazole resistance in other protozoan parasites, resistant amoebae did not substantially down-regulate pyruvate:ferredoxin oxidoreductase or up-regulate P-glycoproteins, but exhibited increased expression of iron-containing superoxide dismutase (Fe-SOD) and peroxiredoxin and decreased expression of flavin reductase and ferredoxin 1. Episomal transfection and overexpression of the various antioxidant enzymes revealed significant reduction in susceptibility to metronidazole only in those cells overexpressing Fe-SOD. Reduction was highest in transfected cells simultaneously overexpressing Fe-SOD and peroxiredoxin. Although induced overexpression of Fe-SOD did not confer metronidazole resistance to the extent found in drug-selected cells, transfected cells quickly adapted to constant exposures of otherwise lethal metronidazole concentrations. Moreover, metronidazole selection of transfected amoebae favored retention of the Fe-SOD-containing plasmid. These results strongly suggest that peroxiredoxin and, in particular, Fe-SOD together with ferredoxin 1 are important components involved in the mechanism of metronidazole resistance in E. histolytica.

Animals↗
PubMed2005

A vertebrate-type ferredoxin domain in the Na+-translocating NADH dehydrogenase from Vibrio cholerae.

The Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae contains a single Fe-S cluster localized in subunit NqrF. Here we study the electronic properties of the Fe-S center in a truncated version of the NqrF subunit comprising only its ferredoxin-like Fe-S domain. Mössbauer spectroscopy of the Fe-S domain in the oxidized state is consistent with a binuclear Fe-S cluster with tetrahedral sulfur coordination by the cysteine residues Cys(70), Cys(76), Cys(79), and Cys(111). Important sequence motifs surrounding these cysteines are conserved in the Fe-S domain and in vertebrate-type ferredoxins. The magnetic circular dichroism spectra of the photochemically reduced Fe-S domain exhibit a striking similarity to the magnetic circular dichroism spectra of vertebrate-type ferredoxins required for the in vivo assembly of iron-sulfur clusters. This study reveals a novel function for vertebrate-type [2Fe-2S] clusters as redox cofactors in respiratory dehydrogenases.

Amino Acid Motifs↗
PubMed2001

Ferredoxin:NADP+ oxidoreductase is a subunit of the chloroplast cytochrome b6f complex.

Purified detergent-soluble cytochrome b6f complex from chloroplast thylakoid membranes (spinach) and cyanobacteria (Mastigocladus laminosus) was highly active, transferring 300-350 electrons per cyt f/s. Visible absorbance spectra showed a red shift of the cytochrome f alpha-band and the Qy chlorophyll a band in the cyanobacterial complex and an absorbance band in the flavin 450-480-nm region of the chloroplast complex. An additional high molecular weight (M(r) approximately 35,000) polypeptide in the chloroplast complex was seen in SDS-polyacrylamide gel electrophoresis at a stoichiometry of approximately 0.9 (cytochrome f)(-1). The extra polypeptide did not stain for heme and was much more accessible to protease than cytochrome f. Electrospray ionization mass spectrometry of CNBr fragments of the 35-kDa polypeptide was diagnostic for ferredoxin:NADP+ oxidoreductase (FNR), as were antibody reactivity to FNR and diaphorase activity. The absence of FNR in the cyanobacterial complex did not impair decyl-plastoquinol-ferricyanide activity. The activity of the FNR in the chloroplast b6f complex was also shown by NADPH reduction, in the presence of added ferredoxin, of 0.8 heme equivalents of the cytochrome b6 subunit. It was inferred that the b6f complex with bound FNR, one equivalent per monomer, provides the membrane protein connection to the main electron transfer chain for ferredoxin-dependent cyclic electron transport.

Amino Acid Sequence↗
PubMed2002

High resolution crystal structures of the wild type and Cys-55-->Ser and Cys-59-->Ser variants of the thioredoxin-like [2Fe-2S] ferredoxin from Aquifex aeolicus.

The [2Fe-2S] ferredoxin (Fd4) from Aquifex aeolicus adopts a thioredoxin-like polypeptide fold that is distinct from other [2Fe-2S] ferredoxins. Crystal structures of the Cys-55 --> Ser (C55S) and Cys-59 --> Ser (C59S) variants of this protein have been determined to 1.25 A and 1.05 A resolution, respectively, whereas the resolution of the wild type (WT) has been extended to 1.5 A. The improved WT structure provides a detailed description of the [2Fe-2S] cluster, including two features that have not been noted previously in any [2Fe-2S] cluster-containing protein, namely, pronounced distortions in the cysteine coordination to the cluster and a Calpha-H-Sgamma hydrogen bond between cluster ligands Cys-55 and Cys-9. These features may contribute to the unusual electronic and magnetic properties of the [2Fe-2S] clusters in WT and variants of this ferredoxin. The structures of the two variants of Fd4, in which single cysteine ligands to the [2Fe-2S] cluster are replaced by serine, establish the metric details of serine-ligated Fe-S active sites with unprecedented accuracy. Both the cluster and its surrounding protein matrix change in subtle ways to accommodate this ligand substitution, particularly in terms of distortions of the Fe(2)S(2) inorganic core from planarity and displacements of the polypeptide chain. These high resolution structures illustrate how the interactions between polypeptide chains and Fe-S active sites reflect combinations of flexibility and rigidity on the part of both partners; these themes are also evident in more complex systems, as exemplified by changes associated with serine ligation of the nitrogenase P cluster.

Bacterial Proteins↗
PubMed2005

Cyanobacterial non-mevalonate pathway: (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase interacts with ferredoxin in Thermosynechococcus elongatus BP-1.

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE), which catalyzes the conversion of 2-C-methyl-D-erythritol cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP), is an essential enzyme of the non-mevalonate (2-C-methyl-D-erythritol-4-phosphate (MEP)) pathway for isoprenoid biosynthesis. The terminal steps of the MEP pathway are still not fully understood, although this pathway is necessary for survival in various organisms such as cyanobacteria, plastids of algae and higher plants, and the apicoplast of human malaria parasites. To determine the efficient redox partner for thermophilic cyanobacterial GcpE, We have expressed the gcpE and petF genes in Escherichia coli and studied the protein-protein interaction of GcpE protein with ferredoxin I (PetF) from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. Recombinant GcpE protein was purified by an N-terminal His(6) tag and reconstituted as a [4Fe-4S](2+) metalloprotein. GcpE was shown to interact strongly with PetF via the bacterial two-hybrid system designed to detect protein-protein interactions. Moreover, a direct protein-protein interaction between PetF and GcpE was confirmed in an in vitro glutathione S-transferase (GST) pull-down assay. To investigate electron transfer activity from PetF to GcpE, we also constructed a NADPH-dependent reducing shuttle system with purified recombinant ferredoxin-NADP(+) oxidoreductase (PetH) and PetF. The result demonstrated that PetF has the ability to transfer electrons to GcpE. Thus, the combined data provide the first evidence that GcpE is a ferredoxin-dependent enzyme in T. elongatus BP-1.

Alkyl and Aryl Transferases↗
PubMed1995

Interference of nucleases in cyanobacterium ferredoxin purification.

Isolation of cyanobacterial ferredoxin is normally carried out using nucleases in order to degrade the nucleic acids that accompany this protein during the purification procedure. However, this practice presents the inconvenience that these proteins remain in trace amounts in the purified ferredoxin preparations, although they are not visible by electrophoretical techniques. Evidence of that fact is shown in this report and an alternative procedure is described for the rapid preparation of ferredoxin from crude extracts of Anabaena PCC 7119. The method involves a treatment of the crude extract with streptomycin sulphate, a high molecular weight polication that precipitates the nucleic acids in the beginning of the purification.

Anabaena↗
PubMed1975

A minor component of ferredoxin from Aphanothece sacrum cells.

About 20% of the total extractable ferredoxin from a blue-green alga, Aphanothece sacrum, was separated as a minor component on a DEAE-cellulose column during the preparation of the major component of the cells. It was always found in cells collected in three different seasons. The chemical properties of the minor component was different from those of the major one. The enzymatic activity and spectral properties were those of typical chloroplast-type ferredoxins. The significance of isozymes in ferredoxin is discussed.

Amino Acids↗
PubMed1976

Amino acid sequence of the major component of Aphanothece sacrum ferredoxin.

The amino acid sequence of the major ferredoxin component isolated from a blue-green alga, Aphanothece sacrum, has been fully determined. Chymotryptic and tryptic peptides of carboxymethyl-ferredoxin and chymotryptic peptides of oxidized ferredoxin were prepared and their sequences were analyzed...

Amino Acid Sequence↗
PubMed1981

The second ferredoxin from spinach leaves.

A minor component of ferredoxin (about 20% of the total content) was separated from the major one on a DEAE Trisacryl M column only after carboxymethylation of the ferredoxin purified from spinach leaves by the conventional procedure. The amino acid compositions and terminal sequences of the two revealed that the major component corresponded to the one previously sequenced (Matsubara, H. & Sasaki, M. (1968) J. Biol. Chem. 243, 1732-1757) and the minor one had a completely different structure from that of the major one. Comparison of the amino(N)-terminal 34 residues showed 11 amino acid substitutions between the two components. Thus the existence of a second ferredoxin was demonstrated in spinach leaves.

Amino Acid Sequence↗
PubMed1982

Analyses of optical absorption and circular dichroism spectra of spinach ferredoxin at alkaline pH.

The whole protein structure and the microenvironments of the iron-sulfur cluster and of the side chains of amino acid residues of spinach ferredoxin were studied by optical absorption and circular dichroism (CD) spectroscopy in the alkaline pH range. From the pH-dependence of the optical absorption changes at 245 nm, the four tyrosyl residues of ferredoxin were classified into three groups: one exposed residue with a normal apparent pK value of 10.1, two exposed residues with abnormal apparent pK values of 12.0, and one buried residue showing time-dependent ionization. The absorption in the visible region disappeared gradually with the ionization of the buried residue rather than that of the three exposed residues. The apparent pK value of 10.0 was obtained from the rapid CD changes at 258 nm caused by pH elevation from neutral to alkaline pH. The structural alteration associated with the CD change had no effect on the secondary structure of the protein moiety other than the iron-sulfur cluster and the microenvironment of the cluster. The rate constants obtained from the time courses of the CD changes in the near-ultraviolet and visible regions were in good agreement with those obtained from the time courses of the optical absorption changes. These results lead to the conclusions that (1) the native ferredoxin structure is maintained through the interaction with the iron-sulfur cluster and (2) the protein structure in the neighborhood of the cluster, important for the physiological activity, is not perturbed even though the exposed tyrosyl residues are ionized.

Circular Dichroism↗
PubMed1983

Main chain fold of a [2Fe-2S]ferredoxin I from Aphanothece sacrum at 2.5 A resolution.

Crystal structure analysis of a [2Fe-2S]ferredoxin I from Aphanothece sacrum, a blue green alga, was carried out at 2.5 A resolution. The phase angle of each reflection was determined by the single isomorphous replacement method coupled with the anomalous dispersion effect for the uranium derivative. The four molecules in an asymmetric unit were clearly seen in a 3.9 A electron density map. The main chains of three molecules were traced at 2.5 A resolution. The structure of the remaining one molecule, however, remains unknown because of the poor electron density of the corresponding region. The three main chain folds exhibit the same topology as that in Spirulina platensis. The structural similarity between A. sacrum and S. platensis ferredoxins, whose amino acid sequences are different from each other by about 30%, strongly suggests that all plant-type ferredoxins have the same main chain fold.

Amino Acid Sequence↗
PubMed1984

Spirulina ferredoxin-NADP+ reductase. The complete amino acid sequence.

The amino acid sequence of ferredoxin-NADP+ oxidoreductase [EC 1.18.1.2, FNR] from Spirulina sp., a blue-green alga, was determined. Spirulina ferredoxin-NADP+ oxidoreductase was composed of 294 amino acid residues and the molecular weight of the holoenzyme was 34,135. An apparent homology of the amino(N)-terminal region was found between ferredoxin-NADP+ reductases from Spirulina and spinach. We also found some sequence similarities in human erythrocyte glutathione reductase and p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, both of which are NADPH-dependent FAD enzymes.

Amino Acid Sequence↗
PubMed1985

Ferredoxin from a liverwort, Marchantia polymorpha. Purification and amino acid sequence.

Marchantia polymorpha ferredoxin was purified by DE-52 and Sephadex G-75 column chromatographies to homogeneity. The complete amino acid sequence of the carboxymethylated (Cm) ferredoxin was determined by conventional methods to be as follows. Thr-Phe-Lys- Val-Thr-Leu-Asn-Thr-Pro-Thr-Gly-Gln-Ser-Val-Ile-Asp-Val-Glu-Asp- Asp-Glu-Tyr-Ile-Leu-Asp-Ala-Ala-Glu-Glu-Ala-Gly-Leu-Ser-Leu-Pro- Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Ser-Cys-Ala-Gly-Lys-Val-Thr- Ala-Gly-Glu-Val-Asp-Gln-Ser-Asp-Glu-Ser-Phe-Leu-Asp-Asp-Asp-Gln- Met-Asp-Glu-Gly-Tyr-Val-Leu-Thr-Cys-Ile-Ala-Tyr-Pro-Thr-Ser-Asp- Leu-Thr-Ile-Asp-Thr-His-Gln-Glu-Glu-Ala-Leu-Ile. The total number of amino acid residues was 95 and the molecular weight was calculated to be 10,174, excluding iron and sulfur atoms. The distribution of the four cysteine residues chelating the two iron atoms was identical to those of other [2Fe-2S] ferredoxins. The relationship between M. polymorpha and other plants was discussed in terms of plant phylogeny.

Amino Acid Sequence↗
PubMed2002

Electron transfer in ferredoxin: are tunneling pathways evolutionarily conserved?

A theoretical study of electron transfer (ET) pathways in a recently crystallized Clostridium acidurici ferredoxin is reported. The electronic structure of the protein complex is treated at the semiempirical extended Hückel level, and the tunneling pathways are calculated with the rigorous quantum mechanical method of tunneling currents. The model predicts two pathways between the two [4Fe-4S] cubanes: a strong one running directly from Cys(14) to Cys(43) and a weaker one from Cys(14) via Ile(23) to Cys(18), whereas other amino acids do not play a significant role in the electron tunneling. The cysteine ligands conduct almost all of the current when Ile(23) is mutated to valine in silico, so that there is no appreciable change in the ET rate. The calculated value of the transfer matrix element is consistent with the experimentally determined rate of transfer. Results of the sequence analysis performed on this ferredoxin reveal that Ile(23) is a highly variable amino acid compared with the cubane-ligating cysteine amino acids, even though Ile(23) lies directly between the donor and acceptor complexes. We further argue that the homologous proteins with a [3Fe-4S] cofactor, which does not have one of the four cysteine ligands, use the same tunneling pathways as those in this ferredoxin, on the basis of the high homology as well as the absolute conservation of Cys(14) and Cys(43) which serve as the main tunneling conduit. Our results explain why mutation of amino acids around and between the donor and acceptor cubane clusters, including that of Ile(23), does not appreciably affect the rate of transfer and add support to the proposal that there exist evolutionarily conserved electron tunneling pathways in biological ET reactions.

Amino Acid Sequence↗
PubMed1997

Synthetic mutants of Clostridium pasteurianum ferredoxin: open iron sites and testing carboxylate coordination.

The entire polypeptide chains for two new Clostridium pasteurianum ferredoxin (Fd) mutants were prepared with the following site-specific substitutions: Cys11Asp and Cys11 alpha-aminobutyric acid (Cys11 alpha-Aba), the latter being a non-naturally occurring amino acid. Standard t-Boc procedures were used for the synthesis and the peptides. The two apoproteins were reconstituted to the 2[4Fe-4S] holoprotein and their spectroscopic, redox and thermal properties were compared with those of native C.pasteurianum Fds. The fully reconstituted Cys11Asp and Cys11 alpha-Aba mutants were initially found to have both clusters intact, i.e. they were 2[4Fe-4S] ferredoxins. The unconventional ligands of Asp and alpha-Aba led to holo-Fds that were not very stable and easily released an iron to form the [3Fe-4S] cluster, presumably through oxidation. The Cys11 alpha-Aba mutant was somewhat more thermally stable than Cys11Asp. In contrast, while both mutants were less stable than the native protein upon exposure to oxygen, the Cys11 alpha-Aba mutant was less stable than Cys11Asp. The Cys11Gly mutant was also prepared, but all attempts, despite repeated and varied experimental conditions, at reconstitution to the Cys11Gly holo 2[4Fe-4S] Fd were unsuccessful, probably because a Gly-Gly sequence is known to break structure. This work, when compared with molecular biological site-specific mutagenesis, shows some of the advantages of chemical/in vitro reconstitution: certain mutants which cannot be detected as holoproteins by site-specific mutagenesis can be formed after all in vitro. Nonetheless, it seems apparent that altering any of the Cys coordination sites of the Fd clusters results in fundamentally more unstable ferredoxins.

Clostridium↗
PubMed1998

A model for the catabolism of rhizopine in Rhizobium leguminosarum involves a ferredoxin oxygenase complex and the inositol degradative pathway.

Rhizopines are nodule-specific compounds that confer an intraspecies competitive nodulation advantage to strains that can catabolize them. The rhizopine (3-O-methyl-scyllo-inosamine, 3-O-MSI) catabolic moc gene cluster mocCABRDE(F) in Rhizobium leguminosarum bv. viciae strain 1a is located on the Sym plasmid. MocCABR are homologous to the mocCABR gene products from Sinorhizobium meliloti. MocD and MocE contain motifs corresponding to a TOL-like oxygenase and a [2Fe-2S] Rieske-like ferredoxin, respectively. The mocF gene encodes a ferredoxin reductase that would complete the oxygenase system, but is not essential for rhizopine catabolism. We propose a rhizopine catabolic model whereby MocB transports rhizopine into the cell and MocDE and MocF (or a similar protein elsewhere in the genome), under the regulation of MocR, act in concert to form a ferredoxin oxygenase system that demethylates 3-O-MSI to form scyllo-inosamine (SI). MocA, an NAD(H)-dependent dehydrogenase, and MocC continue the catabolic process. Compounds formed then enter the inositol catabolic pathway.

Amino Acid Sequence↗
PubMed2002

Expression and biochemical properties of a ferredoxin-dependent heme oxygenase required for phytochrome chromophore synthesis.

The HY1 gene of Arabidopsis encodes a plastid heme oxygenase (AtHO1) required for the synthesis of the chromophore of the phytochrome family of plant photoreceptors. To determine the enzymatic properties of plant heme oxygenases, we have expressed the HY1 gene (without the plastid transit peptide) in Escherichia coli to produce an amino terminal fusion protein between AtHO1 and glutathione S-transferase. The fusion protein was soluble and expressed at high levels. Purified recombinant AtHO1, after glutathione S-transferase cleavage, is a hemoprotein that forms a 1:1 complex with heme. In the presence of reduced ferredoxin, AtHO1 catalyzed the formation of biliverdin IXalpha from heme with the concomitant production of carbon monoxide. Heme oxygenase activity could also be reconstituted using photoreduced ferredoxin generated through light irradiation of isolated thylakoid membranes, suggesting that ferredoxin may be the electron donor in vivo. In addition, AtHO1 required an iron chelator and second reductant, such as ascorbate, for full activity. These results show that the basic mechanism of heme cleavage has been conserved between plants and other organisms even though the function, subcellular localization, and cofactor requirements of heme oxygenases differ substantially.

Arabidopsis↗