Diversification of Ferredoxins across Living Organisms.

Ferredoxins, iron-sulfur (Fe-S) cluster proteins, play a key role in oxidoreduction reactions. To date, evolutionary analysis of these proteins across the domains of life have been confined to observing the abundance of Fe-S cluster types (2Fe-2S, 3Fe-4S, 4Fe-4S, 7Fe-8S (3Fe-4s and 4Fe-4S) and 2[4Fe-4S]) and the diversity of ferredoxins within these cluster types was not studied. To address this research gap, here we propose a subtype classification and nomenclature for ferredoxins based on the characteristic spacing between the cysteine amino acids of the Fe-S binding motif as a subtype signature to assess the diversity of ferredoxins across the living organisms. To test this hypothesis, comparative analysis of ferredoxins between bacterial groups, Alphaproteobacteria and Firmicutes and ferredoxins collected from species of different domains of life that are reported in the literature has been carried out. Ferredoxins were found to be highly diverse within their types. Large numbers of alphaproteobacterial species ferredoxin subtypes were found in Firmicutes species and the same ferredoxin subtypes across the species of Bacteria, Archaea, and Eukarya, suggesting shared common ancestral origin of ferredoxins between Archaea and Bacteria and lateral gene transfer of ferredoxins from prokaryotes (Archaea/Bacteria) to eukaryotes. This study opened new vistas for further analysis of diversity of ferredoxins in living organisms.

Expression of the minor isoform pea ferredoxin in tobacco alters photosynthetic electron partitioning and enhances cyclic electron flow.

Ferredoxins (Fds) are ferrosulfoproteins that function as low-potential electron carriers in plants. The Fd family is composed of several isoforms that share high sequence homology but differ in functional characteristics. In leaves, at least two isoforms conduct linear and cyclic photosynthetic electron transport around photosystem I, and mounting evidence suggests the existence of at least partial division of duties between these isoforms. To evaluate the contribution of different kinds of Fds to the control of electron fluxes along the photosynthetic electron transport chain, we overexpressed a minor pea (Pisum sativum) Fd isoform (PsFd1) in tobacco (Nicotiana tabacum) plants. The transplastomic OeFd1 plants exhibited variegated leaves and retarded growth and developmental rates. Photosynthetic studies of these plants indicated a reduction in carbon dioxide assimilation rates, photosystem II photochemistry, and linear electron flow. However, the plants showed an increase in nonphotochemical quenching, better control of excitation pressure at photosystem II, and no evidence of photoinhibition, implying a better dynamic regulation to remove excess energy from the photosynthetic electron transport chain. Finally, analysis of P700 redox status during illumination confirmed that the minor pea Fd isoform promotes enhanced cyclic flow around photosystem I. The two novel features of this work are: (1) that Fd levels achieved in transplastomic plants promote an alternative electron partitioning even under greenhouse light growth conditions, a situation that is exacerbated at higher light intensity measurements; and (2) that an alternative, minor Fd isoform has been overexpressed in plants, giving new evidence of labor division among Fd isoforms.

Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea.

BACKGROUND: In 2004, we discovered an atypical protein in metagenomic data from marine thaumarchaeotal species. This protein, referred as DnaJ-Fer, is composed of a J domain fused to a Ferredoxin (Fer) domain. Surprisingly, the same protein was also found in Viridiplantae (green algae and land plants). Because J domain-containing proteins are known to interact with the major chaperone DnaK/Hsp70, this suggested that a DnaK protein was present in Thaumarchaeota. DnaK/Hsp70, its co-chaperone DnaJ and the nucleotide exchange factor GrpE are involved, among others, in heat shocks and heavy metal cellular stress responses. RESULTS: Using phylogenomic approaches we have investigated the evolutionary history of the DnaJ-Fer protein and of interacting proteins DnaK, DnaJ and GrpE in Thaumarchaeota. These proteins have very complex histories, involving several inter-domain horizontal gene transfers (HGTs) to explain the contemporary distribution of these proteins in archaea. These transfers include one from Cyanobacteria to Viridiplantae and one from Viridiplantae to Thaumarchaeota for the DnaJ-Fer protein, as well as independent HGTs from Bacteria to mesophilic archaea for the DnaK/DnaJ/GrpE system, followed by HGTs among mesophilic and thermophilic archaea. CONCLUSIONS: We highlight the chimerical origin of the set of proteins DnaK, DnaJ, GrpE and DnaJ-Fer in Thaumarchaeota and suggest that the HGT of these proteins has played an important role in the adaptation of several archaeal groups to mesophilic and thermophilic environments from hyperthermophilic ancestors. Finally, the evolutionary history of DnaJ-Fer provides information useful for the relative dating of the diversification of Archaeplastida and Thaumarchaeota.

cDNA cloning, expression levels and gene mapping of photosynthetic and non-photosynthetic ferredoxin genes in sunflower (Helianthus annuus L.).

Fatty acid desaturation in plastids and chloroplasts depends on the electron-donor activity of ferredoxins. Using degenerate oligonucleotides designed from known photosynthetic and heterotrophic plant ferredoxin sequences, two full-length ferredoxin cDNAs were cloned from sunflower (Helianthus annuus L.) leaves and developing seeds, HaFd1 and HaFd2, homologous to photosynthetic and non-photosynthetic ferredoxins, respectively. Based on these cDNAs, the respective genomic sequences were obtained and the presence of DNA polymorphisms was investigated. Complete sequencing of the HaFd1 and HaFd2 genes in different lines indicated the presence of two haplotypes for HaFd2 and their alignment showed that sequence polymorphisms are restricted to the 5'-NTR intron. In addition, specific DNA markers for the HaFd1 and HaFd2 genes were developed that enabled the genes to be mapped. Accordingly, the HaFd1 locus maps to linkage group 10 of the public sunflower map, while the HaFd2 locus maps to linkage group 11. Both ferredoxins display different spatial-temporal patterns of expression. While HaFd2 is expressed at similar levels in all tissues tested (leaves, stem, roots, cotyledons and developing seeds), HaFd1 is more strongly expressed in green tissues than in all the other tissues tested. Both photosynthetic- and heterotrophic-ferredoxins are present in sunflower seeds and may contribute to fatty acid desaturation during oil accumulation. Nevertheless, the levels of HaFd2 expression during seed formation are distinct in lines that only varied in the HaFd2 haplotypes they expressed.

Characterization of the recombinant Rieske [2Fe-2S] proteins HcaC and YeaW from E. coli.

Three genes within the genome of E. coli K12 are predicted to encode proteins containing the typical Rieske iron-sulfur cluster-binding motifs. Two of these, hcaC and yeaW, were overexpressed in E. coli BL21 and Tuner (DE3) pLacI. The recombinant proteins were purified and analyzed by UV/Vis- and EPR-spectroscopy. HcaC and YeaW display the typical redox-dependent UV/Vis-spectra of iron-sulfur proteins. The EPR spectrum of reduced HcaC shows characteristic g-values of a Rieske cluster whereas the g-values for YeaW are close to the upper limit for this type of iron-sulfur cluster. Both iron-sulfur clusters could be reduced by dithionite, but not by ascorbate, confirming their classification as low-potential Rieske proteins as derived from the amino acid sequences. A phylogenetic analysis of the two proteins reveals that HcaC clearly segregates with the Rieske ferredoxins of class IIB oxygenases whereas the classification of YeaW remains doubtful.

Molecular detection and diversity of novel diterpenoid dioxygenase DitA1 genes from proteobacterial strains and soil samples.

Resin acids are tricyclic diterpenoids naturally synthesized by trees that are released from wood during pulping processes. Using a newly designed primer set, genes similar to that encoding the DitA1 catalytic alpha-subunit of the diterpenoid dioxygenase, a key enzyme in abietane resin acid degradation by Pseudomonas abietaniphila BKME-9, could be amplified from different Pseudomonas strains, whereas ditA1 gene sequence types representing distinct branches in the evolutionary tree were amplified from Burkholderia and Cupriavidus isolates. All isolates harbouring a ditA1-homologue were capable of growth on dehydroabietic acid as the sole source of carbon and energy and reverse transcription polymerase chain reaction analysis in three strains confirmed that ditA1 was expressed constitutively or in response to DhA, demonstrating its involvement in DhA-degradation. Evolutionary analyses indicate that gyrB (as a phylogenetic marker) and ditA1 genes have coevolved under purifying selection from their ancestral variants present in the most recent common ancestor of the genera Pseudomonas, Cupriavidus and Burkholderia. A polymerase chain reaction-single-strand conformation poylmorphism fingerprinting method was established to monitor the diversity of ditA1 genes in environmental samples. The molecular fingerprints indicated the presence ofa broad, previously unrecognized diversity of diterpenoid dioxygenase genes in soils, and suggest that other bacterial phyla may also harbour the genetic potential for DhA-degradation.

Structure of T4moC, the Rieske-type ferredoxin component of toluene 4-monooxygenase.

The structure of the Rieske-type ferredoxin (T4moC) from toluene 4-monooxygenase was determined by X-ray crystallography in the [2Fe-2S](2+) state at a resolution of 1.48 A using single-wavelength anomalous dispersion phasing with the [2Fe-2S] center. The structure consists of ten beta-strands arranged into the three antiparallel beta-sheet topology observed in all Rieske proteins. Trp69 of T4moC is adjacent to the [2Fe-2S] centre, which displaces a loop containing the conserved Pro81 by approximately 8 A away from the [2Fe-2S] cluster compared with the Pro loop in the closest structural and functional homolog, the Rieske-type ferredoxin BphF from biphenyl dioxygenase. In addition, T4moC contains five hydrogen bonds to the [2Fe-2S] cluster compared with three hydrogen bonds in BphF. Moreover, the electrostatic surface of T4moC is distinct from that of BphF. These structural differences are identified as possible contributors to the evolutionary specialization of soluble Rieske-type ferredoxins between the diiron monooxygenases and cis-dihydrodiol-forming dioxygenases.

Amino acid sequences of ferredoxins from Atropa belladonna and Hyoscyamus niger: their similarities to those in other tropane-alkaloid-containing plants.

The complete amino acid sequences of [2Fe-2S] ferredoxin from Atropa belladonna and Hyoscyamus niger have been determined by automated Edman degradation of the entire S-carboxymethylcysteinyl proteins and of the peptides obtained by enzymatic digestion. These two ferredoxins exhibited 1-8 differences in their amino acid sequences compared to those of other tropane-alkaloid-containing plants (Scopolia japonica, Datura stramonium, D. metel, and D. arborea), and only 1 or 4 differences compared to S. japonica and D. arborea. In contrast, 9-23 differences were observed among the other solanaceous ferredoxins. This suggests that tropane-alkaloid-containing plants are closely related taxonomically.

A Rieske ferredoxin typifying a subtype within Rieske proteins: spectroscopic, biochemical and stability studies.

A new subtype of archaeal Rieske ferredoxin (RFd) has been identified in the genome of the thermoacidophilic archaeon Acidianus ambivalens. The gene is inserted in an atypical genomic context in a gene cluster encoding a NiFe hydrogenase. Sequence and phyletic analysis showed that the protein is related to bacterial RFd but not to any of the known archaeal Rieske proteins. The recombinant 14 kDa protein isolated from Escherichia coli behaved as a dimer in solution. It contained approximately 2 Fe/mol and all visible and EPR spectroscopic features typical of Rieske centre-containing proteins. However, its redox potential (+170 mV) was significantly higher than those of canonical RFd. This difference is rationalized in terms of the protein structure environment, as discrete amino acid substitutions in key positions around the metal centre account for the higher potential.

g-Strain, ENDOR, and structure of active centers of two-iron ferredoxins.

Collaborative chemical and spectroscopic work from several laboratories resulted in a qualitative structure for the active center in the two-iron ferredoxins, with each iron being in a distorted tetrahedron of sulfur atoms (two acid-labile sulfurs bridging the two iron atoms and the other two from cysteine sulfurs). Subsequent X-ray data from other laboratories confirmed this structure. Detailed EPR spectral syntheses showed that there is a distribution of structures in any given protein (even in a single crystal) resulting in a distribution of the principal values of the g tensor, which may be described by a statistical distribution of still another tensor whose principal axes are, in general, not coincident with the principal-axis frame of the g tensor.

NMR studies of a ferredoxin from Haloferax mediterranei and its physiological role in nitrate assimilatory pathway.

Haloferax mediterranei is a halophilic archaeon that can grow in aerobic conditions with nitrate as sole nitrogen source. The electron donor in the aerobic nitrate reduction to ammonium was a ferredoxin. This ferredoxin has been purified and characterised. Air-oxidized H. mediterranei ferredoxin has a UV-visible absorption spectra typical of 2Fe-type ferredoxins with an A420/A280 of 0.21. The nuclear magnetic resonance (NMR) spectra of the ferredoxin showed similarity to those of ferredoxins from plant and bacteria, containing a [2Fe-2S] cluster. The physiological function of ferredoxin might be to serve as an electron donor for nitrate reduction to ammonium by assimilatory nitrate (EC 1.6.6.2) and nitrite reductases (EC 1.7.7.1). The apparent molecular weight (Mr) of the ferredoxin was estimated to be 21 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Evidence for lateral transfer of genes encoding ferredoxins, nitroreductases, NADH oxidase, and alcohol dehydrogenase 3 from anaerobic prokaryotes to Giardia lamblia and Entamoeba histolytica.

Giardia lamblia and Entamoeba histolytica are amitochondriate, microaerophilic protists which use fermentation enzymes like those of bacteria to survive anaerobic conditions within the intestinal lumen. Genes encoding fermentation enzymes and related electron transport peptides (e.g., ferredoxins) in giardia organisms and amebae are hypothesized to be derived from either an ancient anaerobic eukaryote (amitochondriate fossil hypothesis), a mitochondrial endosymbiont (hydrogen hypothesis), or anaerobic bacteria (lateral transfer hypothesis). The goals here were to complete the molecular characterization of giardial and amebic fermentation enzymes and to determine the origins of the genes encoding them, when possible. A putative giardia [2Fe-2S]ferredoxin which had a hypothetical organelle-targeting sequence at its N terminus showed similarity to mitochondrial ferredoxins and the hydrogenosomal ferredoxin of Trichomonas vaginalis (another luminal protist). However, phylogenetic trees were star shaped, with weak bootstrap support, so we were unable to confirm or rule out the endosymbiotic origin of the giardia [2Fe-2S]ferredoxin gene. Putative giardial and amebic 6-kDa ferredoxins, ferredoxin-nitroreductase fusion proteins, and oxygen-insensitive nitroreductases each tentatively supported the lateral transfer hypothesis. Although there were not enough sequences to perform meaningful phylogenetic analyses, the unique common occurrence of these peptides and enzymes in giardia organisms, amebae, and the few anaerobic prokaryotes suggests the possibility of lateral transfer. In contrast, there was more robust phylogenetic evidence for the lateral transfer of G. lamblia genes encoding an NADH oxidase from a gram-positive coccus and a microbial group 3 alcohol dehydrogenase from thermoanaerobic prokaryotes. In further support of lateral transfer, the G. lamblia NADH oxidase and adh3 genes appeared to have an evolutionary history distinct from those of E. histolytica.

Ferredoxins of the third kind.

Most low-potential ferredoxins (Fds) are of the well-known [2Fe-2S] plant or [4Fe-4S] bacterial type. Yet, an additional class of [2Fe-2S] Fds has been recognized on the basis of sequence and spectroscopic idiosyncrasies. A recent crystal structure has confirmed the uniqueness of this third kind of Fd, and shown that these proteins display an unexpected structural similarity to thioredoxin. The properties of these thioredoxin-like [2Fe-2S] Fds are summarized, and hypotheses concerning their function are discussed.

A comprehensive phylogenetic analysis of Rieske and Rieske-type iron-sulfur proteins.

The Rieske iron-sulfur center consists of a [2Fe-2S] cluster liganded to a protein via two histidine and two cysteine residues present in conserved sequences called Rieske motifs. Two protein families possessing Rieske centers have been defined. The Rieske proteins occur as subunits in the cytochrome bc1 and cytochrome b6f complexes of prokaryotes and eukaryotes or form components of archaeal electron transport systems. The Rieske-type proteins encompass a group of bacterial oxygenases and ferredoxins. Recent studies have uncovered several new proteins containing Rieske centers, including archaeal Rieske proteins, bacterial oxygenases, bacterial ferredoxins, and, intriguingly, eukaryotic Rieske oxygenases. Since all these proteins contain a Rieske motif, they probably form a superfamily with one common ancestor. Phylogenetic analyses have, however, been generally limited to similar sequences, providing little information about relationships within the whole group of these proteins. The aim of this work is, therefore, to construct a dendrogram including representatives from all Rieske and Rieske-type protein classes in order to gain insight into their evolutionary relationships and to further define the phylogenetic niches occupied by the recently discovered proteins mentioned above.

Identification of a gene for a rubrerythrin/nigerythrin-like protein in Spirillum volutans by using amino acid sequence data from mass spectrometry and NH2-terminal sequencing.

A hydrogen peroxide-resistant mutant of the catalase-negative microaerophile, Spirillum volutans, constitutively expresses a 21.5 kDa protein that is undetectable and non-inducible in the wild-type cells. Part of the gene that encodes the protein was cloned using amino acid sequence data obtained by both mass spectrometry and NH2-terminal sequencing. The deduced 158 amino acid polypeptide shows high relatedness to rubrerythrin and nigerythrin previously described in the anaerobes Clostridium perfringens and Desulfovibrio vulgaris. The protein also shows high similarity to putative rubrerythrin proteins found in the anaerobic archeons Archaeoglobus fulgidus, Methanococcus jannaschii and Methanobacterium thermoautotrophicum. This is the first report of this type of protein in an organism that must respire with oxygen. It seems likely that the novel combination of methodologies used in this study could be applied to the rapid cloning of other genes in bacteria for which no genomic library yet exists.

Structural features of the metal binding site and dynamics of gallium putidaredoxin, a diamagnetic derivative of a Cys4Fe2S2 ferredoxin.

The first reconstitution of an Fe2S2 ferredoxin with a diamagnetic prosthetic group was recently described [Kazanis et al. (1995) J. Am. Chem. Soc., 117, 6625-6626]. The replacement of the iron-sulfur cluster of the bacterial ferredoxin putidaredoxin (Pdx) by gallium (Ga3+) renders the protein diamagnetic and permits the use of high-resolution NMR methods to identify resonances near the metal binding site. We now describe structural features of the metal binding site that are not observable by standard NMR methods in native Pdx due to paramagnetic line broadening. These results provide the first example of high-resolution NMR-derived structural data concerning the metal binding domain of an Fe2S2 ferredoxin, and the first structural information of any sort for the metal binding site of a ferredoxin from this class, which includes adrenodoxin, placental ferredoxin and terpredoxin. Assignments were obtained by applying multidimensional NMR methods to a series of selectively and nonselectively 15N- and 13C/15N-labeled GaPdx samples. For most experiments, a mutant of Pdx was used in which a nonligating Cys85 is replaced by serine. All of the major structural features that were identified in native Pdx are conserved in GaPdx. The overall protein dynamics is considerably faster in GaPdx than in the native protein, as reflected by amide proton exchange rates. The C-terminal residue, Trp106, also exhibits considerable mobility, as indicated by 15N[1H] NOE and 15N T1 values of the C-terminal residue of the protein.

Small structural changes account for the high thermostability of 1[4Fe-4S] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima.

BACKGROUND: The characterization of the structural features that account for the high thermostability of some proteins is of great scientific and biotechnological interest. Proteins from hyperthermophilic organisms with optimum growth temperatures of 80 degrees C and higher generally show high intrinsic stabilities. The comparison of high resolution X-ray structures of these proteins with their counterparts from mesophilic organisms has therefore helped to identify potentially stabilizing forces in a number of cases. Small monomeric proteins which comprise only a single domain, such as ferredoxins, are especially suitable for such comparisons since the search for determinants of protein stability is considerably simplified. RESULTS: The 1.75 A crystal structure of the extremely thermostable 1[4Fe-4S] ferredoxin from Thermotoga maritima (FdTm) was determined and compared with other monocluster-containing ferredoxins with different degrees of thermostability. CONCLUSIONS: A comparison of the three-dimensional structure of FdTm with that of ferredoxins from mesophilic organisms suggests that the very high thermostability of FdTm is unexpectedly achieved without large changes of the overall protein structure. Instead, an increased number of potentially stabilizing features is observed in FdTm, compared with mesophilic ferredoxins. These include stabilization of alpha helices, replacement of residues in strained conformation by glycines, strong docking of the N-terminal methionine and an overall increase in the number of hydrogen bonds. Most of these features stabilize several secondary structure elements and improve the overall rigidity of the polypeptide backbone. The decreased flexibility will certainly play a relevant role in shielding the iron-sulfur cluster against physiologically high temperatures and further improve the functional integrity of FdTm.

Insights into protein adaptation to a saturated salt environment from the crystal structure of a halophilic 2Fe-2S ferredoxin.

Haloarcula marismortui is an archaebacterium that flourishes in the world's saltiest body of water, the Dead Sea. The cytosol of this organism is a supersaturated salt solution in which proteins are soluble and active. The crystal structure of a 2Fe-2S ferredoxin from H. marismortui determined at 1.9 A is similar to those of plant-type 2Fe-2S ferredoxins of known structure, with two important distinctions. The entire surface of the protein is coated with acidic residues except for the vicinity of the iron-sulphur cluster, and there is an insertion of two amphipathic helices near the N-terminus. These form a separate hyperacidic domain whose postulated function to provide extra surface carboxylates for solvation. These data and the fact that bound surface water molecules have on the average 40% more hydrogen bonds than in a typical non-halophilic protein crystal structure support the notion that haloadaptation involves better water binding capacity.

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.

A ferredoxin, designated FdxP, stimulates p-hydroxybenzoate hydroxylase activity in Caulobacter crescentus.

A gene, fdxP, was identified upstream of the rrnA gene in Caulobacter crescentus and shown to encode ferredoxin II (FdII) by insertional inactivation. FdII is homologous to a class of [2Fe-2S] ferredoxins typified by putidaredoxin. Furthermore, reconstitution assays demonstrated that FdII was able to promote p-hydroxybenzoate hydroxylase activity in ferredoxin-depleted extracts. Thus, biodegradation of p-hydroxybenzoate may be ferredoxin dependent in C. crescentus.