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PubMed2000

Electron-nuclear interactions in two prototypical [2Fe-2S] proteins: selective (chiral) deuteration and analysis of (1)H and (2)H NMR signals from the alpha and beta hydrogens of cysteinyl residues that ligate the iron in the active sites of human ferredoxin and Anabaena 7120 vegetative ferredoxin.

A vertebrate ferredoxin (human ferredoxin) and a plant-type ferredoxin (the ferredoxin from the vegetative form of Anabaena 7120) were labeled selectively with deuterium at their active site cysteines. The recombinant proteins were produced in Escherichia coli and labeled by replacing natural abundance cysteine in the defined culture medium with (2)H(alpha)-cysteine, (2)H(beta2), (2)H(beta3)-cysteine, or (2)H(beta2)-cystine. The chiral labeled cystine ((2)H(beta2)-cystine) was prepared by selective hydrogen exchange catalyzed by cystathionine gamma-synthase. NMR spectra of these samples in their oxidized and reduced states support unambiguous identifications by atom type of (1)H and (2)H NMR signals from the cysteine alpha and beta hydrogens. These signals lie outside the normal diamagnetic spectral region as a result of interaction of the hydrogens with unpaired electron density from the iron-sulfur cluster, and their chemical shifts are highly dependent on local conformation at the active site. The very different chemical properties of the iron centers of plant-type and vertebrate ferredoxins reflect relatively small differences in the conformation of the iron-sulfur cluster ligands.

Anabaena↗
PubMed2001

New insights into the thermostability of bacterial ferredoxins: high-resolution crystal structure of the seven-iron ferredoxin from Thermus thermophilus.

The crystal structure of the seven-iron ferredoxin from Thermus thermophilus (FdTt) has been determined at 1.64 A resolution, allowing us to unveil the common mechanisms of thermostabilization within "bacterial-type" ferredoxins. FdTt and other homologous thermophilic seven-iron ferredoxins are smaller than their mesophilic counterparts. Thermostabilizing features are optimized in a minimal structural and functional unit, with an extensive cross-linking of secondary structure elements mediated by improved polar and hydrophobic interactions. Most of the potentially stabilizing features are focused on the vicinity of the functional [3Fe-4S] cluster. The structural [4Fe-4S] cluster is shielded in thermophilic FdTt by an increased number of polar interactions involving the two N-terminal residues. Comparisons with the hyperthermostable ferredoxin from Thermotoga maritima reveal that (1) a reduction in the number of non-glycine residues in strained conformations, (2) improved polar interactions within the common iron-sulfur cluster binding (betaalphabeta)2 motif, and (3) an optimized charge distribution at the protein surface, constitute a common strategy for increasing the thermal stability of these ferredoxins.

Amino Acid Sequence↗
PubMed1987

Interaction between photosystem I and ferredoxin. Identification by chemical cross-linking of the polypeptide which binds ferredoxin.

Ferredoxin has been effectively cross-linked to photosystem I complex by treatment of purified particles or thylakoids with N-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a zero-length cross-linker which stabilizes protein-protein electrostatic interactions. Analysis of photosystem I polypeptide composition after such a treatment showed a specific decrease of the 20-kDa subunit and the appearance of a new component of about 42 kDa which was recognized by the anti-ferredoxin antibody. Cross-linking of ferredoxin to thylakoids allowed the membrane preparation to photoreduce cytochrome c without requiring exogenous ferredoxin, whereas photosystem I particles purified from treated thylakoids were inactivated in the NADP+ photoreduction activity. From these results, it can be inferred that the polypeptide of 20 kDa is the photosystem I subunit which interacts with ferredoxin during the photosynthetic electron transport.

Carbodiimides↗
PubMed2005

Two tandemly arranged ferredoxin genes in the Hydrogenobacter thermophilus genome: comparative characterization of the recombinant [4Fe-4S] ferredoxins.

A thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. A gene encoding a ferredoxin involved in this cycle as an electron donor (HtFd1) was cloned and sequenced. Interestingly, another ferredoxin gene (encoding HtFd2) was found in tandem with the HtFd1 gene. These two ferredoxin genes overlapped by four bp, and transcriptional analysis revealed that they are co-transcribed as an operon. The deduced amino acid sequences of HtFd1 and HtFd2 were 42.9% identical and each contained four cysteine residues that serve as probable ligands to an iron-sulfur cluster. Spectroscopic analyses of the purified recombinant ferredoxins heterologously expressed in Escherichia coli indicated that each ferredoxin contains a single [4Fe-4S]2+/1+ cluster.

Amino Acid Sequence↗
PubMed1983

Ferredoxin from Aphanothece halophitica, a unicellular blue-green alga: close relationship to ferredoxins from filamentous blue-green algae and phylogenetic implications.

The amino acid sequence of a ferredoxin from a unicellular blue-green alga, Aphanothece halophitica, was established by the conventional methods. Total number of residues was 98 lacking only tryptophan. A most probable phylogenetic tree was constructed for 19 algal ferredoxins on the basis of an amino acid difference matrix made from the sequence comparison. A. halophitica has been classified as a unicellular blue-green alga in the same genus to which Aphanothece sacrum belongs, but the tree indicates A. halophitica ferredoxin to be very close to those of the members of filamentous blue-green algae. The tree divides prokaryotic and eukaryotic algal ferredoxins into several groups, suggesting that the ferredoxin phylogenetic tree reflects the evolutionary trails of various algae, which is also reflected in the structural characteristics, particularly in the presence of gaps. Other notable features are presented in considering algal taxonomy.

Amino Acid Sequence↗
PubMed1997

Simultaneous expression of ferredoxin, ferredoxin reductase and P450 in COS7 cells.

cDNA fragments encoding mouse ferredoxin and ferredoxin reductase were simultaneously introduced into COS7 cells by using an expression vector, pUC-SR alpha plasmid. When using the mitochondrial fraction prepared from the transfected cells, cytochrome-c reductase activity was detected. This activity was highest when 7.5 micrograms of the ferredoxin expression plasmid (pSR alpha F) and 2.5 micrograms of the ferredoxin reductase expression plasmid (pSR alpha FR) were transfected into COS7 cells. In this system, NADPH could be replaced by NADH as a cofactor for the reduction of cytochrome-c although the cytochrome-c reductase was more dependent on NADPH than NADH at a low concentration. When CYP24 expression plasmid was transfected into COS7 cells along with both pSR alpha F and pSR alpha FR, the transfected cells revealed a 3-fold higher 25-hydroxyvitamin D3-24-hydroxylase activity than COS7 cells transfected with CYP24 expression plasmid.

Animals↗
PubMed1998

Interaction of positively charged amino acid residues of recombinant, cyanobacterial ferredoxin:NADP+ reductase with ferredoxin probed by site directed mutagenesis.

The petH genes encoding ferredoxin:NADP+ reductase (FNR) from two Anabaena species (PCC 7119 and ATCC 29413) were cloned and overexpressed in E. coli. Several positively charged residues (Arg, Lys) have been implicated to be involved in ferredoxin binding and electron transfer by cross-linking, chemical modification and protection experiments, and crystallographic studies. The following substitutions were introduced by site-directed mutagenesis: R153Q, K209Q, K212Q, R214Q, K275N, K430Q and K431Q in Anabaena 29413 FNR, and R153E, K209E, K212E, R214E, K275E, R401E, K427E, and K431E in Anabaena 7119 FNR. Comparison of the diaphorase activities, the specific rates of ferredoxin dependent NADP(+)-photoreduction and cytochrome c reduction catalyzed by FNR showed that all these amino acid residues were required for efficient electron transfer between FNR and ferredoxin. Replacement of any one of these basic residues produced a much more pronounced effect on the cytochrome c reductase activity, where FNR, reduced by NADPH, acted as electron donor, than in the reduction of NADP+ by photosystem I via FNR. A mutation involving the replacement of positive charge by a neutral amide produced in all cases a smaller inhibitory effect on the activity than a charge reversal mutation. In addition, it has been found that R214 was necessary for stable integration of the non covalently bound FAD-cofactor.

Amino Acids↗
PubMed1991

Purification of ferredoxin-NADP+ reductase, flavodoxin and ferredoxin from a single batch of the cyanobacterium Anabaena PCC 7119.

Methods are described for the simultaneous isolation of ferredoxin-NADP+ reductase, ferredoxin and flavodoxin from large quantities of the cyanobacterium Anabaena PCC 7119 allowing the use of a single batch of cells. The ultraviolet-visible spectra and the extinction coefficients of ferredoxin-NADP+ reductase and ferredoxin were determined. The purification procedure also yields enriched fractions of phycobiliproteins and cytochrome c553.

Anabaena↗
PubMed1990

Arginyl groups involved in the binding of Anabaena ferredoxin--NADP+ reductase to NADP+ and to ferredoxin.

Chemical modification of ferredoxin--NADP+ reductase from the cyanobacteria Anabaena has been performed using the alpha-dicarbonyl reagent phenylglyoxal. Inactivation of both the diaphorase and cytochrome-c reductase activities, characteristic of the enzyme, indicates the involvement of one or more arginyl residues in the catalytic process of the enzyme. The determination of the rate constants for the inactivation process under different conditions, including those in which substrates, NADP+ and ferredoxin, as well as other NADP+ analogs were present, indicates the involvement of two different groups in the inactivation process, one that reacts very rapidly with the reagent (kobs = 8.3 M-1 min-1) and is responsible for the binding of NADP+, and a second less reactive group (kobs = 0.9 M-1 min-1), that is involved in the binding of ferredoxin. Radioactive labeling of the enzyme with [14C]phenylglyoxal confirms that two groups are modified while amino acid analysis of the modified protein indicates that the modified groups are arginine residues. The identification of the amino acid residues involved in binding and catalysis of the substrates of ferredoxin--NADP+ reductase will help to elucidate the mechanism of the reaction catalyzed by this important enzyme.

Arginine↗
PubMed1998

A conserved tryptophan at the ferredoxin-binding site of ferredoxin:nitrite oxidoreductase.

Treatment of spinach leaf ferredoxin-dependent nitrite reductase with N-bromosuccinimide (NBS), under conditions where slightly less than 1 mol of tryptophan is modified per mole of nitrite reductase, inhibits the catalytic activity of the enzyme by ca. 80% without any effect on substrate binding or other enzyme properties. Complex formation between nitrite reductase and ferredoxin completely protects the enzyme against this inhibition. Transient kinetic measurements show that the second-order rate constant for reduction of NBS-modified nitrite reductase by reduced ferredoxin is approximately four-fold larger than that observed for the native, unmodified enzyme. Also, reduction of NBS-modified nitrite reductase by the 5-deazariboflavin radical shows a different kinetic pattern than that observed with the native enzyme, suggesting that tryptophan modification increases access of the radical to the low-potential [4Fe-4S] cluster of the enzyme, decreases the accessibility to the siroheme group of the enzyme, or both. The tryptophan that is modified has been identified as the absolutely conserved W92. A methionine, M73, that is also modified by NBS, has been identified. The ferredoxin-binding site on spinach nitrite reductase thus appears to include W92 and perhaps M73, in addition to the previously identified R375, R556, and K436.

Amino Acid Sequence↗
PubMed1987

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation.

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with [14C]iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.

Catalysis↗
PubMed1997

The D13C variant of Bacillus schlegelii 7Fe ferredoxin is an 8Fe ferredoxin as revealed by 1H-NMR spectroscopy.

The N-terminal cluster binding motif Cys8XXXXXXXCys16....Cys49 of Bacillus schlegelii 7Fe ferredoxin, which provides the ligands to the [Fe3S4]+ cluster, was modified by the mutation Asp13 --> Cys. The mutant D13C is expressed in Escherichia coli as an 8Fe ferredoxin, with NMR properties similar to those of clostridial-type ferredoxins. The full assignment of the hyperfine shifted resonances indicates that Cys13 serves as ligand to the new fourth iron atom in the N-terminal cluster despite the atypical binding sequence CysXXXXCysXXCys....Cys. The C alpha-C beta-S-Fe dihedral angles of all cysteine ligands to the two [Fe4S4]2+ clusters of the D13C variant are similar to those observed in other 8Fe and 4Fe ferredoxins.

Aspartic Acid↗
PubMed1982

Thermoacidophilic archaebacteria contain bacterial-type ferredoxins acting as electron acceptors of 2-oxoacid:ferredoxin oxidoreductases.

Thermoplasma acidophilum and Sulfolobus acidocaldarius contain coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductases similar to those found in halophilic archaebacteria. A common feature of these enzymes is the formation of a free radical intermediate in the course of the catalytic cycle. The electron-accepting ferredoxins and a similar protein from Desulfurococcus mobilis have been purified and characterized. In contrast to the [2Fe-2S] ferredoxin of Halobacterium halobium, the ferredoxins of thermoacidophilic archaebacteria most likely contain two [4Fe-4S]2 + (2 + .1 +) clusters per molecule. Properties of these proteins are compared with respect to the evolution of archaebacteria.

Amino Acids↗
PubMed1996

Paramagnetic NMR analysis of the seven-iron ferredoxin from the hyperthermoacidophilic archaeon Desulfurolobus ambivalens reveals structural similarity to other dicluster ferredoxins.

The seven-iron ferredoxin from the hyperthermophilic archaeon Desulfurolobus ambivalens has been investigated by one-dimensional and two-dimensional 1H-NMR in its oxidized and dithionite-reduced states. All iron atoms of both the three-iron and the four-iron cluster are bound to cysteine residues whose hyperfine-shifted resonances were characterized. The pattern of these resonances is similar to those from three-iron, four-iron and eight-iron ferredoxins previously described in the literature, but the four-iron cluster has a shift pattern different from that in other seven-iron proteins. A second set of hyperfine-shifted resonances clearly indicates sample heterogeneity, which possibly involves the four-iron cluster. The observation of interresidue NOEs between two different cysteine residues proves the existence of close spatial proximity of the two clusters in D. ambivalens ferredoxin and therefore indicates structural homology to other dicluster ferredoxins. Moreover, this feature is crucial for the sequence-specific assignment of the hyperfine-shifted resonances. The C alpha-C beta-S-Fe dihedral angles of the cysteine residues coordinating the four-iron cluster could be estimated, and the electronic structure of the three-iron cluster is discussed.

Ferredoxins↗
PubMed1994

Characterization of the genome region encoding an fdxH-type ferredoxin and a new 2[4Fe-4S] ferredoxin from the nonheterocystous, nitrogen-fixing cyanobacterium Plectonema boryanum PCC 73110.

A genomic DNA region with four consecutive open reading frames, including an fdxH-type gene, has been sequenced and initially characterized for the nonheterocystous nitrogen-fixing cyanobacterium Plectonema boryanum PCC 73110. The fdxH gene encodes a [2Fe-2S]-type ferredoxin, 98 amino acids in length, with a deduced molecular mass of 10.9 kDa. Conserved residues include two characteristic lysines at positions 10 and 11, shown recently to be important for interaction with nitrogenase reductase (S. Schmitz, B. Schrautermeier, and H. Böhme, Mol. Gen. Genet. 240:455-460, 1993). The gene is transcribed only under anaerobic nitrogenase-inducing conditions, whereas the Plectonema petF gene, encoding a different (type 1) [2Fe-2S] ferredoxin, is only transcribed in cultures growing with combined nitrogen. The fdxH gene was expressed in Escherichia coli as a holoprotein. The purified protein was able to effectively donate electrons to cyanobacterial nitrogenase, whereas PetF from the same organism was not. The occurrence of FdxH in the nonheterocystous genus Plectonema demonstrates for the first time that FdxH-type ferredoxins are not exclusively expressed within heterocysts, as is true for cyanobacteria differentiating these cells for nitrogen fixation under aerobic growth conditions. Two open reading frames that precede fdxH have high similarity to those found at a corresponding location in Anabaena sp. strain PCC 7120. In the latter organism, they are transcribed only under nitrogen-fixing conditions, but the functions of their gene products remain unclear (D. Borthakur, M. Basche, W. J. Buikema, P. B. Borthakur, and R. Haselkorn, Mol. Gen. Genet. 221:227-234, 1990). An fdxB-type gene encoding a 2[4Fe-4S] ferredoxin not previously identified in cyanobacteria is located immediately downstream of fdxH in P. boryanum.

Amino Acid Sequence↗
PubMed1991

A new [2Fe-2S] ferredoxin from Rhodobacter capsulatus. Coexpression with a 2[4Fe-4S] ferredoxin in Escherichia coli.

A 285-base pair open reading frame was found immediately upstream of the fdxN gene (encoding ferredoxin I) of Rhodobacter capsulatus and coded for a 95-amino acid protein with a predicted molecular weight of 10,156. The deduced amino acid sequence contained 5 cysteines, 4 of which exhibited spacing characteristic of [2Fe-2S] plant and cyanobacterial ferredoxins. The amino acid sequence was found to share approximately 25% amino acid similarity with plant-type ferredoxins. The gene was named fdxC. Expression of the fdxC and fdxN genes together in Escherichia coli was accomplished by subcloning the genes in the vector pUC18 downstream of the lac promoter. Cells containing this plasmid produced a red and a brown protein corresponding to the fdxC and fdxN gene products, respectively. EPR and UV-visible absorption spectroscopy confirmed that the FdxC protein contained a [2Fe-2S] cluster and the FdxN protein contained two [4Fe-4S] clusters and that the centers were correctly assembled and inserted in the ferredoxins expressed in E. coli. Transcription (Northern blot) analysis showed that the genes were transcribed only under nitrogen-limiting (nif-derepressing) growth conditions.

Amino Acid Sequence↗
PubMed1996

Molecular and phylogenetic characterization of pyruvate and 2-ketoisovalerate ferredoxin oxidoreductases from Pyrococcus furiosus and pyruvate ferredoxin oxidoreductase from Thermotoga maritima.

Previous studies have shown that the hyperthermophilic archaeon Pyrococcus furiosus contains four distinct cytoplasmic 2-ketoacid oxidoreductases (ORs) which differ in their substrate specificities, while the hyperthermophilic bacterium Thermotoga maritima contains only one, pyruvate ferredoxin oxidoreductase (POR). These enzymes catalyze the synthesis of the acyl (or aryl) coenzyme A derivative in a thiamine PPi-dependent oxidative decarboxylation reaction with reduction of ferredoxin. We report here on the molecular analysis of the POR (por) and 2-ketoisovalerate ferredoxin oxidoreductase (vor) genes from P. furiosus and of the POR gene from T. maritima, all of which comprise four different subunits. The operon organization for P. furiosus POR and VOR was porG-vorDAB-porDAB, wherein the gamma subunit is shared by the two enzymes. The operon organization for T. maritima POR was porGDAB. The three enzymes were 46 to 53% identical at the amino acid level. Their delta subunits each contained two ferredoxin-type [4Fe-4S] cluster binding motifs (CXXCXXCXXXCP), while their beta subunits each contained four conserved cysteines in addition to a thiamine PPi-binding domain. Amino-terminal sequence comparisons show that POR, VOR, indolepyruvate OR, and 2-ketoglutarate OR of P. furiosus all belong to a phylogenetically homologous OR family. Moreover, the single-subunit pyruvate ORs from mesophilic and moderately thermophilic bacteria and from an amitochondriate eucaryote each contain four domains which are phylogenetically homologous to the four subunits of the hyperthermophilic ORs (27% sequence identity). Three of these subunits are also homologous to the dimeric POR from a mesophilic archaeon, Halobacterium halobium (21% identity). A model is proposed to account for the observed phenotypes based on genomic rearrangements of four ancestral OR subunits.

Amino Acid Sequence↗
PubMed1993

Binding of ferredoxin to ferredoxin:NADP+ oxidoreductase: the role of carboxyl groups, electrostatic surface potential, and molecular dipole moment.

The small, soluble, (2Fe-2S)-containing protein ferredoxin (Fd) mediates electron transfer from the chloroplast photosystem I to ferredoxin: NADP+ oxidoreductase (FNR), a flavoenzyme located on the stromal side of the thylakoid membrane. Ferredoxin and FNR form a 1:1 complex, which is stabilized by electrostatic interactions between acidic residues of Fd and basic residues of FNR. We have used differential chemical modification of Fd to locate aspartic and glutamic acid residues at the intermolecular interface of the Fd:FNR complex (both proteins from spinach). Carboxyl groups of free and FNR-bound Fd were amidated with carbodiimide/2-aminoethane sulfonic acid (taurine). The differential reactivity of carboxyl groups was assessed by double isotope labeling. Residues protected in the Fd:FNR complex were D-26, E-29, E-30, D-34, D-65, and D-66. The protected residues belong to two domains of negative electrostatic surface potential on either side of the iron-sulfur cluster. The negative end of the molecular dipole moment vector of Fd (377 Debye) is close to the iron-sulfur cluster, in the center of the area demarcated by the protected carboxyl groups. The molecular dipole moment and the asymmetric surface potential may help to orient Fd in the reaction with FNR. In support, we find complementary domains of positive electrostatic potential on either side of the FAD redox center of FNR. The results allow a binding model for the Fd:FNR complex to be constructed.

Amino Acid Sequence↗