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PubMed1999

A new assignment strategy for the hyperfine-shifted 13C and 15N resonances in Fe2S2 ferredoxins.

Assignment of hyperfine-shifted resonances in paramagnetic metalloproteins such as Fe2S2 ferredoxins poses a major experimental challenge due to hyperfine shifts and/or severe line broadening. We have explored the possibility of using structural data from homologous proteins as part of an assignment strategy for the sequence-specific assignment of hyperfine-shifted backbone carbonyl (13C') and nitrogen resonances (15N) in Fe2S2 ferredoxins. This strategy is based on the assignment of resonances in the paramagnetic region to particular types of amino acid residues using selective isotope labeling. Reduced metal-nuclear distances are then calculated from experimentally determined T1 relaxation times for those resonances and the calculated distances aligned with the distances of nuclei at corresponding amino acid sequence positions in the crystal structure of a structurally homologous protein. The comparative assignment approach has met with success in correctly predicting the 13C' and 15N assignments in Pdx degrees from the crystal structure data of two similar and related ferredoxins, namely bovine adrenodoxin and Anabaena ferredoxin. Sequence-specific assignments made in this fashion were verified by selective 13C'{15N} decoupling experiments.

Amino Acid Sequence↗
PubMed2001

Role of acidic amino acid residues of PsaD subunit on limiting the affinity of photosystem I for ferredoxin.

The PsaD subunit of photosystem I is one of the central polypeptides for the interaction with ferredoxin, its acidic electron acceptor. In the cyanobacterium Synechocystis 6803, this role is partly performed by a sequence extending approximately from histidine 97 to arginine 119, close to the C-terminus. In the present work, acidic amino acids D100, E105, and E109 are shown to moderate the affinity of Photosystem I for ferredoxin. Most single replacements of these residues by neutral amino acids increased the affinity for ferredoxin, resulting in a dissociation constant as low as 0.015 microM for the E105Q mutant (wild-type K(D) = 0.4 microM). This is the first report on the limitation of photosystem I affinity for ferredoxin due to acidic amino acids from PsaD subunit. It highlights the occurrence of a negative control on the binding during the formation of transient complexes between electron carriers.

Amino Acid Sequence↗
PubMed1997

Solution structure of ferredoxin from the thermophilic cyanobacterium Synechococcus elongatus and its thermostability.

The three-dimensional structure of ferredoxin, purified from the thermophilic cyanobacterium Synechococcus elongatus, was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance. In addition to the 946 distance constraints from nuclear Overhauser effect connectivities, we added 241 distance constraints derived from the crystal structure of Spirulina platensis ferredoxin to the 19 residues close to the [2Fe-2S] iron-sulfur center, where crosspeaks disappeared due to paramagnetic effects. The atomic root-mean-square difference of the ten converged structures from the mean structure was 0.61(+/-0.12) A for backbone atoms (N, C(alpha), C'). The main-chain structure was almost the same as the crystal structures of other mesophile ferredoxins, but comparison of the side-chain structures revealed an extension of the hydrophobic core, a unique hydrophobic patch on the surface of the large beta-sheet, and two unique charge networks in this thermostable ferredoxin structure, some of which might contribute to thermostability.

Amino Acid Sequence↗
PubMed1997

Ferredoxin from the hyperthermophile Thermotoga maritima is stable beyond the boiling point of water.

Heat-stable proteins from hyperthermophilic microorganisms are ideally suited for investigating protein stability and evolution. We measured with differential scanning calorimetry and optical absorption spectroscopy the thermal stability of [4Fe-4S] ferredoxin from Thermotoga maritima (tfdx), which is a small electron transfer protein. The results are consistent with two-state unfolding at the record denaturation temperature of 125 degrees C. According to the crystal structure at 1.75 A resolution, T. maritima ferredoxin contains a significantly increased number of hydrogen bonds that involve charged amino acid side-chains, compared to thermolabile ferredoxins. Thus, our results suggest that polar interactions substantially contribute to protein stability at very high temperatures. Moreover, because small [4Fe-4S] ferredoxins seem to have occurred early in evolution, the extreme thermostability of tfdx supports the hypothesis that life originated at high temperatures.

Calorimetry, Differential Scanning↗
PubMed1993

One-step purification of plant ferredoxin-NADP+ oxidoreductase expressed in Escherichia coli as fusion with glutathione S-transferase.

Complementary DNA sequences encoding the mature form of pea ferredoxin-NADP+ reductase were cloned in-frame at the 3' end of the Schistosoma japonicum glutathione S-transferase gene in the expression vector pGEX-3X (Smith and Johnson, Gene 67, 31-40, 1988). A spacer sequence linking the two genes was modified to provide a proteolytic site just before the first amino acid residue of mature pea reductase. When introduced into competent Escherichia coli cells and induced, the resulting plasmid (pGF205) directed the expression of a 60-kDa immunoreactive peptide that results from the fusion between glutathione S-transferase and ferredoxin-NADP+ reductase sequences. The fused protein could be purified in a single step by selective absorption onto glutathione-agarose beads, followed by elution with free glutathione. It showed both transferase and reductase activities. Removal of the transferase portion by cleavage with the restriction protease Xa rendered ferredoxin-NADP+ reductase electrophoretically homogeneous. The purified transgenic enzyme showed kinetic and spectroscopic properties that were similar to those reported for the plant flavoprotein, indicating that, even when fused to the 27-kDa transferase portion, the reductase was still able to assemble FAD and to acquire an active conformation in the bacterial host. The expression-purification protocol employed here allows the isolation of up to 1 mg of active ferredoxin-NADP+ reductase/g of transformed cells. The system is potentially useful for the purification of activity-impaired forms of the flavoprotein.

Amino Acid Sequence↗
PubMed1988

Molecular cloning and nucleotide sequence analysis of the gene coding for heterocyst ferredoxin from the cyanobacterium Anabaena sp. strain PCC 7120.

In heterocysts of the filamentous cyanobacterium Anabaena 7120 a specific [2Fe-2S] ferredoxin is synthesized, serving as immediate electron donor to nitrogenase. The structural gene for this heterocyst ferredoxin, fdxH, was isolated from a recombinant lambda library, using an oligonucleotide probe derived from a unique segment of the N-terminal amino acid sequence of the purified protein. The sequence of the entire fdxH coding region was determined including 3' and 5' flanking sequences. Assuming proteolytic cleavage of the first methionine residue, the molecular weight of Anabaena 7120 heterocyst ferredoxin is 10,806. Compared with the ferredoxin from vegetative cells, 47 out of 98 amino acid residues are different, including conversions within a highly conserved region responsible for binding of the iron-sulfur cluster. Northern hybridization with a 0.64 kb EcoRI DNA fragment containing the entire fdxH gene indicated two major transcripts of 0.59 and 1.85 kb, which are expressed at a late stage of heterocyst differentiation. By S1 nuclease digestion and primer extension a possible start site of transcription was mapped, 132 bp upstream of fdxH; however, neither a typical Escherichia coli nor nif-type promoter sequence was apparent. Southern hybridization detected only one copy of the fdxH gene in the Anabaena 7120 genome. FdxH is located approximately 7 kb downstream from the nifHDK gene cluster.

Amino Acid Sequence↗
PubMed1979

Ferredoxin degradation in growing Clostridium pasteurianum during periods of iron deprivation.

Clostridium pasteurianum was grown in batch cultures on media with an initial iron concentration of 10 micron. The uptake of iron and the synthesis of ferredoxin was followed. All the iron present in the medium was taken up by the cells before 50% of the final cell density was attained. The bacteria then continued to grow in the complete absence of exogenous iron. Ferrodoxin was synthesized during growth until the exogenous iron concentration dropped below 1 micron. During growth in the absence of iron ferredoxin was degraded with the result that at the end of growth the cells did not contain ferredoxin. The specific activity of the iron sulfur protein, pyruvate synthase (E.C. 1.2.7.1), remained constant during growth of C. pasteurianum in the absence of exogenous iron. This finding suggest that ferredoxin was used as an endogenous source of iron for the synthesis of essential iron proteins during periods of iron deprivation.

Biodegradation, Environmental↗
PubMed1992

Functional analysis of the cysteine motifs in the ferredoxin-like protein FdxN of Rhizobium meliloti involved in symbiotic nitrogen fixation.

The Rhizobium meliloti fdxN gene, which is part of the nifA-nifB-fdxN operon, is absolutely required for symbiotic nitrogen fixation. The deduced sequence of the FdxN protein is characterized by two cysteine motifs typical of bacterial-type ferredoxins. The Fix-phenotype of an R. meliloti fdxN::[Tc] mutant could be rescued by the R. leguminosarum fdxN gene, whereas no complementation was observed with nif-associated genes encoding ferredoxins from Bradyrhizobium japonicum, Azotobacter vinelandii, A. chroococcum and Rhodobacter capsulatus. In addition to these heterologous genes, several R. meliloti fdxN mutant genes constructed by site-directed mutagenesis were analyzed. Not only a cysteine residue within the second cysteine motif (position 42), which is known to coordinate the Fe-S cluster in homologous proteins, but also a cysteine located down-stream of this motif (position 61), was found to be essential for the activity of the R. meliloti FdxN protein. Changing the amino acid residue proline in position 56 into methionine resulted in a FdxN mutant protein with decreased activity, whereas changes in positions 35 (Asp35Glu) and 45 (Gly45Glu) had no significant effect on the function of the FdxN mutant proteins. In contrast to bacterial-type ferredoxins, which contain two identical cysteine motifs of the form C-X2-C-X2-C-X3-C, nif-associated ferredoxins, including R. meliloti FdxN, are characterized by two different cysteine motifs. Six "additional" amino acids separate the second (Cys42) and the third cysteine (Cys51) in the C-terminal motif (C-X2-C-X8-C-X3-C).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Examination of protein sequence homologies: IV. Twenty-seven bacterial ferredoxins.

Sequence homologies of 27 bacterial ferredoxins were examined using a computer program that quantitatively evaluates extent of similarity as a correlation coefficient. The results of a similarity search among the sequences demonstrated that the basal sequence consists of a pair of extremely similar segments of 26 amino acids connected by a three-amino acid group. The segment pairs, which would have arisen from gene duplication, are termed the first and second units. Because of the gene duplication, the connector sequence appears to have been introduced as a structurally important chain reversal. Each of the two units contains four cysteine residues, which are inserted one by one among seven, two, two, three, and eight amino acid alignments, respectively. The bacterial ferredoxins were categorized with regard to basal constitution as follows: group 1, in which both units closely conform to the basal structure; group 2, in which the second unit is modified in a characteristic manner among members; group 3, in which the first unit is modified in a characteristic manner, while the conforming second unit is accompanied by a long accessory sequence; group 4, in which there are modifications before and/or after the units, of which the respective central domains remain nearly intact; and group 5, where only the former of two Fe:S cluster ligation sets of four cysteines is estimated to remain intact, whereas the latter set is extremely modified. It is noteworthy that throughout all bacterial ferredoxins, one of two cysteine sets never fails to be completely intact and, moreover, the connector of three amino acids also exists intact. Based on this grouping and on the correspondences among the groups, average correlation coefficients among all members were computed, and the respective evolutionary relationships were examined. The results supported the proposition that transposition had occurred in the Azotobacter-type ferredoxins of group 3.

Amino Acid Sequence↗
PubMed2006

Pyrococcus furiosus 4Fe-ferredoxin, chemisorbed on gold, exhibits gated reduction and ionic strength dependent dimerization.

Pyrococcus furiosus ferredoxin is a small metalloprotein that shuttles electrons between redox enzymes. In its native 4Fe-4S form the protein is highly thermostable. In addition to three cluster-ligating cysteines, two surface cysteine residues (C21 and C48) are present. We used the reactivity of these surface thiols to directly immobilize ferredoxin on a bare gold electrode, with an orientation in which the cluster is exposed to solution. Voltammetry, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM) studies established the immobilization of the 4Fe form. Native and recombinant wild-type ferredoxins were compared with the C48S, C21S, and C21S/C48S mutants. The variants with one and two surface cysteines can be directly chemisorbed on bare gold. Cyclic voltammetry demonstrated that the reduction potentials are similar to those in solution. The interfacial electron transfer kinetics revealed that the reduction is gated by the interconversion between two oxidized species. AFM images showed that dimers are chemisorbed at low ionic strength, while monomers are present at high ionic strength. XPS spectra revealed the presence of S, Fe, C, N, and O at the surface, which are assigned to the corresponding atoms in the peptide and the cofactor. Analysis of the sulfur spectrum corroborates that both C21 and C48 form gold-thiolate bonds. Moreover, two inorganic sulfide and two iron species were identified, suggesting an inhomogeneous charge distribution in the 4Fe-4S cluster. In conclusion, P. furiosus ferredoxin can be directly and vectorially chemisorbed on gold with retention of its properties. This may provide a biocompatible electrode surface with docking sites for redox enzymes.

Cysteine↗
PubMed1990

Characterization of a ferredoxin:NADP+ oxidoreductase from a nonphotosynthetic plant tissue.

A flavoprotein with properties similar to those of ferredoxin:NADP+ oxidoreductases found in the leaves of higher plants has been purified to apparent homogeneity from bean sprouts, a nonphotosynthetic plant tissue. The absorbance and circular dichroism spectra of the bean sprout protein are similar to those of spinach leaf ferredoxin:NADP+ oxidoreductase and an antibody raised against the spinach enzyme recognized the bean sprout enzyme. The bean sprout enzyme catalyzed ferredoxin-dependent electron transfer from NADPH to equine cytochrome c at a high rate but, unlike the spinach enzyme, exhibited little NADPH to 2,6-dichlorophenol indophenol diaphorase activity. The bean sprout enzyme forms a 1:1 electrostatically stabilized complex with ferredoxins isolated from either bean sprouts or spinach leaves.

Chromatography, Affinity↗
PubMed1992

Expression and characterization of human mitochondrial ferredoxin reductase in Escherichia coli.

Ferredoxin reductase (Fd-reductase) supplies reducing equivalents obtained from NADPH to mitochondrial cytochrome P450 enzymes via the small iron-sulfur protein ferredoxin. Two cDNAs (differing by the presence or absence of an 18-bp insert in the coding region) for the human Fd-reductase were subcloned into a newly constructed general purpose expression vector, p delta blue; protein expression under control of the bacteriophage lambda pL promoter was then induced in Escherichia coli. Western blot analysis of subcellular fractions indicated that Fd-reductase protein expressed from both plasmids was present in both inclusion bodies and soluble fractions. However, only the form lacking the insert exhibited Fd-reductase activity. The active material was purified and was found to have electrophoretic, chromatographic, optical, and circular dichroism properties comparable to the bovine homologue. The apparent Km of the expressed protein for NADPH was determined to be 0.7 +/- 0.1 microM and the apparent Km for human ferredoxin was found to be 106 +/- 8 nM. While yields of active enzyme were relatively low (approximately 0.1 mg/liter of culture), the production of Fd-reductase in E. coli will allow structural and mechanistic studies of the enzyme and its interactions with ferredoxin.

Base Sequence↗
PubMed1981

Thioredoxin/fructose-1,6-bisphosphatase affinity in the enzyme activation by the ferredoxin-thioredoxin system.

In this work we analyze the affinity relationship between photosynthetic fructose-1,6-bisphosphatase and ferredoxin and thioredoxin from spinach leaves, two components of the proposed light-activation system of this enzyme, using affinity techniques on ferredoxin- and thioredoxin-Sepharose columns. Oxidized and reduced ferredoxin did not show enzyme affinity, whereas thioredoxin, both the oxidized and the dithiothreitol-reduced form, exhibited a strong bisphosphatase affinity at pH 7.5; this thioredoxin/enzyme affinity appears diminished at pH 8.2. When the affinity experiments were performed in the presence of 5 mM Mg2+, only 30% and 12% of the bisphosphatase remained bound to the thioredoxin-Sepharose at pH 7.5 and 8.0, respectively; these percentages were reduced to 6% when the Mg2+ concentration increased to 10 mM. These results suggest that a rise of stromal pH and Mg2+ concentration can account for a loosening of the thioredoxin/bisphosphatase linkage, which could be of physiological significance in the dark-light transition. Studies on the nature of the chemical groups responsible for the affinity have shown that the thioredoxin/bisphosphatase linkage is concerned with the existence of hydrophobic clusters. We have found no difference in the behaviour of the chloroplastic thioredoxins f and m, and the cytoplasmic ones cf and cm. These results support the existence of an in vivo thioredoxin/fructose-1,6-bisphosphatase interaction, in accordance with the light-activation mechanism by the ferredoxin-thioredoxin system.

Amino Acids↗
PubMed1976

The separation of pyruvate-ferredoxin oxidoreductase from Clostridium pasteurianum into two enzymes catalyzing different reactions.

The ferredoxin requiring cleavage of pyruvate to acetyl-CoA and CO2 is catalyzed by pyruvate ferredoxin oxidoreductase (pyruvate:ferredoxin oxidoreductase (CoA-acetylating):, EC 1.2.7.1). The same enzyme is thought to catalyze the reversal of this reaction, i.e. the synthesis of pyruvate from acetyl-CoA and CO2 in the presence of reduced ferredoxin. Evidence is presented that the forward and reverse reactions are catalyzed not by one, but by two proteins that are clearly separable by Sephadex G-200 gel filtration.

Clostridium↗
PubMed1977

Molecular heterogeneity of ferredoxin-NADP+ reductase from spinach leaves.

Ferredoxin-NADP+ reductase (NADPH: ferredoxin oxidoreductase, EC 1.6.7.1) from spinach leaves has been purified according to a new procedure. The enzyme shows the presence of five molecular forms as identified by isoelectric focusing, namely a, b, c, d and e with pI values of 6.0, 5.5, 5.2, 5.0 and 4.8, respectively. All the bands are catalytically active and are clearly identifiable after the first steps of the purification procedure. The basic pattern of the ferredoxin-NADP+ reductase forms is the same whether extracted from one or many spinach plants and is not affected by the different purification procedures used. Two distinct classes of molecular weight have been found for the isolated forms b, c and d as measured by sodium dodecyl sulphate electrophoresis, with values of 33 000-34 000 for the first and 36 000-38 000 for the later two forms. Gel electrophoresis in non-denaturing media at different gel concentrations gives the same order of molecular weight values, thus ruling out the possibility that the native enzyme is a dimer, as has been reported by Schneeman, R. and Krogmann, D.W. ((1975) J. Biol. Chem. 250, 4965-4971). No significant kinetic differences were detectable for the isolated forms of ferredoxin-NADP+ reductase.

Amino Acids↗
PubMed1978

Isoelectric focusing of ferredoxins, flavordoxins and a rubredoxin.

Isoelectric points of ferredoxins, flavodoxins and a rubredoxin from a range of sources were measured by electrofocusing over the pH range between 2.5 and 5.0 on thin layers of polyacrylamide gel. The pH gradient along the gel was measured directly by a surface electrode. The isoelectric points of the plant-type ferredoxins were between approx. 3.15 and 3.35, and those of the flavodoxins close to 3.5. Ferredoxin, rubredoxin and flavodoxin from Clostridium pasteurianum had isolectric points of the of 2.75, 2.9, and 3.1, respectively. The values for the isoelectric points ferredoxins are significantly lower than previous results in the literature suggest.

Bacteria↗
PubMed1980

Amino acid sequence of 2Fe-2S ferredoxin from an extreme halophile, Halobacterium of the Dead Sea.

The primary structure of the 2Fe-2S ferredoxin from Halobacterium of the Dead Sea was determined and it consisted of 128 amino acid residues including an N epsilon-acetyllysyl residue. Due to a high degree of sequence homology between this ferredoxin and the one from Halobacterium halobium, all tryptic peptides could be aligned in order. Only 20 amino acid differences were observed between these two halobacterial ferredoxins. The distribution of cysteinyl residues involved in the iron chelation was similar to that of chloroplast-type ferredoxins.

Amino Acid Sequence↗
PubMed1989

Experimental evidence of an alpha helix in Desulfovibrio desulfuricans Norway ferredoxin I: a two-dimensional NMR study.

Desulfovibrio ferredoxins are small proteins involved in biological oxido-reduction reactions and contain either one or two (4Fe-4S) clusters. The conformation of D. desulfuricans Norway ferredoxin I in solution was studied by two-dimensional NMR and various conformational parameters (n.O.e. and J-coupling) indicate the presence of an alpha-helix involving residues 41 to 50. These data confirm an earlier proposal (Fukuyama et al, J. Mol. Biol. 199, 183 (1988] in which the space of the missing cluster in monocluster ferredoxins is occupied by an alpha-helix. The evolutionary relevance of this result is discussed in view of published sequences and structures of related ferredoxins.

Amino Acid Sequence↗