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PubMed1975

Evolutionary information involved in primary structures of chloroplast-type ferredoxins.

The complete amino acid sequence of a chloroplast-type ferredoxin from a fresh water prokaryote, Aphanothece sacrum, was determined. The sequence consisted of 96 amino acid residues and was homologous to those of ferredoxins of higher plants. Comparison of eight ferredoxins, including one from a green alga and three from blue-green algae, suggested that the sequences of algal ferredoxins were as different from one another as from those of higher plants. The relationship between the numbers of differences in amino acids and the period since separation from a common ancestor was not linear, even after correction for multiple substitution at an amino acid site in the sequence. It is very likely that the ferredoxins of angiosperms evolved rather rapidly and that those of algae, and particularly blue-green algae evolved rather slowly in the evolutionary scale. Several possible mechanisms of evolution of plants are discussed.

Amino Acid Sequence↗

PubMed1981

Ferredoxin excreted from photosynthetic bacterium, Rhodospirillum rubrum: purification and properties.

When the photoheterotroph, Rhodospirillum rubrum, was grown in the light, ferredoxin was excreted from the cells in a significant amount, as well as hydrogenase. The extracellular ferredoxin was purified to a homogeneous state. The molecular weight was approximately 9,000, and the oxidation-reduction mid-potential was -0.29 V (N=1) at pH 7.0 and 25 degrees C. The amino acid composition was different from those of the intracellular ferredoxins, which were already known. The contents of non-heme iron and acid-labile sulfur were 10.6 and 7.9 mol/mol protein, respectively. The extracellular hydrogenase catalyzed the evolution of hydrogen gas from the ferredoxin in the reduced form. The Km for the ferredoxin was 4.1 micro M, one-seven hundredth as low as that for methyl viologen. There is a possibility that hydrogenase here were functional for evolution of hydrogen gas outside the cells.

Amino Acids↗

PubMed1982

Immobilized ferredoxin-NADP+ reductase: preparation and properties.

Immobilized ferredoxin-NADP+ reductase (FNR) was prepared by coupling reaction of CNBr-Sepharose 4B with the spinach enzyme. The immobilized FNR was found to retain the activity of complex formation with ferredoxin as well as the enzymatic activities such as NADPH-diaphorase and NADPH-cytochrome c reductase activities. The complex formation of immobilized FNR with ferredoxin was investigated by measuring reflex spectra of the immobilized FNR with or without ferredoxin and by titration with ferredoxin. The experimental results obtained for the dissociation constant, pH profile and effect of salts were coincidental with those reported for the free enzyme system.

Enzymes, Immobilized↗

PubMed1997

Plant-type ferredoxin-NADP+ reductases: a basal structural framework and a multiplicity of functions.

Ferredoxin-NADP+ (oxido)reductase (EC 1.18.1.2, FNR) is an FAD-containing enzyme that catalyzes the reversible electron transfer between NADP(H) and electron carrier proteins such as ferredoxin and flavodoxin. Isoforms of this flavoprotein are present in chloroplasts, mitochondria, and bacteria in which they participate in a wide variety of redox metabolic pathways. Although ferredoxin-NADP+ reductases have been thoroughly investigated and their properties reviewed on several occasions, considerable advances in the understanding of these flavoenzymes have occurred in the last few years, including the characterization of cDNA and genomic clones encoding FNR proteins from plants, algae, vertebrates, and bacteria, determination of the atomic structure of a plant FNR at high resolution, and the expression of functional reductases in microorganisms like Escherichia coli and Saccharomyces cerevisiae. The aim of this article is to summarize information gained through these recent developments, including the phylogenetic relationships among ferredoxin reductases and the key structural features of the plant FNR family. Other aspects such as the catalytic mechanism of FNR and the molecular events underlying biogenesis, intracellular sorting, folding, and holoenzyme assembly of this important flavoenzyme are also discussed in some detail. Ferredoxin-NADP+ reductases display several outstanding properties that make them excellent model proteins to address broad biological questions.

Bacteria↗

PubMed2004

The Arabidopsis chloroplastic NifU-like protein CnfU, which can act as an iron-sulfur cluster scaffold protein, is required for biogenesis of ferredoxin and photosystem I.

The biosynthesis of iron-sulfur clusters is a highly regulated process involving several proteins. Among them, so-called scaffold proteins play pivotal roles in both the assembly and delivery of iron-sulfur clusters. Here, we report the identification of two chloroplast-localized NifU-like proteins, AtCnfU-V and AtCnfU-IVb, from Arabidopsis (Arabidopsis thaliana) with high sequence similarity to a cyanobacterial NifU-like protein that was proposed to serve as a molecular scaffold. AtCnfU-V is constitutively expressed in several tissues of Arabidopsis, whereas the expression of AtCnfU-IVb is prominent in the aerial parts. Mutant Arabidopsis lacking AtCnfU-V exhibited a dwarf phenotype with faint pale-green leaves and had drastically impaired photosystem I accumulation. Chloroplasts in the mutants also showed a decrease in both the amount of ferredoxin, a major electron carrier of the stroma that contains a [2Fe-2S] cluster, and in the in vitro activity of iron-sulfur cluster insertion into apo-ferredoxin. When expressed in Escherichia coli cells, AtCnfU-V formed a homodimer carrying a [2Fe-2S]-like cluster, and this cluster could be transferred to apo-ferredoxin in vitro to form holo-ferredoxin. We propose that AtCnfU has an important function as a molecular scaffold for iron-sulfur cluster biosynthesis in chloroplasts and thereby is required for biogenesis of ferredoxin and photosystem I.

Amino Acid Sequence↗

PubMed1986

Ferredoxin reductase levels in the ovaries of pigs and superovulated rats during follicular cell growth and luteinization.

The concentration of ferredoxin reductase, a component of the mitochondrial steroidogenic electron transport chain, was measured in the ovaries of pigs and superovulated rats by a protein blotting procedure using polyclonal antibodies to the purified protein. The concentration of ferredoxin reductase in porcine granulosa cells doubled during growth of follicles from small (1-2 mm diameter) to large (6-12 mm diameter). The concentration doubled again during the period of luteinization. This is in contrast to the rate of cholesterol side-chain cleavage, which showed little change during follicular growth but increased by more than tenfold during luteinization. A similar large increase in cholesterol side-chain cleavage occurs during the period of luteinization in the ovaries of superovulated rats, but as for the pig, only a small increase in ferredoxin reductase was observed. A threefold increase in the yield of mitochondrial protein from tissue homogenates was found between the granulosa cells of small-medium follicles and the cells of the corpora luteum. The increase in ferredoxin reductase during follicular development and luteinization, therefore, correlates well with the increase in mitochondria in the cells, but does not correlate with the dramatic increase in cholesterol side-chain cleavage activity which occurs during luteinization. Based on these results, it is unlikely that the level of ferredoxin reductase limits the expression of the full steroidogenic activity of the granulosa cells of the ovary.

Animals↗

PubMed1992

Purification and characterization of the ferredoxin-glutamate synthase from the unicellular cyanobacterium Synechococcus sp. PCC 6301.

Ferredoxin-glutamate synthase from the unicellular cyanobacterium Synechococcus sp. PCC 6301 has been purified using, as main steps, ethanol fractionation in the presence of high ionic strength, ion-exchange chromatography and ferredoxin-Sepharose affinity chromatography. The overall process yielded an homogeneous enzyme with a specific activity of 30 U/mg protein, after a purification of 2800-fold with a recovery of 43%. The molecular mass of the native protein was 156 kDa, as calculated from its Stokes radius (rS, 4.32 nm) and sedimentation coefficient (S20,w, 8.46 S). The size was also estimated by SDS/PAGE as 160 kDa, indicating that the native protein was a monomer. The enzyme exhibited absorption maxima at 279, 370 and 438 nm and a A279/A438 absorbance ratio of 11. One molecule of FMN, but not FAD, was found/molecule native protein. The addition of dithionite resulted in the loss of the absorption peak at 438 nm, which was restored by the addition of 2-oxoglutarate, thus indicating that the prosthetic group is functional in catalysis. Classical hyperbolic kinetics with substrate inhibition was seen for 2-oxoglutarate. The Km values determined for glutamine and ferredoxin were 0.7 mM and 7 microM, respectively, and the apparent Km for 2-oxoglutarate was estimated to be 1.7 mM. Azaserine and 6-diazo-5-oxo-L-norleucine were potent inhibitors of the activity, while pyridoxal 5-phosphate, known to react with Lys residues, partially inactivated the enzyme. This ferredoxin-dependent glutamate synthase is, as far as we know, the first purified from prokaryotic organisms and resembles its counterpart from chloroplasts, suggesting that cyanobacterial glutamate synthase may have been the ancestor of ferredoxin-glutamate synthase in plants.

Amino Acid Oxidoreductases↗

PubMed1995

Characterization of a ferredoxin from Desulfovibrio vulgaris (Hildenborough) that interacts with RNA.

The purification and characterization of a ferredoxin from Desulfovibrio vulgaris (Hildenborough) is described. The protein can be isolated in two forms; the major form is strongly complexed to RNA, while a minor form is free from nucleic acid. Bound RNA cannot be removed by digestion with nucleases, or by heating to 70 degrees C, and it can only be partially removed by rechromatography. The ultraviolet/visible spectrum shows typical absorption maxima at 280 nm and 400 nm for the RNA-free ferredoxin. The RNA-bound protein exhibits an additional strong peak at 260 nm. The RNA can be extracted from the protein with phenol. The ferredoxin is a dimer of subunits, each of 7.5 kDa; its pI is 3.9. The protein contains a [4Fe-4S](2+;1+) cluster with an EPR spectrum (g = 1.90, 1.93 and 2.05) in the reduced state. A reduction potential of -360 mV was determined for the RNA-free ferredoxin with reversible voltammetry at glassy carbon. From the temperature dependence of the reduction potential, the unusually high standard reaction entropy was calculated as delta S degree = -230 J.K-1.mol-1. No electrochemical response was obtained with the RNA-bound ferredoxin. Binding of RNA appears to require the presence of an intact cluster, since the absence of absorption at 400 nm runs in parallel with the absence of absorption at 260 nm. The possibility is discussed that the binding to the RNA has a regulatory function and is controlled by the state of the cluster.

Desulfovibrio vulgaris↗

PubMed1995

Solution structure of the oxidized 2[4Fe-4S] ferredoxin from Clostridium pasteurianum.

Following the recently developed approach to the solution structure of paramagnetic high-potential iron-sulfur proteins, the three-dimensional structure in solution of the oxidized Clostridium pasteurianum ferredoxin has been solved by 1H-NMR. The X-ray structure is not available. The protein contains 55 amino acids and two [4Fe-4S] clusters. In the oxidized state, the clusters have S = 0 ground states, but are paramagnetic because of thermal population of excited states. Due to the somewhat small size of the protein and to the presence of two clusters, approximately 55% of the residues have at least one proton with a non-selective T1 smaller than 25 ms. The protein has thus been used as a test system to challenge the present paramagnetic NMR methodology both in achieving an extended assignment and in obtaining a suitable number of constraints. 79% of protein protons have been assigned. Analogy with other ferredoxins of known structure has been of help to speed up the final stages of the assignment, although we have shown that this independent information is not necessary. In addition to dipolar connectivities, partially detected through tailored experiments, 3JHN-H alpha, H-bond constraints and dihedral angle constraints on the Cys chi 2 angles have been generated by using a recently derived Karplus-type relationship for the hyperfine shifts of cysteine beta CH2 protons. In total, 456 constraints have been used in distance geometry calculations. The final quality of the structures is satisfactory, with root-mean-square deviation values of 66 pm and 108 pm for backbone and heavy atoms, respectively. The resulting structure is compared with that of Clostridium acidi urici ferredoxin [Duée, E. D., Fanchon, E., Vicat, J., Sieker, L. C., Meyer, J. & Moulis, J.-M. (1994) J. Mol. Biol. 243, 683-695]. The two proteins are very similar in the overall folding, secondary structure elements and side-chain orientations. The C alpha root-mean-square deviation values between the X-ray-determined C. acidi urici ferredoxin structure and the conformer with lowest energy of the C. pasteurianum ferredoxin family is 78 pm (residues 3-53). Discrepancies in residues 26-28 may arise from the disorder observed in the X-ray structure in that region.

Amino Acid Sequence↗

PubMed1996

Identification of residues of Rhodobacter capsulatus ferredoxin I important for its interaction with nitrogenase.

In Rhodobacter capsulatus, ferredoxin I (FdI) serves as natural electron donor to nitrogenase. In order to probe amino acid residues possibly involved in the interaction with dinitrogenase reductase, FdI was subjected to site-specific mutagenesis. A three-dimensional structure of FdI was designed by computer modelling and used for selecting target residues. Mutant ferredoxins bearing substitutions of surface residues, as well as a variant having a Met2 --> Tyr replacement in the vicinity of one cluster, have been constructed. All FdI variants were expressed to similar levels both in Escherichia coli and in a FdI-deleted mutant of the natural host. Once purified, the mutant ferredoxins exhibited molecular and spectroscopic properties almost identical to wild-type FdI. Determination of the reduction potential of FdI by cyclic voltammetry gave an E'o of -510 mV (pH 7.6) for both clusters, which is one of the lowest values reported for a 2[4Fe-4S] ferredoxin. Only the [Tyr2]FdI variant showed a significant difference in redox potential (delta E'o = -15 mV). Based on in vitro assays, a [Glu27, Glu28]FdI double mutant exhibited a twofold decrease in the electron transfer rate to dinitrogenase reductase while the affinity of this mutant for the enzyme was barely affected. On the other hand, an Asp36 --> His substitution resulted in a sevenfold increase of the apparent Km for dinitrogenase reductase. Unlike FdI and the other mutant ferredoxins, the [His36]FdI variant also failed to form a cross-linked complex with dinitrogenase reductase upon incubation with a carbodiimide. It is concluded that Asp36 in FdI probably participates in the interaction between the two protein partners. Nevertheless, all the FdI mutants proved competent in restoring a wild-type phenotype when expressed in a FdI-deleted mutant background, indicating that none of the studied residues was absolutely critical for electron transfer to nitrogenase.

Amino Acid Sequence↗

PubMed1984

Ferredoxin-linked reduction of metronidazole in Clostridium pasteurianum.

Clostridium pasteurianum cell-free extracts enzymatically reduced metronidazole when coupled by hydrogenase via reduced ferredoxin. A 5 mM concentration of methyl viologen, flavin adenine dinucleotide, or flavin mononucleotide could completely replace ferredoxin (0.05 mM) in the in vitro reduction assay system, whereas 5 mM benzyl viologen was less effective. However, when these electron carriers were used at a concentration of 0.05 mM, there was a drastic loss in their abilities to couple the metronidazole reduction system compared with the comparable concentration of ferredoxin. It is not understood why these flavin coenzymes participate in this enzymatic reaction. NAD and NADP had no activity when substituted for ferredoxin in the enzyme system. Two reduced ferredoxin-linked pathways, "metronidazole reductase" and the inducible dissimilatory sulfite reductase system, when combined in a single in vitro competition experiment demonstrated a preferential flow of electrons to metronidazole away from sulfite. A proposed bactericidal mechanism for metronidazole against C. pasteurianum incorporating the above findings is discussed.

Clostridium↗

PubMed1984

Characterization of ferredoxin, flavodoxin, and rubredoxin from Clostridium formicoaceticum grown in media with high and low iron contents.

Ferredoxin, flavodoxin, and rubredoxin were purified to homogeneity from Clostridium formicoaceticum and characterized. Variation of the iron concentration of the growth medium caused substantial changes in the concentrations of ferredoxin and flavodoxin but not of rubredoxin. The ferredoxin has a molecular weight of 6,000 and is a four iron-four sulfur protein with eight cysteine residues. The spectrum is similar to that of other ferredoxins. The molar extinction coefficients are 22.6 X 10(3) and 17.6 X 10(3) at 280 and 390 nm, respectively. From 100 g wet weight of cells grown with 3.6 microM iron and with 40 microM iron, 5 and 20 mg offerredoxin were isolated, respectively. The molecular weight of rubredoxin is 5,800 and it contains one iron and four cysteines. The UV-visible absorption spectrum is dissimilar to those of other rubredoxins in that the 373 nm absorption peak is quite symmetric, lacking the characteristic 350-nm shoulder found in other rubredoxins. The flavodoxin is a 14,500-molecular-weight protein which contains 1 mol of flavin mononucleotide per mol of protein. It forms a stable, blue semiquinone upon light irradiation in the presence of EDTA or during enzymatic reduction. When cells were grown in low-iron medium, flavodoxin constituted at least 2% of the soluble cell protein; however, it was not detected in extracts of cells grown in high-iron medium. The rubredoxin and ferredoxin expressed during growth in low-iron and high-iron media are identical as judged by iron, inorganic sulfide, and amino acid analysis, as well as light absorption spectroscopy.

Clostridium↗

PubMed1989

Purification and properties of ferredoxin and rubredoxin from Butyribacterium methylotrophicum.

A ferredoxin and a rubredoxin from Butyribacterium methylotrophicum, which displays a carbonyl-dependent acetyl-coenzyme A synthesis, were purified to electrophoretic homogeneity. The two electron carriers showed absorption spectra similar to those in Clostridium species. The ferredoxin displayed absorption peaks at 280 and 391 nm, while rubredoxin displayed absorption peaks at 279, 382, and 482 nm. Minimum molecular weights calculated from the respective amino acid compositions were 5,727 for ferredoxin and 5,488 for rubredoxin, excluding iron and inorganic sulfur atoms. Both electron carriers were isolated as monomers, according to gel-filtration data. Electron spin resonance analysis revealed that the ferredoxin was a 2[4Fe-4S]-type and that both clusters had a midpoint redox potential value of -410 mV, whereas rubredoxin contained one acid-stable iron and had a redox value of -40 mV. The coupling of these electron carriers to hydrogenase and carbon monoxide dehydrogenase activities was investigated. Rubredoxin showed higher activity towards carbon monoxide dehydrogenase, whereas ferredoxin showed higher activity towards hydrogenase.

Aldehyde Oxidoreductases↗

PubMed2000

Characterization of three XylT-like [2Fe-2S] ferredoxins associated with catabolism of cresols or naphthalene: evidence for their involvement in catechol dioxygenase reactivation.

The xylT gene product, a component of the xylene catabolic pathway of Pseudomonas putida mt2, has been recently characterized as a novel [2Fe-2S] ferredoxin which specifically reactivates oxygen-inactivated catechol 2,3-dioxygenase (XylE). In this study, three XylT-like proteins potentially involved in the catabolism of naphthalene (NahT) or cresols (PhhQ and DmpQ) have been overexpressed in Escherichia coli, purified, and compared with respect to their biochemical properties and interaction with XylE. The three XylT analogues show general spectroscopic characteristics common to plant-type [2Fe-2S] ferredoxins as well as distinctive features that appear to be typical for the XylT subgroup of these proteins. The midpoint redox potentials of the PhhQ and DmpQ proteins were -286 mV and -323 mV, respectively. Interestingly, all purified XylT-like proteins promoted in vitro reactivation of XylE almost as efficiently as XylT. The interaction of XylE with XylT and its analogues was studied by cross-linking experiments using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. A polypeptide band with an M(r) of 46,000, which corresponded to the cross-linked product between one XylE subunit and one molecule of ferredoxin, was obtained in all cases. The formation of the complex was affected by ionic strength, indicating that electrostatic forces are involved in the dioxygenase-ferredoxin interaction. In complementation experiments, plasmids expressing xylT or its analogues were introduced into an XylT-null mutant of P. putida which is unable to grow on p-methylbenzoate. All transconjugants regained the wild-type phenotype, indicating that all analogues can substitute for XylT in the in vivo reactivation of XylE. Our results provide evidence for a subgroup of [2Fe-2S] ferredoxins with distinct biochemical properties whose specific function is to reactivate intrinsically labile extradiol ring cleavage dioxygenases involved in the catabolism of various aromatic hydrocarbons.

Amino Acid Sequence↗

PubMed1979

Immunofluorescent localization of 25-hydroxyvitamin-D3-1 alpha-hydroxylase ferredoxin in the renal tissue of chick.

The chick renal mitochondrial 25-hydroxyvitamin-D3-1 alpha-hydroxylase is composed of three proteins, namely, cytochrome P-450, iron-sulfur protein (ferredoxin) and flavoprotein. Antibodies were raised in rabbits against homogeneous preparations of the ferredoxin. The antibodies were used in indirect immunofluorescence studies to localize the ferrdoxin along the nephron of renal tissues obtained either from vitamin D3-deficient or vitamin D3-sufficient chicks. The ferredoxin is predominantly localized in the glomerulus and proximal convoluted tubules. These results suggest that, in addition to the mitochondrial localization of the 1-hydroxylase, the enzyme may also be present in renal nuclei. The amount of the ferredoxin in kidney, as evidenced by the intensity of fluorescence, appeared to be independent of the vitamin D status of the chick. This finding indicated that changes in the concentration of the renal ferredoxin is not a major factor in the regulation of the 1-hydroxylase activity.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed1993

The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate stimulates the dephosphorylation of mitochondrial ferredoxin in cultured chick kidney cells.

The tumor promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), presumably through activation of protein kinase C, decreases the production of 1 alpha, 25-dihydroxyvitamin D3 [1,25(OH)2D3] and increases that of 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3] by primary cultures of chick kidney cells. We have previously shown that the regulation of the cellular output of 1,25(OH)2D3 and 24,25(OH)2D3 by PTH and 1,25(OH)2D3 can be quantitatively accounted for by altered hydroxylase activities within isolated mitochondria. In the present paper, we examined the effects of TPA and 1-oleoyl-2-acetyl-glycerol (OAG) on the state of mitochondrial protein phosphorylation and on 25-hydroxyvitamin D3 [25(OH)D3] metabolism. There was a good correlation between 25(OH)D3- 1 alpha- and 24-hydroxylase activities in mitochondria isolated from cells pretreated with either TPA or OAG and the pattern of 1- and 24-hydroxylation of 25(OH)D3. The most notable change in protein phosphorylation in the molecular mass range of 10-60 kilodaltons (kDa) was a dramatic decrease in the phosphorylation of a 12.5-kDa mitochondrial matrix protein after treatment of kidney cells with TPA or OAG. The amino acid composition of the 12.5-kDa protein was similar to bovine and human ferredoxins and it comigrated with bovine and human ferredoxins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 12.5-kDa phosphoprotein was immunoprecipitated specifically by an antipeptide polyclonal antibody for chick ferredoxin. The dephosphorylation of ferredoxin in response to TPA was both rapid and transient, with the phosphate content of the 12.5-kDa protein reduced by 70% after a 5-min exposure and returning to control levels by 20 min. A similar transience was observed with regard to the rapid effects of TPA on 1 alpha-hydroxylase activity, again showing maximal inhibition at 5 min. The results of our studies are consistent with the idea that ferredoxin phosphorylation plays a role in the regulation of steroid hydroxylation.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed1992

NMR studies of Azotobacter vinelandii and Pseudomonas putida seven-iron ferredoxins. Direct assignment of beta-cysteinyl carbon NMR resonances and further proton NMR assignments of cysteinyl and aromatic resonances.

Ferredoxins are proteins which contain iron and inorganic sulfide and are capable of electron transport. They are found in a wide range of organisms, from anaerobic bacteria, to plants and mammals. Although NMR spectroscopy has been used to study ferredoxins since the 1970s, little important structural or biochemical information has resulted from these investigations. The major difficulty has been the effect of the paramagnetic iron-sulfur clusters on the peptide resonances, hindering nuclear Overhauser effect (NOE) studies and causing broad line widths. These effects are most pronounced on resonances arising from the nuclei closest to the iron-sulfur center. Unfortunately, these are likely to be the most interesting nuclei, as they report the events and geometry in the vicinity of the active sites. In this paper, the first direct assignment of beta-cysteinyl 13C resonances for any iron-sulfur protein is reported for the spectrum of Pseudomonas putida ferredoxin. These resonances are of special significance, as they arise from the atoms on the protein closest to the iron centers, with the exception of the directly bound cysteinyl sulfur atoms. In addition, cysteinyl and ring system 1H NMR resonance assignments are made for the spectra of P. putida ferredoxin and Azotobacter vinelandii ferredoxin I.

Azotobacter vinelandii↗

PubMed1990

Spectroscopic characterization of the novel iron-sulfur cluster in Pyrococcus furiosus ferredoxin.

Pyrococcus furiosus ferredoxin is the only known example of a ferredoxin containing a single [4Fe-4S] cluster that has non-cysteinyl ligation of one iron atom, as evidenced by the replacement of a ligating cysteine residue by an aspartic acid residue in the amino acid sequence. The properties of the iron-sulfur cluster in both the aerobically and anaerobically isolated ferredoxin have been characterized by EPR, magnetic circular dichroism, and resonance Raman spectroscopies. The anaerobically isolated ferrodoxin contains a [4Fe-4S]+,2+ cluster with anomalous properties in both the oxidized and reduced states which are attributed to aspartate and/or hydroxide coordination of a specific iron atom. In the reduced form, the cluster exists with a spin mixture of S = 1/2 (20%) and S = 3/2 (80%) ground states. The dominant S = 3/2 form has a unique EPR spectrum that can be rationalized by an S = 3/2 spin Hamiltonian with E/D = 0.22 and D = +3.3 +/- 0.2 cm-1. The oxidized cluster has an S = 0 ground state, and the resonance Raman spectrum is characteristic of a [4Fe-4S]2+ cluster except for the unusually high frequency for the totally symmetric breathing mode of the [4Fe-4S] core, 342 cm-1. Comparison with Raman spectra of other [4Fe-4S]2+ centers suggests that this behavior is diagnostic of anomalous coordination of a specific iron atom. The iron-sulfur cluster is shown to undergo facile and quantitative [4Fe-4S] in equilibrium [3Fe-4S] interconversion, and the oxidized and reduced forms of the [3Fe-4S] cluster have S = 1/2 and S = 2 ground states, respectively. In both redox states the [3Fe-4S]0,+ cluster exhibits spectroscopic properties analogous to those of similar clusters in other bacterial ferredoxins, suggesting non-cysteinyl coordination for the iron atom that is removed by ferricyanide oxidation. Aerobic isolation induces partial degradation of the [4Fe-4S] cluster to yield [3Fe-4S] and possibly [2Fe-2S] centers. Evidence is presented to show that only the [4Fe-4S] form of this ferredoxin exists in vivo.

Aerobiosis↗