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.     

Characterization of the selenium-substituted 2 [4Fe-4Se] ferredoxin from Clostridium pasteurianum

The sulfur atoms of the two [4Fe-4S] clusters present in the ferredoxin from Clostridium pasteurianum have been replaced by selenium. The substitution is readily carried out by incubating the apoferredoxin with excess amounts of Fe3+, selenite, and dithiothreitol under anaerobic conditions. The UV-visible absorption spectrum of the Se-substituted ferredoxin, the core extrusion of its active sites, and analyses of its iron and selenium contents show that it contains two [4Fe-4Se] clusters. The Se-substituted ferredoxin is considerably less resistant to oxygen or to acidic and alkaline pH than the native ferredoxin: the half-lives of the former are 20-500 times shorter than those of the latter. The native ferredoxin and the Se-substituted ferredoxin display similar kinetic properties when used as electron donors to the hydrogenase from C. pasteurianum. It is of note, however, that the Km and Vmax values are lower for the 2[4Fe-4Se] ferredoxin than for the 2[4Fe-4S] ferredoxin. Reductive and oxidative titrations with dithionite and with thionine, respectively, show that both ferredoxins are two-electron carriers. The redox potentials of the ferredoxins have been measured by equilibrating them with the H2/H+ couple via hydrogenase: values of -423 and -417 mV have been found for the 2[4Fe-4S] ferredoxin and 2[4Fe-4Se] ferredoxin, respectively. Ferredoxins containing both chalcogenides in their [4Fe-4X] (X = S, Se) clusters have been prepared by reconstitution reactions involving mixtures of sulfide and selenide: the latter experiments show that sulfide and selenide are equally reactive in the incorporation of [4Fe-4X] (X = S, Se) sites into ferredoxin. The present report, together with former studies, establishes the general feasibility of the Se/S substitution in [2Fe-2S] and in [4Fe-4S] clusters of proteins and of synthetic analogues.     

Characterization of the recombinant Clostridium pasteurianum ferredoxin and comparison of its properties with those of the native protein

Ferredoxins (Fds) constitute an important class of nonheme iron-sulfur proteins. One of the most studied Fds is the [8Fe-8S] Fd from Clostridium pasteurianum. The gene for this Fd has previously been cloned and sequenced. We report the expression of this Fd in Escherichia coli, and the characterization and comparison of this recombinant protein to the native Fd. We have found that the purified recombinant protein has the same enzymatic, redox, magnetic and electronic properties as the native Fd isolated from C. pasteurianum, which indicates that the two [4Fe-4S] clusters present in the Fd were correctly formed in E. coli.     

Structural differences between [2Fe-2S] clusters in spinach ferredoxin and in the "red paramagnetic protein" from Clostridium pasteurianum. A resonance Raman study

The [2Fe-2S] ferredoxin ("Red paramagnetic protein", RPP) from C. pasteurianum has been found to be composed of two identical subunits of 10,000 +/- 2 000 daltons, each containing a [2Fe-2S] cluster. Resonance Raman (RR) spectra of RPP have been obtained at 23 degrees K, and compared to those of spinach ferredoxin (Sp Fd). Ten modes of the [2Fe-2S] chromophore were observed in the 100-450 cm-1 range. Assignments of non fundamental modes in the 500-900 cm-1 range allowed correlations between fundamental stretching modes of RPP and Sp Fd. Although assuming a [2Fe-2S] structure, the chromophore of RPP differs from that of Sp Fd by its conformation and by a slight weakening of Fe-S bonds, involving both the inorganic core and the cysteine ligands.     

The [2Fe-2S] protein I (Shetna protein I) from Azotobacter vinelandii is homologous to the [2Fe-2S] ferredoxin from Clostridium pasteurianum

 The [2Fe-2S] protein from Azotobacter vinelandii that was previously known as iron-sulfur protein I, or Shethna protein I, has been shown to be encoded by a gene belonging to the major nif gene cluster. Overexpression of this gene in Escherichia coli yielded a dimeric protein of which each subunit comprises 106 residues and contains one [2Fe-2S] cluster. The sequence of this protein is very similar to that of the [2Fe-2S] ferredoxin from Clostridium pasteurianum (2FeCpFd), and the four cysteine ligands of the [2Fe-2S] cluster occur in the same positions. The A. vinelandii protein differs from the C. pasteurianum one by the absence of the N-terminal methionine, the presence of a five-residue C-terminal extension, and a lesser number of acidic and polar residues. The UV-visible absorption and EPR spectra, as well as the redox potentials of the two proteins, are nearly identical. These data show that the A. vinelandii FeS protein I, which is therefore proposed to be designated 2FeAvFdI, is the counterpart of the [2Fe-2S] ferredoxin from C. pasteurianum. The occurrence of the 2FeAvFdI-encoding gene in the nif gene cluster, together with the previous demonstration of a specific interaction between the 2FeCpFd and the nitrogenase MoFe protein, suggest that both proteins might be involved in nitrogen fixation, with possibly similar roles. Received: 21 December 1998 / Accepted: 1 March 1999     

Characterization and cloning of an extremely thermostable, Pyrococcus furiosus-type 4Fe ferredoxin from Thermococcus profundus.

An extremely thermostable [4Fe-4S] ferredoxin was isolated under anaerobic conditions from a hyperthermophilic archaeon Thermococcus profundus, and the ferredoxin gene was cloned and sequenced. The nucleotide sequence of the ferredoxin gene shows the ferredoxin to comprise 62 amino acid residues with a sequence similar to those of many bacterial and archaeal 4Fe (3Fe) ferredoxins. The unusual Fe-S cluster type, which was identified in the resonance Raman and EPR spectra, has three cysteines and one aspartate as the cluster ligands, as in the Pyrococcus furiosus 4Fe ferredoxin. Under aerobic conditions, a ferredoxin was purified that contains a [3Fe-4S] cluster as the major Fe-S cluster and a small amount of the [4Fe-4S] cluster. Its N-terminal amino acid sequence is the same as that of the anaerobically-purified ferredoxin up to the 26th residue. These results indicate that the 4Fe ferredoxin was degraded to 3Fe ferredoxin during aerobic purification. The aerobically-purified ferredoxin was reversibly converted back to the [4Fe-4S] ferredoxin by the addition of ferrous ions under reducing conditions. The anaerobically-purified [4Fe-4S] ferredoxin is quite stable; little degradtion was observed over 20 h at 100 degrees C, while the half-life of the aerobically-purified ferredoxin is 10 h at 100 degrees C. Both the anaerobically- and aerobically-purified ferredoxins were found to function as electron acceptors for the pyruvate-ferredoxin oxidoreductase purified from the same archaeon.     

Structural similarities between the N-terminal domain of Clostridium pasteurianum hydrogenase and plant-type ferredoxins

An N-terminal domain of Clostridium pasteurianum hydrogenase I, encompassing 76 residues out of the 574 composing the full-size enzyme, had previously been overproduced in Escherichia coli and shown to form a stable fold around a [2Fe-2S] cluster. This domain displays only marginal sequence similarity with [2Fe-2S] proteins of known structure, and therefore, two-dimensional 1H NMR has been implemented to elucidate features of the polypeptide fold. Despite the perturbing presence of the paramagnetic [2Fe-2S] cluster, 57 spin systems were detected in the TOCSY spectra, 52 of which were sequentially assigned through NOE connectivities. Several secondary structure elements were identified. The N terminus of the protein consists of two antiparallel beta strands followed by an alpha helix contacting both strands. Two additional antiparallel beta strands, one of them at the C terminus of the sequence, form a four-stranded beta sheet together with the two N-terminal strands. The proton resonances that can be attributed to this beta2alphabeta2 structural motif undergo no paramagnetic perturbations, suggesting that it is distant from the [2Fe-2S] cluster. In plant- and mammalian-type ferredoxins, a very similar structural pattern is found in the part of the protein farthest from the [2Fe-2S] cluster. This indicates that the N-terminal domain of C. pasteurianum hydrogenase folds in a manner very similar to those of plant- and mammalian-type ferredoxins over a significant part (ca. 50%) of its structure. Even in the vicinity of the metal site, where 1H NMR data are blurred by paramagnetic interactions, the N-terminal domains of hydrogenase and mammalian- and plant-type ferredoxins most likely display significant structural similarity, as inferred from local sequence alignments and from previously reported circular dichroism and resonance Raman spectra. These data afford structural information on a kind of [2Fe-2S] cluster-containing domain that occurs in a number of redox enzymes and complexes. In addition, together with previously published sequence alignments, they highlight the widespread distribution of the plant-type ferredoxin fold in bioenergetic systems encompassing anaerobic metabolism, photosynthesis, and aerobic respiratory chains.     

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.     

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.     

Oxygen disruption of the 2[4Fe-4S] clusters in Clostridium pasteurianum ferredoxin shown by 1H-NMR.

The ferredoxin from Clostridium pasteurianum, which contains two [4Fe-4S] clusters, was investigated in its oxidized and reduced states by two-dimensional (2D) (1)H-(1)H nuclear Overhauser enhancement spectroscopy (NOESY). Comparison of the data from the oxidized ferredoxin with those published previously revealed the same NOE connectivities. No previous (1)H-(1)H NOESY study of the fully reduced ferredoxin has previously been published. However, it was possible to compare our results with those of a 2D exchange spectroscopy investigation of half-reduced C. pasteurianum ferredoxin. The present results with reduced C. pasteurianum ferredoxin confirm many of the (1)H peaks and NOE interactions reported earlier, revise others, and locate resonances previously undetected. When the ferredoxin was slightly exposed to oxygen, several of the hyperfine shifted resonances were irreversibly influenced. A resonance at 34 ppm in the (1)H NMR spectra of both redox states is indicative of oxygen exposure. These results indicate the importance of keeping the ferredoxin strictly anaerobic during purification and solvent exchange.     

Repair of nitric oxide-modified ferredoxin [2Fe-2S] cluster by cysteine desulfurase (IscS)

Iron-sulfur proteins are among the sensitive targets of the nitric oxide cytotoxicity. When Escherichia coli cells are exposed to nitric oxide, iron-sulfur clusters are modified forming protein-bound dinitrosyl iron complexes. Such modified protein dinitrosyl iron complexes are stable in vitro but are efficiently repaired in aerobically growing E. coli cells even without any new protein synthesis. Here we show that cysteine desulfurase encoded by the gene iscS of E. coli can directly convert the ferredoxin dinitrosyl iron complex to the ferredoxin [2Fe-2S] cluster in the presence of L-cysteine in vitro. A reassembly of the [2Fe-2S] cluster in the ferredoxin dinitrosyl iron complex does not require any addition of iron or other protein components. Furthermore, a complete removal of the dinitrosyl iron complex from ferredoxin prevents reassembly of the [2Fe-2S] cluster in the protein. The results suggest that cysteine desulfurase (IscS) together with L-cysteine can efficiently repair the nitric oxide-modified ferredoxin [2Fe-2S] cluster and that the iron center in the dinitrosyl iron complex may be recycled for the reassembly of iron-sulfur clusters in proteins.     

A novel phenanthrene dioxygenase from Nocardioides sp. Strain KP7: expression in Escherichia coli

Nocardioides sp. strain KP7 grows on phenanthrene but not on naphthalene. This organism degrades phenanthrene via 1-hydroxy-2-naphthoate, o-phthalate, and protocatechuate. The genes responsible for the degradation of phenanthrene to o-phthalate (phd) were found by Southern hybridization to reside on the chromosome. A 10.6-kb DNA fragment containing eight phd genes was cloned and sequenced. The phdA, phdB, phdC, and phdD genes, which encode the alpha and beta subunits of the oxygenase component, a ferredoxin, and a ferredoxin reductase, respectively, of phenanthrene dioxygenase were identified. The gene cluster, phdAB, was located 8. 3 kb downstream of the previously characterized phdK gene, which encodes 2-carboxybenzaldehyde dehydrogenase. The phdCD gene cluster was located 2.9 kb downstream of the phdB gene. PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the alpha and beta subunits of other ring-hydroxylating dioxygenases. The PhdC sequence showed features of a [3Fe-4S] or [4Fe-4S] type of ferredoxin, not of the [2Fe-2S] type of ferredoxin that has been found in most of the reported ring-hydroxylating dioxygenases. PhdD also showed moderate (less than 40%) sequence identity to known reductases. The phdABCD genes were expressed poorly in Escherichia coli, even when placed under the control of strong promoters. The introduction of a Shine-Dalgarno sequence upstream of each initiation codon of the phdABCD genes improved their expression in E. coli. E. coli cells carrying phdBCD or phdACD exhibited no phenanthrene-degrading activity, and those carrying phdABD or phdABC exhibited phenanthrene-degrading activity which was significantly less than that in cells carrying the phdABCD genes. It was thus concluded that all of the phdABCD genes are necessary for the efficient expression of phenanthrene-degrading activity. The genetic organization of the phd genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component suggest phenanthrene dioxygenase in Nocardioides sp. strain KP7 to be a new class of aromatic ring-hydroxylating dioxygenases.     

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.     

A [2Fe-2S] protein from the hyperthermophilic bacterium Aquifex aeolicus.

Overexpression in Escherichia coli of the fdx4 gene from Aquifex aeolicus has allowed isolation and characterization of the first hyperthermophilic [2Fe-2S](Scys)(4) protein, a homodimer of M = 2 x 12.4 kDa with one [2Fe-2S] cluster per subunit. This protein is undamaged by heating to 100 degrees C for at least three hours. The primary structure, in particular the characteristic distribution of the four cysteine ligands of the metal site, and the spectroscopic properties of the A. aeolicus protein relate it to well characterized [2Fe-2S] proteins from Clostridium pasteurianum and Azotobacter vinelandii. These proteins are also homologous to subunits or domains of hydrogenases and NADH-ubiquinone oxidoreductase (Complex I) of respiratory chains. The A. aeolicus [2Fe-2S] protein is thus representative of a presumably novel protein fold involved in a variety of functions in very diverse cellular backgrounds.     

An Isc-type extremely thermostable [2Fe-2S] ferredoxin from Aquifex aeolicus. Biochemical,spectroscopic, and unfolding studies

Analysis of the genome of the hyperthermophilic bacterium Aquifex aeolicus has revealed the presence of a previously undetected gene potentially encoding a plant- and mammalian-type [2Fe-2S] ferredoxin. Expression of that gene in Escherichia coli has yielded a novel thermostable [2Fe-2S] ferredoxin (designated ferredoxin 5) whose sequence is most similar to those of ferredoxins involved in the assembly of iron-sulfur clusters (Isc-Fd). It nevertheless differs from the latter proteins by having deletions near its N- and C-termini, and no cysteine residues other than those involved in [2Fe-2S] cluster coordination. Resonance Raman, low-temperature MCD and EPR studies show close spectral similarities between ferredoxin 5 and the Isc-Fd from Azotobacter vinelandii. M?ssbauer spectra of the reduced protein were analyzed with an S = 1/2 spin Hamiltonian and interpreted in the framework of the ligand field model proposed by Bertrand and Gayda. The redox potential of A. aeolicus ferredoxin 5 (-390 mV) is in keeping with its relatedness to Isc-Fd. Unfolding experiments showed that A. aeolicus ferredoxin 5 is highly thermostable (T(m) = 106 degrees C at pH 7), despite being devoid of features (e.g., high content of charged residues) usually associated with extreme thermal stability. Searches for genes potentially encoding plant-type [2Fe-2S] ferredoxins have been performed on the sequenced genomes of hyperthermophilic organisms. None other than the two proteins from A. aeolicus were retrieved, indicating that this otherwise widely distributed group of proteins is barely represented among hyperthermophiles.     

Determination of cooperative interaction between clusters in Clostridium pasteurianum 2 (4Fe-4S) ferredoxin.

The effect of reducing one 4Fe-4S cluster in Clostridium pasteurianum 2 (4Fe-4S) ferredoxin on the reduction potential of the unreduced cluster has been investigated. While such an effect is suggested by both the x-ray structure of Peptococcus aerogenes 2 (4F-4S) ferredoxin and the polypeptide conformational change on reduction present in clostridial-type 2 (4Fe-4S) ferredoxins, present studies indicate that cluster-cluster cooperative interaction is not strong enough to be of functional importance in these proteins.     

Resonance Raman studies of the [4Fe-4S] to [2Fe-2S] cluster conversion in the iron protein of nitrogenase

Resonance Raman spectroscopy has been used to investigate the Fe-S stretching modes of the [4Fe-4S]2+ cluster in the oxidized iron protein of Clostridium pasteurianum nitrogenase. The results are consistent with a cubane [4Fe-4S] cluster having effective Td symmetry with cysteinyl coordination for each iron. In accord with previous optical and EPR studies [(1984) Biochemistry 23, 2118-2122], treatment with the iron chelator alpha, alpha'-dipyridyl in the presence of MgATP is shown to effect cluster conversion to a [2Fe-2S]2+ cluster. Resonance Raman data also indicate that partial conversion to a [2Fe-2S]2+ cluster is induced by thionine-oxidation in the presence of MgATP in the absence of an iron chelator. This result suggests new explanations for the dramatic change in the CD spectrum that accompanies MgATP-binding to the oxidized Fe protein and the anomalous resonance Raman spectra of thionine-oxidized Clostridium pasteurianum bidirectional hydrogenase.