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PubMed2002

Amino acid sequences of ferredoxins from Scopolia japonica and Lycium chinense: their similarities to that of Datura arborea.

The complete amino acid sequences of [2Fe-2S] ferredoxins from Scopolia japonica and Lycium chinense have been determined by automated Edman degradation of the entire Cm-proteins and of the peptides obtained by enzymatic digestions. These two ferredoxins exhibited only 2-7 differences in the amino acid sequence when compared to the Datura-ferredoxins (D. stramonium, D. metel, and D. arborea), and especially only 2 or 3 differences compared to D. arborea. On the contrary, 8-19 differences were observed among the other solanaceous ferredoxins. This suggests that S. japonica and L. chinense are closely related taxonomically to Datura plants, especially to D. arborea.

Amino Acid Sequence↗
PubMed1995

Protein chemotaxonomy of genus Datura. IV. Amino acid sequence of Datura ferredoxins depends not on the species but the section of Datura plants from which it comes.

The complete amino acid sequences of [2Fe-2S] ferredoxins from Datura quercifolia (section Stramonium) and D. fastuosa (section Dutra) have been determined by automated Edman degradation of the entire Cm-protein and of the peptides obtained by tryptic digestion and CNBr treatment. The D. quercifolia and D. fastuosa ferredoxins exhibited identical amino acid sequences to D. stramonium (section Stramonium) and D. metal (section Dutra) ferredoxins, respectively. This result suggests that the amino acid sequence of Datura ferredoxins depends on the section but not the species of Datura plants.

Amino Acid Sequence↗
PubMed1992

Assembly of plant ferredoxin-NADP+ oxidoreductase in Escherichia coli requires GroE molecular chaperones.

We have recently reported the expression in Escherichia coli of an enzymatically competent ferredoxin-NADP+ oxidoreductase from cloned pea genes encoding either the mature enzyme or its precursor protein (Ceccarelli, E. A., Viale, A. M., Krapp, A. R., and Carrillo, N. (1991) J. Biol. Chem. 266, 14283-14287). Processing to the mature form by bacterial protease(s) and FAD assembly occurred in the bacterial cytosol. Expression of ferredoxin-NADP+ reductase in chaperonin-deficient (groE-) mutants of E. coli resulted in partial reductase assembly at permissive growth temperatures (i.e. 30 degrees C), and in total breakdown of holoenzyme assembly, and accumulation as aggregated inclusion bodies at non-permissive temperatures (i.e. 42 degrees C). Coexpression in these mutants of a cloned groESL operon from the phototrophic bacterium Chromatium vinosum resulted in partial or total recoveries of ferredoxin-NADP+ reductase assembly. The overall results indicate that bacterial chaperonins are required for the productive folding/assembly of eucaryotic ferredoxin-NADP+ reductase expressed in E. coli.

Bacterial Proteins↗
PubMed1979

Studies on nitrate reductase of Clostridium perfringens. II. Purification and some properties of ferredoxin.

A ferredoxin was purified from Clostridium perfringens by DEAE-cellulose chromatography and Sephadex G-50 gel filtration. It had absorption maxima at 390 and 280 nm. The molecular weight was estimated to be 6,000 by Sephadex gel filtration and from the results of amino acid analysis. The isoelectric point was 3.0. It contained four atoms of iron, four atoms of labile sulfur, and six cysteine residues. This ferredoxin as well as ferredoxin from C. pasteurianum acted as an electron donor for nitrate reductase from C. perfringens. The ferredoxin could also act as an electron donor for the hydrogenase from C. pasteurianum in hydrogen evolution.

Amino Acids↗
PubMed1990

Structure-function relationship in spinach ferredoxin-NADP+ reductase as studied by limited proteolysis.

Studies of limited proteolysis on purified ferredoxin-NADP+ reductase with various proteases were performed in the presence and absence of the flavoprotein ligands. Both the diaphorase and the ferredoxin-dependent activities of the enzyme were followed as well as the proteolytic pattern in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with further characterization of the polypeptides produced. These experiments revealed that only two regions of the flavoprotein are susceptible to the attack of the proteases used: (a) the N-terminal chain which can be cleaved only up to Lys35 and (b) the sequence segment 235-250. It can be inferred that these regions are on the surface of the protein molecule and presumably have a very flexible conformation adaptable to the protease active site. The deletion of the N-terminal region up to Thr36 of the native reductase (Mr 35,000) produced a truncated form (Mr about 31,000) which had full diaphorase activity but lost the capacity to catalyze the ferredoxin-dependent reaction. Proteolytic cleavage at the 235-250 segment of the sequence yielded a nicked protein (Mr about 30,000 by gel filtration; 23,000 plus 7,000 in denaturing electrophoresis) devoid of both activities. Protection by the flavoprotein ligands implies that the 23-35 region of the sequence is part of the binding site for ferredoxin and the 235-250 polypeptide segment is in the NADP(+)-binding site.

Amino Acid Sequence↗
PubMed1990

The downfield resonances in the 1H NMR spectra of Azotobacter vinelandii and Pseudomonas putida seven-iron ferredoxins.

Pseudomonas putida and Azotobacter vinelandii ferredoxins each contain one [4Fe-4S] cluster and one [3Fe-4S] cluster. Their polypeptide chains are nearly identical, differing by only 15 residues out of a total of 106. T1 measurements and temperature dependence studies of the 1H NMR spectrum of each ferredoxin demonstrate that all six resolved downfield resonances are near an iron-sulfur center. The five most downfield resonances are shown to arise from protons on cysteinyl beta-carbons by incorporation of cysteine deuterated at the beta-carbon into cell protein. The sixth peak (10.5 ppm) is shown to be a non-cysteinyl proton. This peak resolves into two resonances of approximately equal intensity at temperatures below 15 degrees or above 25 degrees C. A nuclear Overhauser effect observed between the two downfield-most resonances of A. vinelandii ferredoxin indicates that they originate from a geminal pair of beta-cysteinyl protons. An Overhauser effect observed between the resonances at 22.3 and 15.7 ppm, in conjunction with other results, implies that the resonance at 22.3 ppm arises from a beta-proton on the 3Fe-center-bound Cys16, while the resonance at 15.7 ppm arises from Cys45 beta-proton, which is bound to the 4Fe center. The five most downfield resonances are pH-dependent. The sixth peak (10.5 ppm in P. putida ferredoxin) is pH-independent. Possible origins for the observed pH dependencies are discussed.

Azotobacter↗
PubMed1980

[Role of ferredoxin in pseudo-cyclic electron transport in isolated pea chloroplasts].

The pseudo-cyclic electron transport in isolated pea chloroplasts in a medium without exogenous cofactors but containing methylviologen and ferredoxin was studied. In order to establish the steps limiting the electron transport, the rates of electron transport a different pH values and under phosphorylating and non-phosphorylating conditions in the presence of the uncoupler--gramicidin D--were determined. It was shown that in the absence of exogenous cofactors the total electron transport is limited by a stage of electron transfer from the acceptor site of photosystem I to oxygen. An addition of ferredoxin or methylviologen stimulates oxygen consumption. It was shown that the plot of the dependence of the rate of oxygen consumption on ferredoxin concentration is two-phase in contrast to that of methylviologen concentration, which is indicative of the existence of two competitive reactions of oxygen reduction involving ferredoxin (with different Km values for the protein).

Chloroplasts↗
PubMed1982

Amino acid sequence of a 3Fe:3S ferredoxin from the "archaebacterium" Methanosarcina barkeri (DSM 800).

The complete amino acid sequence for a 3Fe:3S ferredoxin from the "archaebacterium" Methanosarcina barkeri (DSM 800) was determined by repetitive Edman degradation on the whole protein and peptides derived from trypsin, thermolysin, and Staphylococcus aureus protease digestion. The protein has 59 residues of which 8 are cysteines. The latter have the same spacing and distribution as found for the clostridial-type 2 x 4Fe:4S ferredoxins. Also, the sequence had evidence of internal homology which is indicative of gene duplication prior to the divergence of the archaebacteria and the eubacteria. This is the first sequence to be reported for a methanogen ferredoxin and only the fourth for a 3Fe:3S ferredoxin from any source.

Amino Acid Sequence↗
PubMed1982

Core dimensions in the 3Fe cluster of Desulfovibrio gigas ferredoxin II by extended X-ray absorption fine structure spectroscopy.

We have obtained the iron K-edge extended X-ray adsorption fine structure spectra of the 3Fe ferredoxin II of Desulfovibrio gigas in the oxidized and reduced states. For both states, interpretation of the EXAFS data suggests that the Fe-S first shell coordination distance is near 2.25 A, in agreement with crystallographic studies of model compounds and proteins containing 2Fe-2S and 4Fe-4S centers, as well as with a recent crystallographic study of Azotobacter vinelandii ferredoxin I (Ghosh, D., Furey, W., Jr., O'Donnell, S., and Stout, C. D. (1981) J. Biol. Chem. 256, 4185-4192). The apparent Fe-Fe distance we obtain for the desulfovibrio protein (2.7 A) also agrees with similar distances seen in other Fe-S centers, except with the 3Fe cluster in the Azotobacter vinelandii ferredoxin I structure, for which an Fe-Fe distance of 4.2 A was reported. We conclude that either the two 3Fe ferredoxins have substantially different core dimensions, a possibility apparently unique to 3Fe centers among known Fe-S systems in proteins, or that one (or more) of the structural studies is in substantial error.

Desulfovibrio↗
PubMed1994

Mutational analysis of photosystem I polypeptides in Synechocystis sp. PCC 6803. Subunit requirements for reduction of NADP+ mediated by ferredoxin and flavodoxin.

The subunit requirements for NADP+ reduction by photosystem I were assessed in mutants of Synechocystis sp. PCC 6803 created by targeted inactivation of the psaD, psaE, psaF, and psaL genes. The PsaE-less, PsaF-PsaJ-less, and PsaL-less mutants showed normal photoautotrophic growth, while the growth of PsaD-less mutants was slower without glucose. In isolated wild-type membranes, the rate of flavodoxin reduction and flavodoxin-mediated NADP+ reduction were 800 and 480 mumol/mg of chlorophyll/h, respectively. The rate of ferredoxin-mediated NADP+ photoreduction was 460 mumol/mg of chlorophyll/h. There was no diminution in NADP+ photoreduction in membranes isolated from the PsaF-less and PsaL-less mutants. The rates of ferredoxin-mediated NADP+ photoreduction in membranes of the PsaE-less mutants were 25 mumol/mg of chlorophyll/h. However, the rate of flavodoxin reduction was 380 mumol/mg of chlorophyll/h, and that of flavodoxin-mediated NADP+ photoreduction was 170 mumol/mg of chlorophyll/h. PsaD-less membranes showed < 20% of the wild-type rates of flavodoxin-mediated NADP+ photoreduction, but were completely deficient in ferredoxin-mediated NADP+ photoreduction. Therefore, the roles of PsaE and PsaD are more crucial for "docking" of ferredoxin than of flavodoxin. Proteolysis studies showed that while PsaD was susceptible to rapid in vitro degradation by thermolysin, the number and sizes of protease-resistant fragments were not affected by the absence of PsaE. Protease accessibility studies further indicated that the C-terminal domain of PsaD is surface-exposed on the n-side. These results suggest that PsaE and the C-terminal domain of PsaD generate the docking site for the electron acceptors of photosystem I.

Bacterial Proteins↗
PubMed1993

The transit sequence mediates the specific interaction of the precursor of ferredoxin with chloroplast envelope membrane lipids.

The interaction of the precursor of the chloroplast protein ferredoxin with membrane lipids was studied in monolayer experiments in order to investigate the possible involvement of membrane lipids in the protein translocation process. The precursor efficiently and specifically inserts into a total lipid extract of its biological target the outer envelope membrane of chloroplasts. This interaction is mediated by the transit sequence as it can also be observed for the chemically prepared transit peptide of ferredoxin but neither for the ferredoxin apoprotein nor holoprotein. Interactions with the individual chloroplast lipids, monogalactosyl-diacylglycerol, sulfoquinovosyl-diacylglycerol, and phosphatidylglycerol are predominantly involved which corresponds to the results obtained for transit peptide fragments of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (van't Hof, R., Demel, R. A., Keegstra, K., and De Kruijff, B. (1991) FEBS Lett. 291, 350-354). No efficient interaction was obtained with digalactosyl-diacylglycerol and phosphatidylcholine, suggesting that a loose lipid headgroup packing due to small lipid headgroups and/or electrostatic repulsions facilitates efficient insertion. The observed preferences for interaction of the precursor and transit peptide of ferredoxin for the chloroplast outer envelope membrane lipid extract and the presequence of cytochrome c oxidase subunit IV for the mitochondrial outer membrane lipid extract indicate that targeting sequence-lipid interactions contribute to organelle-specific protein targeting.

Amino Acid Sequence↗
PubMedDate not supplied

Structural features correlated with the extreme thermostability of 1[4Fe-4S] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima.

Understanding the molecular mechanisms behind extreme temperature stability is of relevance for the protein folding problem and for designing proteins for industrial and medical applications. A powerful approach for understanding the structural basis of thermostability is the comparison of high resolution structures of homologous proteins from mesophiles and thermophiles. The 1.75 A crystal structure of Thermotoga maritima 1[4Fe-4S] ferredoxin was compared with those of mesophilic ferredoxins. Detailed analysis of structural differences reveals that thermostability is achieved without large changes of the overall polypeptide chain folding. The most striking differences include the formation of additional hydrogen bonding networks involving both side-chain and main-chain atoms. These networks are mainly connecting turns and strongly fix the N-terminus to the central core of the protein, increasing the overall rigidity of Thermotoga maritima ferredoxin. Other possibly stabilizing factors are the shortening of a solvent exposed surface loop, the increased content of alanines in the second alpha-helix, and the replacement of three residues close to the iron-sulfur cluster, which are in energetically unfavourable conformations in other ferredoxins, by glycines.

Amino Acid Sequence↗
PubMed1986

Second gene (nifH*) coding for a nitrogenase iron protein in Azotobacter chroococcum is adjacent to a gene coding for a ferredoxin-like protein.

Azotobacter chroococcum MCD1 contains a cluster of nitrogen fixation (nif) genes coding for the structural polypeptides for nitrogenase (nifH for the Fe-protein and nifD and nifK for the MoFe protein) and a second sequence in the genome homologous to nifH. DNA fragments bearing this second nifH-like sequence were cloned and the DNA sequence around the homologous region determined. Two open reading frames were identified in this region. One codes for a protein of 289 amino acid residues and is highly homologous to other Fe-proteins but is different from the gene adjacent to the nifDK genes in A. chroococcum. This putative gene we call nifH*. The following open reading frame codes for a protein of 63 amino acids, nine of which are cysteine residues. The protein is homologous to the small low-potential ferredoxins found in anaerobic bacteria, and in particular those from Chlorobium limicola. Linkage between a structural gene for nitrogenase and a small ferredoxin has not previously been observed. Sequence analysis suggests that the two genes form an operon. Transcription of the ferredoxin gene on a 1320-bp transcript was only detectable under conditions in which A. chroococcum MCD1155, which carries a chromosomal deletion of 6.3 kb removing the entire nifHDK cluster, is capable of fixing N2, i.e. in media containing no added molybdenum or high levels of NH3. The size of the observed transcript agrees well with the predicted size for a transcript encoding nifH* and the ferredoxin genes. Expression of the nifH* promoter was not significantly activated in Escherichia coli even when nifA, the positive activator of nif genes in Klebsiella pneumoniae, was supplied in multiple copies. The results are discussed in relation to an alternative pathway for N2 fixation in A. chroococcum.

Amino Acid Sequence↗
PubMed1997

Indolepyruvate ferredoxin oxidoreductase from Pyrococcus sp. KOD1 possesses a mosaic structure showing features of various oxidoreductases.

Indolepyruvate ferredoxin oxidoreductase (IOR) catalyzes the oxidative decarboxylation of arylpyruvates. Gene cloning and sequencing analysis of the IOR gene from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was performed. Two genes, iorA and iorB. encoding alpha and beta subunits of IOR were found to be tandemly arranged, which suggests that gene expression is translationally coupled. Sequence analysis showed the C-terminal region of the alpha subunit to have a typical ferredoxin-type [4Fe-4S] cluster motif (CXXCXXCXXCXXXCP), which is similar to that present in the delta subunits of other oxidoreductases such as pyruvate ferredoxin oxidoreductase (POR) and 2-ketoisovalerate ferredoxin oxidoreductase (VOR). We suggest that the alpha subunit of KOD1-IOR has a mosaic structure composed of features characteristic of the alpha, beta and delta subunits from POR and VOR. KOD1-IOR was overproduced in anaerobically incubated Escherichia coli cells and the crude enzyme was extracted under anaerobic conditions. The optimal temperature for activity of recombinant IOR was 70 degrees C and the half-life of this enzyme in the presence of air was 15 min at 25 degrees C.

Amino Acid Sequence↗
PubMed1996

Characterisation and purification of pyruvate:ferredoxin oxidoreductase from Giardia duodenalis.

The major 2-oxoacid oxidoreductase (2-OR), pyruvate:ferredoxin oxidoreductase (PFOR) from Giardia duodenalis has been purified to apparent homogeneity. A second 2-OR with a preference for alpha-ketobutyrate as substrate was identified and was removed from PFOR containing fractions during purification. Only PFOR and the second 2-OR were identified in gels of crude Giardia extracts assayed for 2-OR activity. The native form of PFOR which is membrane associated, is a homodimer of 138 kDa subunits. Pyruvate is the preferred substrate: alpha-ketobutyrate and oxaloacetate, but not phenyl-pyruvate or alpha-ketoglutarate, are decarboxylated. PFOR from Giardia is more stable than PFOR from most other organisms and purified PFOR can be stored without deterioration at -70 degrees C. Purified PFOR donates electrons to Giardia ferredoxin (Fd I) with concomitant reduction of metronidazole. However, two other Giardia ferredoxins did not accept electrons from PFOR. Consistent with the involvement of PFOR in metronidazole activation, the activity of pyruvate dependent 2-OR activity was decreased in all metronidazole-resistant lines tested but not in furazolidone-resistant lines. The presence of three different ferredoxins and two 2-ORs in Giardia suggests that a number of different electron transport pathways operate in this organism providing unusual metabolic flexibility for a eukaryote.

Animals↗
PubMed1993

Purification and characterization of pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus grows optimally at 100 degrees C by carbohydrate fermentation. It is thought to contain a novel tungsten-dependent, NAD(P)-independent glycolytic pathway in which one of the oxidation steps is catalyzed by a tungsten-containing aldehyde ferredoxin oxidoreductase. The enzyme that catalyzes the terminal oxidation step, pyruvate ferredoxin oxidoreductase (POR), has now been purified. POR has a molecular mass of 100 kDa and is comprised of three subunits (45, 31 and 24 kDa). It lacks tungsten but contains thiamine pyrophosphate (TPP) and two ferredoxin-type [4Fe-4S] clusters per molecule which, by EPR spectroscopy, can be differentiated by their relaxation properties. The enzyme requires CoASH but not TPP for pyruvate oxidation activity and will not use 2-oxoglutarate, phenyl pyruvate or indole pyruvate as substrates. POR is virtually inactive at 25 degrees C and shows a temperature optimum for pyruvate oxidation above 90 degrees C. The apparent Km values for pyruvate, CoASH and P. furiosus ferredoxin at 80 degrees C are 460, 100 and 70 microM, respectively. Carbon monoxide was a potent inhibitor of pyruvate oxidation (apparent Ki = 7 microM). The half-life of activity (t50%) in air at 25 degrees C was 15 min and the t50% value at 80 degrees C (under anaerobic conditions) was 23 min. Based on molecular comparisons with PORs from mesophilic organisms, it is proposed that P. furiosus POR may represent an ancestral form of a pyruvate-oxidizing enzyme.

Amino Acids↗
PubMed1982

The pyruvate: ferredoxin oxidoreductase in heterocysts of the cyanobacterium Anabaena cylindrica.

Heterocyst preparations have been obtained which actively perform nitrogen fixation (C2H2 reduction) and contain the enzymes of glycolysis and some of the tricarboxylic acid cycle. Pyruvate: ferredoxin oxidoreductase has been unambiguously demonstrated in extracts from heterocysts by the formation of acetylcoenzyme A, CO2 and reduced methyl viologen (ferredoxin) from pyruvate, coenzyme A and oxidized methyl viologen (ferredoxin) as well as by the synthesis of pyruvate from CO2, acetylcoenzyme A and reduced methyl viologen. Pyruvate supports C2H2 reduction by isolated heterocysts, however, with lower activity than Na2S2O4 and H2. alpha-Ketoglutarate: ferredoxin oxidoreductase is absent in Anabaena cylindrica, confirming that the organism has an incomplete tricarboxylic acid cycle.

Carbon Radioisotopes↗
PubMed2005

Crystal structure of phycocyanobilin:ferredoxin oxidoreductase in complex with biliverdin IXalpha, a key enzyme in the biosynthesis of phycocyanobilin.

Phytobilins (light harvesting and photoreceptor pigments in higher plants, algae, and cyanobacteria) are synthesized from biliverdin IXalpha (BV) by ferredoxin-dependent bilin reductases (FDBRs). Phycocyanobilin:ferredoxin oxidoreductase (PcyA), one such FDBR, is a new class of radical enzymes that require neither cofactors nor metals and serially reduces the vinyl group of the D-ring and A-ring of BV using four electrons from ferredoxin to produce phycocyanobilin, one of the phytobilins. We have determined the crystal structure of PcyA from Synechocystis sp. PCC 6803 in complex with BV, revealing the first tertiary structure of an FDBR family member. PcyA is folded in a three-layer alpha/beta/alpha sandwich structure, in which BV in a cyclic conformation is positioned between the beta-sheet and C-terminal alpha-helices. The basic patch on the PcyA surface near the BV molecule may provide a binding site for acidic ferredoxin, allowing direct transfer of electrons to BV. The orientation of BV is definitely fixed in PcyA by several hydrophilic interactions and the shape of the BV binding pocket of PcyA. We propose the mechanism by which the sequential reduction of the D- and A-rings is controlled, where Asp-105, located between the two reduction sites, would play the central role by changing its conformation during the reaction. Homology modeling of other FDBRs based on the PcyA structure fits well with previous genetic and biochemical data, thereby providing a structural basis for the reaction mechanism of FDBRs.

Amino Acid Sequence↗