A vertebrate-type ferredoxin domain in the Na+-translocating NADH dehydrogenase from Vibrio cholerae

The Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae contains a single Fe-S cluster localized in subunit NqrF. Here we study the electronic properties of the Fe-S center in a truncated version of the NqrF subunit comprising only its ferredoxin-like Fe-S domain. M?ssbauer spectroscopy of the Fe-S domain in the oxidized state is consistent with a binuclear Fe-S cluster with tetrahedral sulfur coordination by the cysteine residues Cys(70), Cys(76), Cys(79), and Cys(111). Important sequence motifs surrounding these cysteines are conserved in the Fe-S domain and in vertebrate-type ferredoxins. The magnetic circular dichroism spectra of the photochemically reduced Fe-S domain exhibit a striking similarity to the magnetic circular dichroism spectra of vertebrate-type ferredoxins required for the in vivo assembly of iron-sulfur clusters. This study reveals a novel function for vertebrate-type [2Fe-2S] clusters as redox cofactors in respiratory dehydrogenases.     

Characterization of MOCS1A, an oxygen-sensitive iron-sulfur protein involved in human molybdenum cofactor biosynthesis

The human proteins MOCS1A and MOCS1B catalyze the conversion of a guanosine derivative to precursor Z during molybdenum cofactor biosynthesis. MOCS1A shares homology with S-adenosylmethionine (AdoMet)-dependent radical enzymes, which catalyze the formation of protein and/or substrate radicals by reductive cleavage of AdoMet through a [4Fe-4S] cluster. Sequence analysis of MOCS1A showed two highly conserved cysteine motifs, one near the N terminus and one near the C terminus. MOCS1A was heterologously expressed in Escherichia coli and purified under aerobic and anaerobic conditions. Individual mutations of the conserved cysteines to serine revealed that all are essential for synthesis of precursor Z in vivo. The type and properties of the iron-sulfur (FeS) clusters were investigated using a combination of UV-visible absorption, variable temperature magnetic circular dichroism, resonance Raman, M?ssbauer, and EPR spectroscopies coupled with iron and acid-labile sulfide analyses. The results indicated that anaerobically purified MOCS1A is a monomeric protein containing two oxygen-sensitive FeS clusters, each coordinated by only three cysteine residues. A redox-active [4Fe-4S](2+,+) cluster is ligated by an N-terminal CX(3)CX(2)C motif as is the case with all other AdoMet-dependent radical enzymes investigated thus far. A C-terminal CX(2)CX(13)C motif that is unique to MOCS1A and its orthologs primarily ligates a [3Fe-4S](0) cluster. However, MOCS1A could be reconstituted in vitro under anaerobic conditions to yield a form containing two [4Fe-4S](2+) clusters. The N-terminal [4Fe-4S](2+) cluster was rapidly degraded by oxygen via a semistable [2Fe-2S](2+) cluster intermediate, and the C-terminal [4Fe-4S](2+) cluster was rapidly degraded by oxygen to yield a semistable [3Fe-4S](0) cluster intermediate.     

Site-specific mutational analysis of a novel cysteine motif proposed to ligate the 4Fe-4S cluster in the iron-sulfur flavoprotein of the thermophilic methanoarchaeon Methanosarcina thermophila

Isf (iron-sulfur flavoprotein) from Methanosarcina thermophila has been produced in Escherichia coli as a dimer containing two 4Fe-4S clusters and two FMN (flavin mononucleotide) cofactors. The deduced sequence of Isf contains six cysteines (Cys 16, Cys 47, Cys 50, Cys 53, Cys 59, and Cys 180), four of which (Cys 47, Cys 50, Cys 53, and Cys 59) comprise a motif with high identity to a motif (CX(2)CX(2)CX(4-7)C) present in all homologous Isf sequences available in the databases. The spacing of the motif is highly compact and atypical of motifs coordinating known 4Fe-4S clusters; therefore, all six cysteines in Isf from M. thermophila were altered to either alanine or serine to obtain corroborating biochemical evidence that the motif coordinates the 4Fe-4S cluster and to further characterize properties of the cluster dependent on ligation. All except the C16S variant were produced in inclusion bodies and were void of iron-sulfur clusters and FMN. Reconstitution of the iron-sulfur cluster and FMN was attempted for each variant. The UV-visible spectra of all reconstituted variants indicated the presence of iron-sulfur clusters and FMN. The reduced C16A/S variants showed the same electron paramagnetic resonance (EPR) spectra as wild-type Isf, whereas the reduced C180A/S variants showed EPR spectra identical to those of one of the two 4Fe-4S species present in the wild-type Isf spectrum. Conversely, EPR spectra of the oxidized C50A and C59A variants showed g values characteristic of a 3Fe-4S cluster. The spectra of the C47A and C53A variants indicated a 4Fe-4S cluster with g values and linewidths different from those for the wild type. The combined results of this study support a role for the novel CX(2)CX(2)CX(4-7)C motif in ligating the 4Fe-4S clusters in Isf and Isf homologues.     

Properties of the cysteine residues and iron-sulfur cluster of the assimilatory 5'-adenylyl sulfate reductase from Pseudomonas aeruginosa

APS reductase from Pseudomonas aeruginosa has been shown to contain a [4Fe-4S] cluster. Thiol determinations and site-directed mutagenesis studies indicate that the single [4Fe-4S] cluster contains only three cysteine ligands, instead of the more typical arrangement in which clusters are bound to the protein by four cysteines. Resonance Raman studies in the Fe-S stretching region are also consistent with the presence of a redox-inert [4Fe-4S](2+) cluster with three cysteinate ligands and indicate that the fourth ligand is likely to be an oxygen-containing species. This conclusion is supported by resonance Raman and electron paramagnetic resonance (EPR) evidence for near stoichiometric conversion of the cluster to a [3Fe-4S](+) form by treatment with a 3-fold excess of ferricyanide. Site-directed mutagenesis experiments have identified Cys139, Cys228, and Cys231 as ligands to the cluster. The remaining two cysteines present in the enzyme, Cys140 and Cys256, form a redox-active disulfide/dithiol couple (E(m) = -300 mV at pH 7.0) that appears to play a role in the catalytic mechanism of the enzyme.     

Characterization of the evolutionarily conserved iron-sulfur cluster of sirohydrochlorin ferrochelatase from Arabidopsis thaliana

Sirohaem is a cofactor of nitrite and sulfite reductases, essential for assimilation of nitrogen and sulfur. Sirohaem is synthesized from the central tetrapyrrole intermediate uroporphyrinogen III by methylation, oxidation and ferrochelation reactions. In Arabidopsis thaliana, the ferrochelation step is catalysed by sirohydrochlorin ferrochelatase (SirB), which, unlike its counterparts in bacteria, contains an [Fe-S] cluster. We determined the cluster to be a [4Fe-4S] type, which quickly oxidizes to a [2Fe-2S] form in the presence of oxygen. We also identified the cluster ligands as four conserved cysteine residues located at the C-terminus. A fifth conserved cysteine residue, Cys(135), is not involved in ligating the cluster directly, but influences the oxygen-sensitivity of the [4Fe-4S] form, and possibly the affinity for the substrate metal. Substitution mutants of the enzyme lacking the Fe-S cluster or Cys(135) retain the same specific activity in vitro and dimeric quaternary structure as the wild-type enzyme. The mutant variants also rescue a defined Escherichia coli sirohaem-deficient mutant. However, the mutant enzymes cannot complement Arabidopsis plants with a null AtSirB mutation, which exhibits post-germination arrest. These observations suggest an important physiological role for the Fe-S cluster in Planta, highlighting the close association of iron, sulfur and tetrapyrrole metabolism.     

X-ray structure of the complete ABC enzyme ABCE1 from Pyrococcus abyssi

The ATP binding cassette enzyme ABCE1 (also known as RNase-L (ribonuclease L) inhibitor, Pixie, and HP68), one of the evolutionary most sequence-conserved enzymes, functions in translation initiation, ribosome biogenesis, and human immunodeficiency virus capsid assembly. However, its structural mechanism and biochemical role in these processes have not been revealed. We determined the crystal structure of Pyrococcus abyssi ABCE1 in complex with Mg(2+) and ADP to 2.8A resolution. ABCE1 consists of four structural domains. Two nucleotide binding domains are arranged in a head-to-tail orientation by a hinge domain, suggesting that these domains undergo the characteristic tweezers-like powerstroke of ABC enzymes. In contrast to all other known ABC enzymes, ABCE1 has a N-terminal iron-sulfur-cluster (FeS) domain. The FeS domain contains two [4Fe-4S] clusters and is structurally highly related to bacterial-type ferredoxins. However, one cluster is coordinated by an unusual CX(4)CX(3/4)C triad. Surprisingly, intimate interactions of the FeS domain with the adenine and ribose binding Y-loop on nucleotide binding domain 1 suggest a linkage between FeS domain function and ATP-induced conformational control of the ABC tandem cassette. The structure substantially expands the functional architecture of ABC enzymes and raises the possibility that ABCE1 is a chemomechanical engine linked to a redox process.     

The iron-sulfur center of biotin synthase: site-directed mutants

Biotin synthase contains an essential [4Fe-4S]+ cluster that is thought to provide an electron for the cleavage of S-adenosylmethionine, a cofactor required for biotin formation. The conserved cysteine residues Cys53, Cys57 and Cys60 have been proposed as ligands to the [4Fe-4S] cluster. These residues belong to a C-X3-C-X2-C motif which is also found in pyruvate formate lyase-activating enzyme, lysine 2,3-aminomutase and the anaerobic ribonucleotide reductase-activating component. To investigate the role of the cysteine residues, Cys-->Ala mutants of the eight cysteine residues of Escherichia coli biotin synthase were prepared and assayed for activity. Our results show that six cysteines are important for biotin formation. Only two mutant proteins, C276A and C288A, closely resembled the wild-type protein, indicating that the corresponding cysteines are not involved in iron chelation and biotin formation. The six other mutant proteins, C53A, C57A, C60A, C97A, C128A and C188A, were inactive but capable of assembling a [4Fe-4S] cluster, as shown by M?ssbauer spectroscopy. The C53A, C57A and C60A mutant proteins are unique in that their cluster could not undergo reduction to the [4Fe-4S]+ state, as shown by EPR and M?ssbauer spectroscopy. On this basis and by analogy with pyruvate formate lyase-activating enzyme and the anaerobic ribonucleotide reductase-activating component, it is suggested that the corresponding cysteines coordinate the cluster even though one cannot fully exclude the possibility that other cysteines play that role as well. Therefore it appears that for activity biotin synthase absolutely requires cysteines that are not involved in iron chelation.     

The asymmetric IscA homodimer with an exposed [2Fe-2S] cluster suggests the structural basis of the Fe-S cluster biosynthetic scaffold

It has been shown that the so-called scaffold proteins are vital in Fe-S cluster biosynthesis by providing an intermediate site for the assembly of Fe-S clusters. However, since no structural information on such scaffold proteins with bound Fe-S cluster intermediates is available, the structural basis of the core of Fe-S cluster biosynthesis remains poorly understood. Here we report the first Fe-S cluster-bound crystal structure of a scaffold protein, IscA, from Thermosynechococcus elongatus, which carries three strictly conserved cysteine residues. Surprisingly, one partially exposed [2Fe-2S] cluster is coordinated by two conformationally distinct IscA protomers, termed alpha and beta, with asymmetric cysteinyl ligation by Cys37, Cys101, Cys103 from alpha and Cys103 from beta. In the crystal, two alphabeta dimers form an unusual domain-swapped tetramer via central domains of beta protomers. Together with additional biochemical data supporting its physiologically relevant configuration, we propose that the unique asymmetric Fe-S cluster coordination and the resulting distinct conformational stabilities of the two IscA protomers are central to the function of IscA-type Fe-S cluster biosynthetic scaffold.     

Crystal structure of Escherichia coli SufA involved in biosynthesis of iron-sulfur clusters: implications for a functional dimer

IscA and SufA are paralogous proteins that play crucial roles in the biosynthesis of Fe-S clusters, perhaps through a mechanism involving transient Fe-S cluster formation. We have determined the crystal structure of E. coli SufA at 2.7A resolution. SufA exists as a homodimer, in contrast to the tetrameric organization of IscA. Furthermore, a C-terminal segment containing two essential cysteine residues (Cys-Gly-Cys), which is disordered in the IscA structure, is clearly visible in one molecule (the alpha1 subunit) of the SufA homodimer. Although this segment is disordered in the other molecule (the alpha2 subunit), computer modeling of this segment based on the well-defined conformation of alpha1 subunit suggests that the four cysteine residues (Cys114 and Cys116 in each subunit) in the Cys-Gly-Cys motif are positioned in close proximity at the dimer interface. The arrangement of these cysteines together with the nearby Glu118 in SufA dimer may allow coordination of an Fe-S cluster and/or an Fe atom.     

FeoC from Klebsiella pneumoniae Contains a [4Fe-4S] Cluster

Iron is essential for pathogen survival, virulence, and colonization. Feo is suggested to function as the ferrous iron (Fe²⁺) transporter. The enterobacterial Feo system is composed of 3 proteins: FeoB is the indispensable component and is a large membrane protein likely to function as a permease; FeoA is a small Src homology 3 (SH3) domain protein that interacts with FeoB; FeoC is a winged-helix protein containing 4 conserved Cys residues in a sequence suitable for harboring a putative iron-sulfur (Fe-S) cluster. The presence of an iron-sulfur cluster on FeoC has never been shown experimentally. We report that under anaerobic conditions, the recombinant Klebsiella pneumoniae FeoC (KpFeoC) exhibited hyperfine-shifted nuclear magnetic resonance (NMR) and a UV-visible (UV-Vis) absorbance spectrum characteristic of a paramagnetic center. The electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) results were consistent only with the [4Fe-4S] clusters. Substituting the cysteinyl sulfur with oxygen resulted in significantly reduced cluster stability, establishing the roles of these cysteines as the ligands for the Fe-S cluster. When exposed to oxygen, the [4Fe-4S] cluster degraded to [3Fe-4S] and eventually disappeared. We propose that KpFeoC may regulate the function of the Feo transporter through the oxygen- or iron-sensitive coordination of the Fe-S cluster.     

Crystal structure of Escherichia coli Fdx, an adrenodoxin-type ferredoxin involved in the assembly of iron-sulfur clusters

Escherichia coli ferredoxin (Fdx) is an adrenodoxin-type [2Fe-2S] ferredoxin. Recent genetic analyses show that it has an essential role in the maturation of various iron-sulfur (Fe-S) proteins. Fdx probably functions as a component of the complex machinery responsible for the biogenesis of Fe-S clusters. Its crystal structure was determined by the multiple-wavelength anomalous dispersion method using the iron atoms in the [2Fe-2S] cluster of the protein and then refined to R and R(free) values of 0.255 and 0.278, respectively, at 1.7 A resolution. The structure of Fdx is similar to the structures of bovine adrenodoxin (Adx) and Pseudomonas putida putidaredoxin (Pdx) whose respective root-mean-square deviations of the corresponding Calpha atoms are 1.8 and 2.2 A. This analysis also revealed the structure of the C-terminal residues protruding into the solvent, which is missing in Adx and Pdx. The [2Fe-2S] cluster is located at the edge of the molecule and bonds with the Sgamma atoms of Cys42, Cys48, Cys51, and Cys87. Electrostatic potential analysis showed that the surface of Fdx has two negatively charged areas separated by a hydrophobic lane. One is conserved on the surface of Adx which is an area of interaction with adrenodoxin reductase. Cys46 is located on the molecular surface in the vicinity of the [2Fe-2S] cluster, an indication that it may be involved in Fe-S cluster formation.     

A bridging [4Fe-4S] cluster and nucleotide binding are essential for function of the Cfd1-Nbp35 complex as a scaffold in iron-sulfur protein maturation

The essential P-loop NTPases Cfd1 and Nbp35 of the cytosolic iron-sulfur (Fe-S) protein assembly machinery perform a scaffold function for Fe-S cluster synthesis. Both proteins contain a nucleotide binding motif of unknown function and a C-terminal motif with four conserved cysteine residues. The latter motif defines the Mrp/Nbp35 subclass of P-loop NTPases and is suspected to be involved in transient Fe-S cluster binding. To elucidate the function of these two motifs, we first created cysteine mutant proteins of Cfd1 and Nbp35 and investigated the consequences of these mutations by genetic, cell biological, biochemical, and spectroscopic approaches. The two central cysteine residues (CPXC) of the C-terminal motif were found to be crucial for cell viability, protein function, coordination of a labile [4Fe-4S] cluster, and Cfd1-Nbp35 hetero-tetramer formation. Surprisingly, the two proximal cysteine residues were dispensable for all these functions, despite their strict evolutionary conservation. Several lines of evidence suggest that the C-terminal CPXC motifs of Cfd1-Nbp35 coordinate a bridging [4Fe-4S] cluster. Upon mutation of the nucleotide binding motifs Fe-S clusters could no longer be assembled on these proteins unless wild-type copies of Cfd1 and Nbp35 were present in trans. This result indicated that Fe-S cluster loading on these scaffold proteins is a nucleotide-dependent step. We propose that the bridging coordination of the C-terminal Fe-S cluster may be ideal for its facile assembly, labile binding, and efficient transfer to target Fe-S apoproteins, a step facilitated by the cytosolic iron-sulfur (Fe-S) protein assembly proteins Nar1 and Cia1 in vivo.     

IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU

Iron-sulfur cluster biosynthesis in both prokaryotic and eukaryotic cells is known to be mediated by two highly conserved proteins, termed IscS and IscU in prokaryotes. The homodimeric IscS protein has been shown to be a cysteine desulfurase that catalyzes the reductive conversion of cysteine to alanine and sulfide. In this work, the time course of IscS-mediated Fe-S cluster assembly in IscU was monitored via anaerobic anion exchange chromatography. The nature and properties of the clusters assembled in discrete fractions were assessed via analytical studies together with absorption, resonance Raman, and M?ssbauer investigations. The results show sequential cluster assembly with the initial IscU product containing one [2Fe-2S](2+) cluster per dimer converting first to a form containing two [2Fe-2S](2+) clusters per dimer and finally to a form that contains one [4Fe-4S](2+) cluster per dimer. Both the [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU are reductively labile and are degraded within minutes upon being exposed to air. On the basis of sequence considerations and spectroscopic studies, the [2Fe-2S](2+) clusters in IscU are shown to have incomplete cysteinyl ligation. In addition, the resonance Raman spectrum of the [4Fe-4S](2+) cluster in IscU is best interpreted in terms of noncysteinyl ligation at a unique Fe site. The ability to assemble both [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU supports the proposal that this ubiquitous protein provides a scaffold for IscS-mediated assembly of clusters that are subsequently used for maturation of apo Fe-S proteins.     

Characterization of cluster N5 as a fast-relaxing [4Fe-4S] cluster in the Nqo3 subunit of the proton-translocating NADH-ubiquinone oxidoreductase from Paracoccus denitrificans

The NADH-quinone oxidoreductase from Paracoccus denitrificans consists of 14 subunits (Nqo1-14) and contains one FMN and eight iron-sulfur clusters. The Nqo3 subunit possesses fully conserved 11 Cys and 1 His in its N-terminal region and is considered to harbor three iron-sulfur clusters; however, only one binuclear (N1b) and one tetranuclear (N4) were previously identified. In this study, the Nqo3 subunit containing 1x[2Fe-2S] and 2x[4Fe-4S] clusters was expressed in Escherichia coli. The second [4Fe-4S](1+) cluster is detected by EPR spectroscopy below 6 K, exhibiting very fast spin relaxation. The resolved EPR spectrum of this cluster is broad and nearly axial. The subunit exhibits an absorption-type EPR signal around g approximately 5 region below 6 K, most likely arising from an S = 3/2 ground state of the fast-relaxing [4Fe-4S](1+) species. The substitution of the conserved His(106) with Cys specifically affected the fast-relaxing [4Fe-4S](1+) cluster, suggesting that this cluster is coordinated by His(106). In the cholate-treated NDH-1-enriched P. denitrificans membranes, we observed EPR signals arising from a [4Fe-4S] cluster below 6 K, exhibiting properties similar to those of cluster N5 detected in other complex I/NDH-1 and of the fast-relaxing [4Fe-4S](1+) cluster in the expressed Nqo3 subunit. Hence, we propose that the His-coordinated [4Fe-4S] cluster corresponds to cluster N5.     

Avian glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing and activity are dependent upon essential cysteine residues.

Avian glutamine phosphoribosylpyrophosphate amidotransferase contains an NH2-terminal propetide-like sequence. NH2-terminal sequence analysis of immunoaffinity purified enzyme from chicken liver indicates that the propeptide is processed and the mature enzyme starts with Cys. Propeptide processing was investigated by site-directed mutagenesis using a system for expression in HeLa cells. Glutamine-dependent activity and processing were abolished by replacement of the conserved cysteine at position 1, whereas NH3-dependent activity was retained. Cys1 is thus inferred to have a role in glutamine-dependent activity and in propeptide processing. Inactive, insoluble enzymes in which the propeptide was not processed were obtained as a result of replacements of cysteines 415 and 488. Cysteine residues at positions 415 and 488 are inferred to be ligands to an Fe-S cluster on the basis of sequence similarity to the enzyme from Bacillus subtilis. Mutation of Cys269 and Cys295 led to loss of enzyme activity and propeptide processing, although solubility was unchanged. The results suggest that incorporation of an Fe-S cluster is needed for native structure, resultant propeptide processing, and glutamine-dependent activity.     

Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.     

Enterococcus faecalis SufU scaffold protein enhances SufS desulfurase activity by acquiring sulfur from its cysteine-153

Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups that play essential roles in all living organisms. In vivo [Fe-S] cluster biogenesis requires enzymes involved in iron and sulfur mobilization, assembly of clusters, and delivery to their final acceptor. In these systems, a cysteine desulfurase is responsible for the release of sulfide ions, which are incorporated into a scaffold protein for subsequent [Fe-S] cluster assembly. Although three machineries have been shown to be present in Proteobacteria for [Fe-S] cluster biogenesis (NIF, ISC, and SUF), only the SUF machinery has been found in Firmicutes. We have recently described the structural similarities and differences between Enterococcus faecalis and Escherichia coli SufU proteins, which prompted the proposal that SufU is the scaffold protein of the E. faecalis sufCDSUB system. The present work aims at elucidating the biological roles of E. faecalis SufS and SufU proteins in [Fe-S] cluster assembly. We show that SufS has cysteine desulfurase activity and cysteine-365 plays an essential role in catalysis. SufS requires SufU as activator to [4Fe-4S] cluster assembly, as its ortholog, IscU, in which the conserved cysteine-153 acts as a proximal sulfur acceptor for transpersulfurization reaction.     

Engineering a three-cysteine, one-histidine ligand environment into a new hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin from Sulfolobus solfataricus

We heterologously overproduced a hyperthermostable archaeal low potential (E(m) = -62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on the properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys(42) and Cys(61)) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Rieske-type ferredoxin appear to be inequivalent as indicated by much higher stability of the His(64) --> Cys variant (H64C) than the His(44) --> Cys variant (H44C). The x-ray absorption and resonance Raman spectra of the H64C variant firmly established the formation of a novel, oxidized [2Fe-2S] cluster with one histidine and three cysteine ligands in the archaeal Rieske-type protein moiety. Comparative resonance Raman features of the wild-type, natural abundance and uniformly (15)N-labeled ARF and its H64C variant showed significant mixing of the Fe-S and Fe-N stretching characters for an oxidized biological [2Fe-2S] cluster with partial histidine ligation.