Glutathione-complexed [2Fe-2S] clusters function in Fe–S cluster storage and trafficking

Glutathione-coordinated [2Fe-2S] complex is a non-protein-bound [2Fe-2S] cluster that is capable of reconstituting the human iron-sulfur cluster scaffold protein IscU. This complex demonstrates physiologically relevant solution chemistry and is a viable substrate for iron-sulfur cluster transport by Atm1p exporter protein. Herein, we report on some of the possible functional and physiological roles for this novel [2Fe-2S](GS₄) complex in iron-sulfur cluster biosynthesis and quantitatively characterize its role in the broader network of Fe–S cluster transfer reactions. UV–vis and circular dichroism spectroscopy have been used in kinetic studies to determine second-order rate constants for [2Fe-2S] cluster transfer from [2Fe-2S](GS₄) complex to acceptor proteins, such as human IscU, Schizosaccharomyces pombe Isa1, human and yeast glutaredoxins (human Grx2 and Saccharomyces cerevisiae Grx3), and human ferredoxins. Second-order rate constants for cluster extraction from these holo proteins were also determined by varying the concentration of glutathione, and a likely common mechanism for cluster uptake was determined by kinetic analysis. The results indicate that the [2Fe-2S](GS₄) complex is stable under physiological conditions, and demonstrates reversible cluster exchange with a wide range of Fe–S cluster proteins, thereby supporting a possible physiological role for such centers.     

Desulfovibrio gigas ferredoxin II: redox structural modulation of the [3Fe–4S] cluster

Desulfovibrio gigas ferredoxin II (DgFdII) is a small protein with a polypeptide chain composed of 58 amino acids, containing one Fe₃S₄ cluster per monomer. Upon studying the redox cycle of this protein, we detected a stable intermediate (FdII_int) with four ¹H resonances at 24.1, 20.5, 20.8 and 13.7 ppm. The differences between FdII_ox and FdII_int were attributed to conformational changes resulting from the breaking/formation of an internal disulfide bridge. The same ¹H NMR methodology used to fully assign the three cysteinyl ligands of the [3Fe–4S] core in the oxidized state (DgFdII_ox) was used here for the assignment of the same three ligands in the intermediate state (DgFdII_int). The spin-coupling model used for the oxidized form of DgFdII where magnetic exchange coupling constants of around 300 cm⁻¹ and hyperfine coupling constants equal to 1 MHz for all the three iron centres were found, does not explain the isotropic shift temperature dependence for the three cysteinyl cluster ligands in DgFdII_int. This study, together with the spin delocalization mechanism proposed here for DgFdII_int, allows the detection of structural modifications at the [3Fe-4S] cluster in DgFdII_ox and DgFdII_int.     

A combined computational and experimental investigation of the [2Fe–2S] cluster in biotin synthase

Biotin synthase was the first example of what is now regarded as a distinctive enzyme class within the radical S-adenosylmethionine superfamily, the members of which use Fe/S clusters as the sulphur source in radical sulphur insertion reactions. The crystal structure showed that this enzyme contains a [2Fe–2S] cluster with a highly unusual arginine ligand, besides three normal cysteine ligands. However, the crystal structure is at such a low resolution that neither the exact coordination mode nor the role of this exceptional ligand has been elucidated yet, although it has been shown that it is not essential for enzyme activity. We have used quantum refinement of the crystal structure and combined quantum mechanical and molecular mechanical calculations to explore possible coordination modes and their influences on cluster properties. The investigations show that the protonation state of the arginine ligand has little influence on cluster geometry, so even a positively charged guanidinium moiety would be in close proximity to the iron atom. Nevertheless, the crystallised enzyme most probably contains a deprotonated (neutral) arginine coordinating via the NH group. Furthermore, the Fe···Fe distance seems to be independent of the coordination mode and is in perfect agreement with distances in other structurally characterised [2Fe–2S] clusters. The exceptionally large Fe···Fe distance found in the crystal structure could not be reproduced.     

Purification and characterization of a fix ABCX-linked 2[4Fe-4S] ferredoxin from Azotobacter vinelandii

 Ferredoxins that contain 2[4Fe-4S]^2+/+ clusters can be divided into two classes. The "clostridial-type" ferredoxins have two CysXXCysXXCysXXXCysPro motifs. The "photosynthetic bacterial and nif-related" ferredoxins have one motif of that type and one more unusual CysXXCysX_7–9CysXXXCysPro motif. In Azotobacter vinelandii three gene sequences have been reported that contain the latter motif, but until now none of the gene products has been purified. Here we report the purification of a small anionic [Fe-S] protein with yields of ∼3 mg per 500 g cell paste. NH₂-terminal sequence analysis shows that this protein is the product of a previously sequenced A. vinelandii gene that is found upstream of fixA and is cotranscribed with fixABCX. That gene was originally named fixP, but since that gene designation is now commonly used for a very different cb-type cytochrome oxidase we have renamed the gene fixFd and its product Fix Fd. Its sequence places Fix Fd in the class of "photosynthetic bacterial and nif-related" 2[4Fe-4S]^2+/1+ ferredoxins that includes Chromatium vinosum ferredoxin. Studies of the purified protein by Fe analysis, absorption, CD and EPR spectroscopies and electrochemistry confirm this characterization; the reduction potentials of the two clusters are –440 mV vs SHE. The fact that A. vinelandii synthesizes three different proteins with the same sequence motif, each of which is likely to have a different function, shows that although sequence motifs may be used reliably to classify ferredoxins by cluster type they cannot yet be used reliably for classifying ferredoxins by function. Received: 31 January 1997 / Accepted: 9 June 1997     

Preliminary X-ray diffraction studies on a [4Fe4S] ferredoxin from Bacillus thermoproteolyticus

A [4Fe4S] ferredoxin from Bacillus thermoproteolyticus has been crystallized. The space group is P1 with two molecules in the unit cell, with the dimensions $\text{a = 32.96}\text{A}\text{?}\text{, b = 37.83}\text{A}\text{?}\text{, c = 39.82}\text{A}\text{?}\text{, α = 118.1 °, β = 104.2 °}\text{and}\text{γ = 89.7 °}$. The Bijvoet-difference Patterson map of the native crystal shows up a prominent peak of [4Fe4S] cluster.     

Determination of ligand conformation in reduced [2Fe-2S] ferredoxin from cysteine β-proton hyperfine couplings

 The isotropic hyperfine couplings of cysteine β-protons in iron-sulfur clusters of proteins provide information about the structure and conformation of the clusters if their magnetic resonance peaks can be resolved and assigned. The application of two-dimensional ESEEM (HYSCORE) spectroscopy to the reduced [2Fe-2S] cluster in ferredoxin from red marine algae Porfira umbilicalis is described. After deuterium substitution of the exchangeable protons, highly-resolved, orientationally-selected HYSCORE spectra show cross-peaks from strongly coupled, nonexchangeable protons. When cross-peaks from all the HYSCORE spectra are linearized and transformed to a common nuclear Zeeman frequency, they fall along five straight lines. Four of these sets of peaks are assigned to β-protons of the cysteine ligands. The isotropic and anisotropic hyperfine couplings for these protons are extracted from the slopes and intercepts of these lines. Two rescaling procedures are examined for the conversion of the experimentally measured isotropic couplings from different irons in [2Fe-2S] and [4Fe-4S] clusters. The couplings from P. umbilicalis appear to fit the same empirical dependence on Fe-S-C-H dihedral angle as do the couplings from a [4Fe-4S] model cluster. A method to assign protons for proteins of unknown structure is proposed that yields the correct assignment as derived from the crystal structure of the highly homologous protein from Spirulina platensis. The conformations of the cysteines in the reduced protein, derived without any adjustable parameters from this procedure and the empirical functions, are consistent with those reported for the latest refinement of the crystal structure of the oxidized protein. Received: 24 September 1997 / Accepted: 28 October 1997     

The CCG-domain-containing subunit SdhE of succinate:quinone oxidoreductase from Sulfolobus solfataricus P2 binds a [4Fe–4S] cluster

In type E succinate:quinone reductase (SQR), subunit SdhE (formerly SdhC) is thought to function as monotopic membrane anchor of the enzyme. SdhE contains two copies of a cysteine-rich sequence motif (CX _n CCGX _m CXXC), designated as the CCG domain in the Pfam database and conserved in many proteins. On the basis of the spectroscopic characterization of heterologously produced SdhE from Sulfolobus tokodaii, the protein was proposed in a previous study to contain a labile [2Fe–2S] cluster ligated by cysteine residues of the CCG domains. Using UV/vis, electron paramagnetic resonance (EPR), ⁵⁷Fe electron–nuclear double resonance (ENDOR) and Mössbauer spectroscopies, we show that after an in vitro cluster reconstitution, SdhE from S. solfataricus P2 contains a [4Fe–4S] cluster in reduced (2+) and oxidized (3+) states. The reduced form of the [4Fe–4S]²⁺ cluster is diamagnetic. The individual iron sites of the reduced cluster are noticeably heterogeneous and show partial valence localization, which is particularly strong for one unique ferrous site. In contrast, the paramagnetic form of the cluster exhibits a characteristic rhombic EPR signal with g _zyx  = 2.015, 2.008, and 1.947. This EPR signal is reminiscent of a signal observed previously in intact SQR from S. tokodaii with g _zyx  = 2.016, 2.00, and 1.957. In addition, zinc K-edge X-ray absorption spectroscopy indicated the presence of an isolated zinc site with an S₃(O/N)₁ coordination in reconstituted SdhE. Since cysteine residues in SdhE are restricted to the two CCG domains, we conclude that these domains provide the ligands to both the iron–sulfur cluster and the zinc site.     

Ferric uptake regulators (Fur) from Vibrio cholerae and Helicobacter pylori bind a [2Fe–2S] cluster in response to elevation of intracellular free iron content

BioMetals - Intracellular iron homeostasis in bacteria is primarily regulated by ferric uptake regulator (Fur). Since its discovery, Fur has been assumed to bind ferrous iron and regulate...     

Protection of scaffold protein Isu from degradation by the Lon protease Pim1 as a component of Fe–S cluster biogenesis regulation

Iron–sulfur (Fe–S) clusters, essential protein cofactors, are assembled on the mitochondrial scaffold protein Isu and then transferred to recipient proteins via a multistep process in which Isu interacts sequentially with multiple protein factors. This pathway is in part regulated posttranslationally by modulation of the degradation of Isu, whose abundance increases >10-fold upon perturbation of the biogenesis process. We tested a model in which direct interaction with protein partners protects Isu from degradation by the mitochondrial Lon-type protease. Using purified components, we demonstrated that Isu is indeed a substrate of the Lon-type protease and that it is protected from degradation by Nfs1, the sulfur donor for Fe–S cluster assembly, as well as by Jac1, the J-protein Hsp70 cochaperone that functions in cluster transfer from Isu. Nfs1 and Jac1 variants known to be defective in interaction with Isu were also defective in protecting Isu from degradation. Furthermore, overproduction of Jac1 protected Isu from degradation in vivo, as did Nfs1. Taken together, our results lead to a model of dynamic interplay between a protease and protein factors throughout the Fe–S cluster assembly and transfer process, leading to up-regulation of Isu levels under conditions when Fe–S cluster biogenesis does not meet cellular demands.     

Proton magnetic resonance studies of 7 Fe ferredoxins: Lower potential of the [4 Fe–4 S] redox center than the [3 Fe–3 S] cluster

Two types of iron-sulfur clusters, [3 Fe–3 S] and [4 Fe–4 S], were identified by ¹H-NMR in ferredoxins from Thermus thermophilus, Mycobacterium smegmatis and Pseudomonas ovalis. The [4 Fe–4 S] clusters always showed the redox couples which had potentials lower than that of the [3 Fe–3 S] clusters.     

Iron–sulfur cluster biosynthesis: characterization of IscU–IscS complex formation and a structural model for sulfide delivery to the [2Fe–2S] assembly site

Recent work on the bacterial iron–sulfur cluster (isc) family of gene products, and eukaryotic homologs, has advanced the molecular understanding of cellular mechanisms of iron–sulfur cluster biosynthesis. Members of the IscS family are pyridoxyl-5′-phosophate dependent proteins that deliver inorganic sulfide during assembly of the [2Fe–2S] cluster on the IscU scaffold protein. Herein it is demonstrated through calorimetry, fluorescence, and protein stability measurements that Thermotoga maritima IscS forms a 1:1 complex with IscU in a concentration-dependent manner (K _D varying from 6 to 34 μM, over an IscS concentration range of approximately 2–50 μM). Docking simulations of representative IscU and IscS proteins reveal critical contact surfaces at the N-terminal helix of IscU and a C-terminal loop comprising a chaperone binding domain. Consistent with the isothermal titration calorimetry results described here, an overall dominant contribution of charged surfaces with a change in the molar heat capacity of binding, ΔC _p ~ 199.8 kcal K⁻¹ mol⁻¹, is observed that accounts for approximately 10% of the total accessible surface area at the binding interface. Both apo and holo IscUs and homologs were found to bind to IscS in an enthalpically driven reaction with comparable K _D values. Both helix and loop regions are highly conserved among phylogenetically diverse organisms from a pool of archael, bacterial, fungal, and mammalian representatives.     

Characterization of a novel DNA glycosylase from S.?sahachiroi involved in the reduction and repair of azinomycin B induced DNA damage

Azinomycin B is a hybrid polyketide/nonribosomal peptide natural product and possesses antitumor activity by interacting covalently with duplex DNA and inducing interstrand crosslinks. In the biosynthetic study of azinomycin B, a gene (orf1) adjacent to the azinomycin B gene cluster was found to be essential for the survival of the producer, Streptomyces sahachiroi ATCC33158. Sequence analyses revealed that Orf1 belongs to the HTH_42 superfamily of conserved bacterial proteins which are widely distributed in pathogenic and antibiotic-producing bacteria with unknown functions. The protein exhibits a protective effect against azinomycin B when heterologously expressed in azinomycin-sensitive strains. EMSA assays showed its sequence nonspecific binding to DNA and structure-specific binding to azinomycin B-adducted sites, and ChIP assays revealed extensive association of Orf1 with chromatin in vivo. Interestingly, Orf1 not only protects target sites by protein–DNA interaction but is also capable of repairing azinomycin B-mediated DNA cross-linking. It possesses the DNA glycosylase-like activity and specifically repairs DNA damage induced by azinomycin B through removal of both adducted nitrogenous bases in the cross-link. This bifunctional protein massively binds to genomic DNA to reduce drug attack risk as a novel DNA binding protein and triggers the base excision repair system as a novel DNA glycosylase.     

Reactions of thiols and selenols with the FeMoS cluster [Fe(MoS4)2]3−

The known complex [Et₄N]₃[Fe(MoS₄)₂] has been shown by EPR and visible spectral studies to react with both thiophenol and selenophenol. The reaction results in a change in the characteristic S=3/2 EPR spectrum of this species from a complex rhombic pattern to one of a very simple axial appearance. Although this effect is similar to that observed for reaction of these species with the iron- molybdenum cofactor of nitrogenase, a moiety known to consist of a FeMoS cluster species, the large excesses of reagents and the long reaction times required for complete formation of product indicate that these reactions are of questionable direct relevance to the biological system. The reaction corresponding to the EPR spectral change from rhombic to axial in the [Fe(MoS₄)₂]³⁻/PhSeH system has also been partially characterized by product isolation which indicates that attack by selenol of the two terminal MoS₂ moieties in the starting material has occurred.     

HdrC2 from Acidithiobacillus ferrooxidans Owns Two Iron–Sulfur Binding Motifs But Binds Only One Variable Cluster Between [4Fe–4S] and [3Fe–4S]

The heterodisulfide reductase complex HdrABC from Acidithiobacillus ferrooxidans was suggested to own novel features that act in reverse to convert the sulfane sulfur of GS _n H species (n > 1) into sulfite in sulfur oxidation. The HdrC subunit is potentially encoded by two different highly upregulated genes sharing only 29 % identity in A. ferrooxidans grown in sulfur-containing medium, which were named as HdrC1 and HdrC2, respectively and had been confirmed to contain iron–sulfur cluster by expression and characterization, especially the HdrC1 which had been showed to bind only one [4Fe–4S] cluster by mutations. However, the mutations of the HdrC2 remain to be done and the detailed binding information of it is still unclear. Here, we report the expression, mutations, and molecular modeling of the HdrC2 from A. ferrooxidans. This HdrC2 had two identical motifs (Cx₂Cx₂Cx₃C) containing total of eight cysteine residues potentially for iron–sulfur cluster binding. This purified HdrC2 was exhibited to contain one variable cluster converted between [4Fe–4S] and [3Fe–4S] according to different conditions by the UV-scanning and EPR spectra. The site-directed mutagenesis results of these eight residues further confirmed that the HdrC2 in reduction with Fe²⁺ condition loaded only one [4Fe–4S]⁺ with spin S = 1/2 ligated by the residues of Cys73, Cys109, Cys112, and Cys115; the HdrC2 in natural aeration condition lost the Fe atom ligated by the residue of Cys73 and loaded only one [3Fe–4S]⁰ with spin S = 0; the HdrC2 in oxidation condition loaded only one [3Fe–4S]⁺ with spin S = 1/2. Molecular modeling results were also in line with the experiment results.