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).     

Amino acid sequence of a four-iron-four-sulphur ferredoxin isolated from Bacillus stearothermophilus.

1. The primary structure of a 4Fe-4S ferredoxin from Bacillus stearothermophilus was determined and shown to consist of a single polypeptide chain of 81 amino acid residues. The molecular weight of the holoprotein is about 9120. 2. There are only four cysteine residues in the molecule; three of these are located near the N-terminus as a Cys-X-X-Cys-X-X-Cys segment, and the fourth cysteine residue is followed by a proline and located in the C-terminal half. 3. The Fe-S chromophore in B. stearothermophilus ferredoxin was previously well characterized and was shown to consist of a single 4Fe-4S cluster. This ferredoxin sequence establishes for the first time the relative location of the four cysteine residues necessary to bind the 4Fe-4S cluster of a 4Fe ferredoxin, and is in agreement with the criteria for the relative positions of the cysteines proposed from X-ray-crystallographic studies on an 8Fe (two 4Fe-4S clusters) ferredoxin. 4. The sequence of B. stearothermophilus ferredoxin is homologous in many segments to that of other bacterial ferredoxins, the degree of homology being greater towards ferredoxins from Desulfovibrio gigas and photosynthetic bacteria than to Clostridial ferredoxins. 5. The presence of a relatively higher number of glutamic acid and lower number of cysteine residues in the molecule may explain the greater thermal stability and oxygen-insenstivity of this ferredoxin.     

Two distinct ferredoxins from Rhodobacter capsulatus: complete amino acid sequences and molecular evolution

Two distinct ferredoxins were purified from Rhodobacter capsulatus SB1003. Their complete amino acid sequences were determined by a combination of protease digestion, BrCN cleavage and Edman degradation. Ferredoxins I and II were composed of 64 and 111 amino acids, respectively, with molecular weights of 6,728 and 12,549 excluding iron and sulfur atoms. Both contained two Cys clusters in their amino acid sequences. The first cluster of ferredoxin I and the second cluster of ferredoxin II had a sequence, CxxCxxCxxxCP, in common with the ferredoxins found in Clostridia. The second cluster of ferredoxin I had a sequence, CxxCxxxxxxxxCxxxCM, with extra amino acids between the second and third Cys, which has been reported for other photosynthetic bacterial ferredoxins and putative ferredoxins (nif-gene products) from nitrogen-fixing bacteria, and with a unique occurrence of Met. The first cluster of ferredoxin II had a CxxCxxxxCxxxCP sequence, with two additional amino acids between the second and third Cys, a characteristics feature of Azotobacter-[3Fe-4S] [4Fe-4S]-ferredoxin. Ferredoxin II was also similar to Azotobacter-type ferredoxins with an extended carboxyl (C-) terminal sequence compared to the common Clostridium-type. The evolutionary relationship of the two together with a putative one recently found to be encoded in nifENXQ region in this bacterium [Moreno-Vivian et al. (1989) J. Bacteriol. 171, 2591-2598] is discussed.     

Purification and characterization of a 7Fe ferredoxin from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii.

A ferredoxin (Fd) was purified from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii. This ferredoxin was a monomer with apparent molecular weight of 13,000 and contained 7 mol Fe/mol ferredoxin. The oxidized ferredoxin showed the characteristic EPR spectrum for [3Fe-4S]1+ (1.2 spin/mol Fd). This signal disappeared upon reduction with dithionite and new signals due to [3Fe-4S]0 and [4Fe-4S]1+ (0.7 spin/mol Fd) appeared. The quantitation of EPR signals and the iron content reveal that B. schlegelii ferredoxin contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster. The ferredoxin has the characteristic distribution of cysteines (-Cys8-X7-Cys16-X3-Cys20-Pro-) for 7Fe ferredoxins in the N-terminus.     

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.     

Sequence, assembly and evolution of a primordial ferredoxin from Thermotoga maritima.

A gene coding for the ferredoxin of the primordial, strictly anaerobic and hyperthermophilic bacterium Thermotoga maritima was cloned, sequenced and expressed in Escherichia coli. The ferredoxin gene encodes a polypeptide of 60 amino acids that incorporates a single 4Fe-4S cluster. T. maritima ferredoxin expressed in E. coli is a heat-stable, monomeric protein, the spectroscopic properties of which show that its 4Fe-4S cluster is correctly assembled within the mesophilic host, and that it remains stable during purification under aerobic conditions. Removal of the iron-sulfur cluster results in an apo-ferredoxin that has no detectable secondary structure. This observation indicates that in vivo formation of the ferredoxin structure is coupled to the insertion of the iron-sulfur cluster into the polypeptide chain. Sequence comparison of T. maritima ferredoxin with other 4Fe-4S ferredoxins revealed high sequence identities (75% and 50% respectively) to the ferredoxins from the hyperthermophilic members of the Archaea, Thermococcus litoralis and Pyrococcus furiosus. The high sequence similarity supports a close relationship between these extreme thermophilic organisms from different phylogenetic domains and suggests that ferredoxins with a single 4Fe-4S cluster are the primordial representatives of the whole protein family. This observation suggests a new model for the evolution of ferredoxins.     

Ferredoxins from the archaeon Acidianus ambivalens: overexpression and characterization of the non-zinc-containing ferredoxin FdB.

Two ferredoxin genes, fdA and fdB, from the extremely thermoacidophilic crenarchaeon Acidianus ambivalens have been sequenced; the sequences share 86% similarity. Whereas the deduced protein sequence of the ferredoxin FdA clearly contains a zinc-binding motif, the corresponding sequence of the FdB is devoid of this motif. Thus far, only the zinc-containing ferredoxin, FdA, from A. ambivalens has been chemically and functionally characterized from its native source. Using RT-PCR and Northern blot analysis, we show that both ferredoxins are expressed by A. ambivalens under either anaerobic or aerobic growth conditions. The zinc-free ferredoxin, FdB, was overexpressed in E. coli and purified to homogeneity. Using EPR spectroscopy, we could demonstrate that FdB contains one [3Fe-4S](1+/0) and one [4Fe-4S](2+/1+) cluster. The reduction potential of the [3Fe-4S](1+/0) cluster was determined as -235+/-10 mV, at pH 6.5, by EPR-monitored redox titration. The high melting temperature of 108+/-2 degrees C of FdB determined by CD spectroscopy reveals that it is not the binding of the Zn2+ that induces the extreme thermostability of these ferredoxins.     

Synthesis of the Caulobacter ferredoxin protein, FdxA, is cell cycle controlled.

The fdxA gene was identified upstream of and in the opposite direction from the Caulobacter crescentus cysC gene. Analyses of the nucleotide sequence and the deduced amino acid sequence of the fdxA gene demonstrated that it encodes a ferredoxin with a molecular mass of 12,080 Da. This ferredoxin has common structural features with ferredoxins that contain a [3Fe-4S] and a [4Fe-4S] cluster, including seven conserved cysteines responsible for the binding of the two clusters. A mutation in the fdxA gene was obtained, and the resulting strain did not produce one of the two ferredoxins (FdI) found in C. crescentus. Further experiments demonstrated that the fdxA gene is temporally expressed in C. crescentus and that FdI is required for completion of the cell cycle at 37 degrees C.     

Refinement of the 7 Fe ferredoxin from Azotobacter vinelandii at 1.9 A resolution

The recently redetermined structure of the 7 Fe ferredoxin from Azotobacter vinelandii has been refined against a new 1.9 A data set. The crystallographic R-factor is 0.215 for all 9586 observed reflections 8.0 to 1.9 A. The model contains 106 amino acid residues, two Fe-S clusters and 21 water molecules. The root-mean-square deviations from ideality of bonds and angles are 0.014 A and 3.3 degrees, respectively. The refinement confirms the presence of two free cysteines: the thiol of C11 is in association with the side-chain of K100; the thiol of C24 is 3.35 A from inorganic sulfur of the [4 Fe-4 S] cluster. The refinement confirms a [3 Fe-4 S] model for the 3 Fe cluster. The two Fe-S clusters have similar bond distances and angles. The structure of the protein for residues 1 to 57 superposes within 0.85 A on residues 1 to 53 of the 8 Fe ferredoxin structure for main-chain N, CA and C atoms, if residues 9, 10, 29 and 30 of 7 Fe ferredoxin are omitted. These residues are part of two loops in contact with residues of the extended C-terminal chain of 7 Fe ferredoxin.     

Di-cluster, seven-iron ferredoxins from hyperthermophilic Sulfolobales

 Seven-iron ferredoxins from the thermoacidophilic archaea Acidianus ambivalens, A. infernus, Metalosphaera prunae and Sulfolobus metallicus were extensively characterised, allowing study of their expression under aerobic and anaerobic growth conditions as well as the putative role in thermal stability of a recently described zinc centre. The archaeon S. metallicus was found to express, under the same growth conditions, two ferredoxins in almost identical amounts, a novelty among Archaea. Most interestingly, these two ferredoxins differ at the N-terminal amino acid sequence in that one has a zinc binding motif (FdA) and the other does not (FdB); in agreement with these findings, FdA contains a zinc ion and FdB does not. These two ferredoxins have identical thermal stabilities, indicating that the zinc atom is not determinant in the protein thermostability. Further, the presence of the additional zinc centre does not interfere with the redox properties of the iron-sulfur clusters since their reduction potentials are almost identical. From the other three archaea, independently of the growth mode in respect to oxygen, only a single zinc-containing ferredoxin was found. EPR studies on the purified proteins, both in the oxidised and dithionite reduced states, allowed the identification of one [3Fe-4S]^1+/0 centre and one [4Fe-4S]^2+/1+ centre in all proteins studied. The complete sequence of A. ambivalens ferredoxin is reported. Together with the data gathered in this study, the properties of the seven-iron ferredoxins from Sulfolobales so far known are re-discussed. Received: 10 June 1998 / Accepted: 25 June 1998     

Amino acid sequence of the [4Fe-4S] ferredoxin isolated from Desulfovibrio desulfuricans Norway

The complete amino acid sequence of the [4Fe-4S] ferredoxin from Desulfovibrio desulfuricans Norway was determined by repetitive Edman degradation of the whole protein and peptides derived from tryptic digestion. The protein has 59 residues. Four of the six cysteine residues are involved in the binding of the [4Fe-4S] cluster in the same arrangement as in clostridial ferredoxins. This sequence is compared to various Desulfovibrio ferredoxin sequences and to the sequence and three-dimensional structure of Peptococcus aerogenes ferredoxin. Evidence of gene duplication is indicated. The requirement of some sequence features in the ferredoxin for an interaction process with its electron transfer partner, cytochrome c3, is postulated in the discussion.     

Structure of [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus refined at 2.3 A resolution. Structural comparisons of bacterial ferredoxins

The structure of a low-potential ferredoxin isolated from Bacillus thermoproteolyticus has been refined by a restrained least-squares method. The final crystallographic R factor is 0.204 for 2906 reflections with F greater than 3 sigma F in the 6.0 to 2.3 A resolution range. The model contains 81 amino acid residues, one [4Fe-4S] cluster, and 59 water molecules. The root-mean-square deviation from ideal values for bond lengths is 0.018 A, and the mean coordinate error is estimated to be 0.25 A. The present ferredoxin is similar in the topology of the polypeptide backbone to the dicluster-type ferredoxins from Peptococcus aerogenes and Azotobacter vinelandii, but has considerable insertions and deletions of the peptide segments as well as different secondary structures. Although all but the C-terminal C zeta atoms of P. aerogenes ferredoxin superpose on the C alpha atoms of A. vinelandii ferredoxin, only 60% superpose on the C alpha atoms of B. thermoproteolyticus ferredoxin, with a root-mean-square distance of 0.82 A for each pair. The conformations of the peptide segments surrounding the [4Fe-4S] clusters in these three ferredoxins are all conserved. Moreover, the schemes for the NH...S hydrogen bonds in these ferredoxins are nearly identical. The site of the aromatic ring of Tyr27 in B. thermoproteolyticus ferredoxin is close spatially to that of Tyr28 in P. aerogenes ferredoxin with reference to the cluster, but these residues do not correspond in the spatial alignment of their polypeptide backbones. We infer that in monocluster-type ferredoxins, the side-chain at the 27th residue has a crucial effect on the stability of the cluster. Of the four cysteine residues that bind to the second Fe-S cluster in the dicluster-type ferredoxins, two are conserved in the monocluster-type ferredoxins from Desulfovibrio gigas. D. desulfuricans Norway, and Clostridium thermoaceticum. The tertiary structure of B. thermoproteolyticus ferredoxin suggests that in such monocluster-type ferredoxins these two cysteine residues, which in it correspond to Ala21 and Asp53, form a disulfide bridge.     

Ferredoxins from two sulfonylurea herbicide monooxygenase systems in Streptomyces griseolus.

We have purified and characterized two ferredoxins, designated Fd-1 and Fd-2, from the soluble protein fraction of sulfonylurea herbicide induced Streptomyces griseolus. These cells have previously been shown to contain two inducible cytochromes P-450, P-450SU1 (CYP105A1) and P-450SU2 (CYP105B1), responsible for herbicide metabolism [O'Keefe, D. P., Romesser, J. A., & Leto, K. J. (1988) Arch. Microbiol. 149, 406-412]. Although Fd-2 is more effective, either ferredoxin can restore sulfonylurea monooxygenase activity to an aerobic mixture of NADPH, spinach ferredoxin:NADP oxidoreductase, purified cytochrome P-450SU1, and herbicide substrate. The gene for Fd-1 is located in the genome just downstream of the gene for cytochrome P-450SU1; the gene for Fd-2 follows the gene for P-450SU2. The deduced amino acid sequences of the two ferredoxins show that, if monomeric, each has a molecular mass of approximately 7 kDa, and alignment of the two sequences demonstrates that they are approximately 52% positionally identical. The spectroscopic properties and iron and acid-labile sulfide contents of both ferredoxins suggest that, as isolated, each contains a single [3Fe-4S] cluster. The presence of only three cysteines in Fd-1 and comparisons with three [4Fe-4S] ferredoxins with high sequence similarity suggest that both Fd-1 and Fd-2 have an alanine in the position where these [4Fe-4S] proteins have a fourth cysteine ligand to the cluster. Transformation of Streptomyces lividans, a strain unable to metabolize sulfonylureas, with DNA encoding both P-450SU1 and Fd-1 results in cells capable of herbicide metabolism. S. lividans transformants encoding only cytochrome P-450SU1 do not metabolize herbicide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unusual spectroscopic and electrochemical properties of the 2[4Fe-4S] ferredoxin of Thauera aromatica

A reduced ferredoxin serves as the natural electron donor for key enzymes of the anaerobic aromatic metabolism in the denitrifying bacterium Thauera aromatica. It contains two [4Fe-4S] clusters and belongs to the Chromatium vinosum type of ferredoxins (CvFd) which differ from the "clostridial" type by a six-amino acid insertion between two successive cysteines and a C-terminal alpha-helical amino acid extension. The electrochemical and electron paramagnetic resonance (EPR) spectroscopic properties of both [4Fe-4S] clusters from T. aromatica ferredoxin have been investigated using cyclic voltammetry and multifrequency EPR. Results obtained from cyclic voltammetry revealed the presence of two redox transitions at -431 and -587 mV versus SHE. X-band EPR spectra recorded at potentials where only one cluster was reduced (greater than -500 mV) indicated the presence of a spin mixture of S = (3)/(2) and (5)/(2) spin states of one reduced [4Fe-4S] cluster. No typical S = (1)/(2) EPR signals were observed. At lower potentials (less than -500 mV), the more negative [4Fe-4S] cluster displayed Q-, X-, and S-band EPR spectra at 20 K which were typical of a single S = (1)/(2) low-spin [4Fe-4S] cluster with a g(av) of 1.94. However, when the temperature was decreased stepwise to 4 K, a magnetic interaction between the two clusters gradually became observable as a temperature-dependent splitting of both the S = (1)/(2) and S = (5)/(2) EPR signals. At potentials where both clusters were reduced, additional low-field EPR signals were observed which can only be assigned to spin states with spins of >(5)/(2). The results that were obtained establish that the common typical amino acid sequence features of CvFd-type ferredoxins determine the unusual electrochemical properties of the [4Fe-4S] clusters. The observation of different spin states in T. aromatica ferredoxin is novel among CvFd-type ferredoxins.     

Amino acid sequence of [2Fe-2S] ferredoxin from Clostridium pasteurianum

The complete amino acid sequence of the [2Fe-2S] ferredoxin from the saccharolytic anaerobe Clostridium pasteurianum has been determined by automated Edman degradation of the whole protein and of peptides obtained by tryptic and by staphylococcal protease digestion. The polypeptide chain consists of 102 amino acids, including 5 cysteine residues in positions 11, 14, 24, 56, and 60. The sequence has been analyzed for hydrophilicity and for secondary structure predictions. In its native state the protein is a dimer, each subunit containing one [2Fe-2S] cluster, and it has a molecular weight of 23,174, including the four iron and inorganic sulfur atoms. The extinction coefficient of the native protein is 19,400 M-1 cm-1 at 463 nm. The positions of the cysteine residues, four of which are most probably the ligands of the [2Fe-2S] cluster, on the polypeptide chain of this protein are very different from those found in other [2Fe-2S] proteins, and in other ferredoxins in general. In addition, whole sequence comparisons of the [2Fe-2S] ferredoxin from C. pasteurianum with a number of other ferredoxins did not reveal any significant homologies. The likely occurrence of several phylogenetically unrelated ferredoxin families is discussed in the light of these observations.     

Thermococcus profundus 2-ketoisovalerate ferredoxin oxidoreductase, a key enzyme in the archaeal energy-producing amino acid metabolic pathway

2-Ketoisovalerate ferredoxin oxidoreductase (VOR) is a key enzyme in hyperthermophiles catalyzing the coenzyme A-dependent oxidative decarboxylation of mainly aliphatic amino acid-derived 2-keto acids. The very oxygen-labile enzyme purified under anaerobic conditions from a hyperthermophilic archaeon, Thermococcus profundus, is a hetero-octamer (alphabetagammadelta)(2) consisting of four different subunits, alpha = 45,000, beta = 31,000, gamma = 22,000 and delta = 13,000, respectively. Electron paramagnetic resonance and resonance Raman spectra of the purified enzyme indicate the presence of approximately three [4Fe-4S] clusters per alphabetagammadelta-protomer, although one of the clusters has a tendency to be converted to a [3Fe-4S] form during purification. The optimal temperature for the enzyme activity is 93 +/- 2 degrees C and the cognate [4Fe-4S] ferredoxin serves as an electron acceptor of the enzyme. The purified enzyme is highly oxygen-labile (t(1/2), approximately 5 min at 25 degrees C), and is partly protected in the presence of magnesium ions, thiamine pyrophosphate and sodium chloride (t(1/2), approximately 25 min at 25 degrees C).     

Spectopotentiometric properties and salt-dependent thermotolerance of a [2Fe-2S] ferredoxin-involved nitrate assimilation in Haloferax mediterranei

Haloferax mediterranei is a halophilic archaeon that can grow using nitrate as the sole nitrogen source. A ferredoxin that serves as the physiological electron donor to the nitrate and nitrite reductases in this assimilatory process has been characterized. The ferredoxin was found to contain approximately two atoms of iron and two atoms of sulphur, indicative of the binding of a [2Fe-2S] cluster. The electron paramagnetic resonance spectrum of the reduced form of the protein displayed a rhombic signal, with g(x)=1.91, g(y)=1.98, g(z)=2.07, that shows considerable similarity to plant and algal [2Fe-2S] ferredoxins. UV-visible spectropotentiometric analysis determined a midpoint redox potential for the [2Fe-2S](2+/1+) transition of around -285 mV vs. SHE that was independent of salt concentration. UV-visible spectroscopy was also used to establish that the [2Fe-2S] cluster integrity of this protein was maintained over the pH range 5-11. Significantly, the Haloferax mediterranei ferredoxin was shown to be a highly thermostable protein. It was stable up to 60 degrees C in a low-salt (0.2 M) medium and this increased to 80 degrees C in a high-salt (4 M) medium. This thermostability at high salt concentration is an essential physiological characteristic because haloarchaea are mainly found in environments where high temperatures and concentrated salt water occur.