Key players and their role during mitochondrial iron-sulfur cluster biosynthesis

Iron-sulfur clusters are multifaceted iron-containing cofactors coordinated and utilized by numerous proteins in nearly all biological systems. Fe-S-cluster-containing proteins help direct pathways essential for cell viability and participate in biological applications ranging from nucleotide biosynthesis and stability, protein translation, enzyme catalysis, and mitochondrial metabolism. Fe-S-containing proteins function by utilizing the unique electronic and chemical properties inherent in the Fe containing cofactor. Fe-S clusters are constructed of inorganic iron and sulfide arranged in a distinct caged structural makeup ranging from [Fe(2) -S(2) ], [Fe(3) -S(4) ], [Fe(4) -S(4) ], up to [Fe(8) -S(8) ] clusters. In eukaryotes, cluster activity is controlled in part at the assembly level and the major pathway for cluster production exists within the mitochondria. Recent insight into the pathway of mitochondrial cluster assembly has come from new in vivo and in vitro reports that provided direct insight into how all protein partners within the assembly pathway interact. However, we are only just beginning to understand the role of each protein within this complex pageant that is mitochondrial Fe-S cluster assembly. In this report we present results, using the yeast model for mitochondrial assembly, to describe the molecular details of how important proteins in the pathway coordinate for cluster assembly.     

Iron-sulfur cluster biosynthesis. Characterization of frataxin as an iron donor for assembly of [2Fe-2S] clusters in ISU-type proteins

ISU (eukaryotes) and IscU (prokaryotes) are a homologous family of proteins that appear to provide a platform for assembly of [2Fe-2S] centers prior to delivery to an apo target protein. The intermediate [2Fe-2S] ISU-bound cluster is formed by delivery of iron and sulfur to the apo ISU, with the latter delivered through an IscS-mediated reaction. The identity of the iron donor has thus far not been established. In this paper we demonstrate human frataxin to bind from six to seven iron ions. Iron binding to frataxin has been quantitated by iron-dependent fluorescence measurements [K(D)(Fe(3+)) approximately 11.7 microM; (K(D)(Fe(2+)) approximately 55.0 microM] and isothermal titration calorimetry (ITC) [K(D)(Fe(3+)) approximately 10.2 microM]. Enthalpies and entropies for ferric ion binding were determined from calorimetric measurements. Both fluorescence (K(D) 0.45 microM) and ITC measurements (K(D) 0.15 microM) demonstrate holo frataxin to form a complex with ISU with sub-micromolar binding affinities. Significantly, apo frataxin does not bind to ISU, suggesting an important role for iron in cross-linking the two proteins and/or stabilizing the structure of frataxin that is recognized by ISU. Holo frataxin is also shown to mediate the transfer of iron from holo frataxin to nucleation sites for [2Fe-2S] cluster formation on ISU. We have demonstrated elsewhere [J. Am. Chem. Soc. 2002, 124, 8774-8775] that this iron-bound form of ISU is viable for assembly of holo ISU, either by subsequent addition of sulfide or by NifS-mediated sulfur delivery. Provision of holo frataxin and inorganic sulfide is sufficient for cluster assembly in up to 70% yield. With NifS as a sulfur donor, yields in excess of 70% of holo ISU were obtained. Both UV-vis and CD spectroscopic characteristics were found to be consistent with those of previously characterized ISU proteins. The time course for cluster assembly was monitored from the 456 nm absorbance of holo ISU formed during the [2Fe-2S] cluster assembly reaction. A kinetic rate constant k(obs) approximately 0.075 min(-)(1) was determined with 100 microM ISU, 2.4 mM Na(2)S, and 40 microM holo frataxin in 50 mM Tris-HCl (pH 7.5) with 4.3 mM DTT. Similar rates were obtained for NifS-mediated sulfur delivery, consistent with iron release from frataxin as a rate-limiting step in the cluster assembly reaction.     

Iron-sulfur cluster biosynthesis: characterization of iron nucleation sites for assembly of the [2Fe-2S]2+ cluster core in IscU proteins

ISU (eukaryotes) and IscU (prokaryotes) are a homologous family of proteins that appear to provide a platform for assembly of [2Fe-2S] centers prior to delivery to a target apoprotein. The intermediate [2Fe-2S] IscU-bound cluster is formed by delivery of iron and sulfur to the apo-IscU, with the latter delivered through an IscS-mediated reaction. The identity of the iron donor is not yet established. In this report we characterize iron-binding sites on IscU that appear to nucleate [2Fe-2S] cluster assembly. This iron-bound form of IscU is shown to be viable for subsequent IscS-mediated assembly of holo-IscU. Following on recent reports, we demonstrate the persulfide form of IscU to be a dead-end complex that is incapable of forming holoprotein after addition of ferrous or ferric ion. The latter observation reflects the low binding affinity of persulfido IscU for iron ion.     

Nature's polyoxometalate chemistry: X-ray structure of the Mo storage protein loaded with discrete polynuclear Mo-O clusters

Some N(2)-fixing bacteria prolong the functionality of nitrogenase in molybdenum starvation by a special Mo storage protein (MoSto) that can store more than 100 Mo atoms. The presented 1.6 ? X-ray structure of MoSto from Azotobacter vinelandii reveals various discrete polyoxomolybdate clusters, three covalently and three noncovalently bound Mo(8), three Mo(5-7), and one Mo(3) clusters, and several low occupied, so far undefinable clusters, which are embedded in specific pockets inside a locked cage-shaped (αβ)(3) protein complex. The structurally identical Mo(8) clusters (three layers of two, four, and two MoO(n) octahedra) are distinguishable from the [Mo(8)O(26)](4-) cluster formed in acidic solutions by two displaced MoO(n) octahedra implicating three kinetically labile terminal ligands. Stabilization in the covalent Mo(8) cluster is achieved by Mo bonding to Hisα156-N(ε2) and Gluα129-O(ε1). The absence of covalent protein interactions in the noncovalent Mo(8) cluster is compensated by a more extended hydrogen-bond network involving three pronounced histidines. One displaced MoO(n) octahedron might serve as nucleation site for an inhomogeneous Mo(5-7) cluster largely surrounded by bulk solvent. In the Mo(3) cluster located on the 3-fold axis, the three accurately positioned His140-N(ε2) atoms of the α subunits coordinate to the Mo atoms. The formed polyoxomolybdate clusters of MoSto, not detectable in bulk solvent, are the result of an interplay between self- and protein-driven assembly processes that unite inorganic supramolecular and protein chemistry in a host-guest system. Template, nucleation/protection, and catalyst functions of the polypeptide as well as perspectives for designing new clusters are discussed.     

Facile assembly of a Cu9 amido complex: a new tripodal ligand design that promotes transition metal cluster formation

A tripodal amido ligand with a central non-chelating phosphorus donor allows for the facile assembly of a pentane soluble organometallic copper cluster with a central copper atom surrounded by a nonplanar chain of eight copper atoms and two terminal amido-copper bonds.     

Assembly of the water-oxidizing complex in photosystem II

The efficient incorporation and assembly of calcium, chloride and manganese followed by photoactivation of the water-oxidizing complex (WOC) is a prerequisite for the unique water-splitting activity of photosystem II. This minireview summarizes the recent results on incorporation and storage of the inorganic cofactors, photoactivation of the WOC and assembly of the protein environment at the donor site of PSII in cyanobacteria with a special focus on the role of the Psb27 protein.     

A Histidine Residue and a Tetranuclear Cuprous-thiolate Cluster Dominate the Copper Loading Landscape of a Copper Storage Protein from Streptomyces lividans

The chemical basis for protecting organisms against the toxic effect imposed by excess cuprous ions is to constrain this through high-affinity binding sites that use cuprous-thiolate coordination chemistry. In bacteria, a family of cysteine rich four-helix bundle proteins utilise thiolate chemistry to bind up to 80 cuprous ions. These proteins have been termed copper storage proteins (Csp). The present study investigates cuprous ion loading to the Csp from Streptomyces lividans (SlCsp) using a combination of X-ray crystallography, site-directed mutagenesis and stopped-flow reaction kinetics with either aquatic cuprous ions or a chelating donor. We illustrate that at low cuprous ion concentrations, copper is loaded exclusively into an outer core region of SlCsp via one end of the four-helix bundle, facilitated by a set of three histidine residues. X-ray crystallography reveals the existence of polynuclear cuprous-thiolate clusters culminating in the assembly of a tetranuclear [Cu₄(μ₂-S-Cys)₄(Ν^δ1-His)] cluster in the outer core. As more cuprous ions are loaded, the cysteine lined inner core of SlCsp fills with cuprous ions but in a fluxional and dynamic manner with no evidence for the assembly of further intermediate polynuclear cuprous-thiolate clusters as observed in the outer core. Using site-directed mutagenesis a key role for His107 in the efficient loading of cuprous ions from a donor is established. A model of copper loading to SlCsp is proposed and discussed.     

Changing the ligation of the distal [4Fe4S] cluster in NiFe hydrogenase impairs inter- and intramolecular electron transfers

In NiFe hydrogenases, electrons are transferred from the active site to the redox partner via a chain of three Iron-Sulfur clusters, and the surface-exposed [4Fe4S] cluster has an unusual His(Cys)3 ligation. When this Histidine (H184 in Desulfovibrio fructosovorans) is changed into a cysteine or a glycine, a distal cubane is still assembled but the oxidative activity of the mutants is only 1.5 and 3% of that of the WT, respectively. We compared the activities of the WT and engineered enzymes for H2 oxidation, H+ reduction and H/D exchange, under various conditions: (i) either with the enzyme directly adsorbed onto an electrode or using soluble redox partners, and (ii) in the presence of exogenous ligands whose binding to the exposed Fe of H184G was expected to modulate the properties of the distal cluster. Protein film voltammetry proved particularly useful to unravel the effects of the mutations on inter and intramolecular electron transfer (ET). We demonstrate that changing the coordination of the distal cluster has no effect on cluster assembly, protein stability, active-site chemistry and proton transfer; however, it slows down the first-order rates of ET to and from the cluster. All-sulfur coordination is actually detrimental to ET, and intramolecular (uphill) ET is rate determining in the glycine variant. This demonstrates that although [4Fe4S] clusters are robust chemical constructs, the direct protein ligands play an essential role in imparting their ability to transfer electrons.     

Pyrazine-N,N′-dioxide/tetracyanoethylene electron donor-acceptor bonding and the effect of donor steric demand and symmetry on the cocrystal assembly

The synthesis of a sterically hindered pyrazine dioxide electron donor (2,5-diethyl-3,6-dimethylpyrazine-N,N′-dioxide) and its cocrystallization with tetracyanoethylene (TCNE) are reported. The resulting DA assembly in the cocrystal is compared to that observed for a smaller pyrazine-N-oxide donor. Increased steric demand and lower donor symmetry yield a new, more complex two-dimensional DA assembly with slightly longer DA bonds and higher TCNE content in the cocrystal.     

Biosynthesis of isoprenoids. purification and properties of IspG protein from Escherichia coli

[Structure: see text]. The IspG protein is known to catalyze the transformation of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate in the nonmevalonate pathway of isoprenoid biosynthesis. We have found that the apparent IspG activity in the cell extracts of recombinant Escherichia coli cells as observed by a radiochemical assay can be enhanced severalfold by coexpression of the isc operon which is involved in the assembly of iron-sulfur clusters. The recombinant protein was isolated by affinity chromatography under anaerobic conditions. With a mixture of flavodoxin, flavodoxin reductase, and NADPH as the reducing agent, stringent assay methods based on photometry or on 13C NMR detection of multiply 13C-labeled substrate/product ratios afforded catalytic activities greater than 60 nmol mg(-1) min(-1) for the protein "as isolated" (i.e., without reconstitution of any kind). Lower apparent activities were found using photoreduced deazaflavin as an artifactual electron donor, whereas dithionite was unable to serve as an artificial electron donor. The apparent Michaelis constant for 2-C-methyl-D-erythritol 2,4-cyclodiphosphate was 700 microM. The enzyme was inactivated by EDTA and could be reactivated by Mn2+. The pH optimum was at 9.0. The protein contained 2.4 iron ions and 4.4 sulfide ions per subunit. The replacement of any of the three conserved cysteine residues afforded mutant proteins which were devoid of catalytic activity and contained less than 6% of Fe2+ and less than 23% of S2- as compared to the wild-type protein. Sequence comparison indicates that putative IspG proteins of plants, the apicomplexan protozoan Plasmodium falciparum, and bacteria from the Bacteroidetes/Chlorobi group contain an insert of about 170-320 amino acid residues as compared with eubacterial enzymes.     

The Nitrogenase FeMo‐Cofactor Precursor Formed by NifB Protein: A Diamagnetic Cluster Containing Eight Iron Atoms

The biological activation of N₂ occurs at the FeMo‐cofactor, a 7Fe–9S–Mo–C–homocitrate cluster. FeMo‐cofactor formation involves assembly of a Fe_6–8–S_X–C core precursor, NifB‐co, which occurs on the NifB protein. Characterization of NifB‐co in NifB is complicated by the dynamic nature of the assembly process and the presence of a permanent [4Fe–4S] cluster associated with the radical SAM chemistry for generating the central carbide. We have used the physiological carrier protein, NifX, which has been proposed to bind NifB‐co and deliver it to the NifEN protein, upon which FeMo‐cofactor assembly is ultimately completed. Preparation of NifX in a fully NifB‐co‐loaded form provided an opportunity for Mössbauer analysis of NifB‐co. The results indicate that NifB‐co is a diamagnetic (S=0) 8‐Fe cluster, containing two spectroscopically distinct Fe sites that appear in a 3:1 ratio. DFT analysis of the ⁵⁷Fe electric hyperfine interactions deduced from the Mössbauer analysis suggests that NifB‐co is either a 4Fe²⁺–4Fe³⁺ or 6Fe²⁺–2Fe³⁺ cluster having valence‐delocalized states.     

Structural and functional diversity of glutaredoxins in yeast

Glutaredoxins are defined as thiol disulfide oxidoreductases that reduce disulfide bonds employing reduced glutathione as electron donor. They constitute a complex family of proteins with a diversity of enzymatic and functional properties. Thus, dithiol glutaredoxins are able to reduce disulfide bonds and deglutathionylate mixed disulfides between glutathione and cysteine protein residues. They could act regulating the redox state of sulfhydryl residues of specific proteins, while thioredoxins (another family of thiol disulfide oxidoreductases which employ NADPH as electron donor) would be the general sulfhydryl reductants. Some dithiol glutaredoxins such as human Grx2 form dimers bridged by one iron-sulfur cluster, which acts as a sensor of oxidative stress, therefore regulating the activity of the glutaredoxin. The ability to interact with iron-sulfur clusters as ligands is also characteristic of monothiol glutaredoxins with a CGFS-type active site. These do not display thiol oxidoreductase activity, but have roles in iron homeostasis. The three members of this subfamily in Saccharomyces cerevisiae participate in the synthesis of the iron-sulfur clusters in mitochondria (Grx5), or in signalling the iron status inside the cell for regulation of iron uptake and intracellular iron relocalization (Grx3 and Grx4). Such a role in iron metabolism seems to be evolutionary conserved. Fungal cells also contain membrane-associated glutaredoxins structurally and enzymatically similar to dithiol glutaredoxins, which may act as redox regulators at the early stages of the protein secretory machinery.     

A simulation of key aspects of a primary process in natural photosynthesis by a Langmuir-Blodgett film assembly

An advanced Langmuir-Blodgett (LB) assembly designed to replicate the three key aspects (antenna, reaction center, and quinone pool) of a primary process in natural photosynthesis was successfully fabricated by alternate deposition of a mixed monolayer of an artificial reaction center and an antenna pigment and a pure bilayer of a second donor.     

Computational studies on optoelectronic and charge transfer properties of some perylene‐based donor‐π‐acceptor systems for dye sensitized solar cell applications

We report DFT studies on some perylene‐based dyes for their electron transfer properties in solar cell applications. The study involves modeling of different donor‐π‐acceptor type sensitizers, with perylene as the donor, furan/pyrrole/thiophene as the π‐bridge and cyanoacrylic group as the acceptor. The effect of different π‐bridges and various substituents on the perylene donor was evaluated in terms of opto‐electronic and photovoltaic parameters such as HOMO‐LUMO energy gap, λ_max, light harvesting efficiency(LHE), electron injection efficiency (Ø_inject), excited state dye potential (E^dye*), reorganization energy(λ), and free energy of dye regeneration (). The effect of various substituents on the dye–I₂ interaction and hence recombination process was also evaluated. We found that the furan‐based dimethylamine derivative exhibits a better balance of the various optical and photovoltaic properties. Finally, we evaluated the overall opto‐electronic and transport parameters of the TiO₂‐dye assembly after anchoring the dyes on the model TiO₂ cluster assembly.     

Dynamics of vesicle self-assembly and dissolution

The dynamics of membranes is studied on the basis of a particle-based meshless surface model, which was introduced earlier [Phys. Rev. E 73, 021903 (2006)]. The model describes fluid membranes with bending energy and-in the case of membranes with boundaries-line tension. The effects of hydrodynamic interactions are investigated by comparing Brownian dynamics with a particle-based mesoscale solvent simulation (multiparticle collision dynamics). Particles self-assemble into vesicles via disk-shaped membrane patches. The time evolution of assembly is found to consist of three steps: particle assembly into discoidal clusters, aggregation of clusters into larger membrane patches, and finally vesicle formation. The time dependence of the cluster distribution and the mean cluster size is evaluated and compared with the predictions of Smoluchowski rate equations. On the other hand, when the line tension is suddenly decreased (or the temperature is increased), vesicles dissolve via pore formation in the membrane. Hydrodynamic interactions are found to speed up the dynamics in both cases. Furthermore, hydrodynamics makes vesicle more spherical in the membrane-closure process.     

Lyngbyatoxin biosynthesis: sequence of biosynthetic gene cluster and identification of a novel aromatic prenyltransferase

The lyngbyatoxins are potent skin irritants produced by Lyngbya majuscula and cause a condition known as "Swimmer's Itch" off Honolulu, HI. Reported is the molecular cloning of the lyngbyatoxin (ltx) biosynthetic gene cluster from L. majuscula using a strategy based on its predicted nonribosomal peptide synthetase (NRPS) assembly. The biosynthetic gene cluster spans 11.3 kilobase pairs and encodes for a two-module NRPS (LtxA), a P450 monooxygenase (LtxB), an aromatic prenyltransferase (LtxC), and an oxidase/reductase protein (LtxD). LtxC was heterologously produced and purified from E. coli and shown to catalyze the transfer of a geranyl group to (-)-indolactam V as the final step in the biosynthesis of lyngbyatoxin A.     

Coordination Chemistry of Lanthanides at “High” pH: Synthesis and Structure of the Pentadecanuclear Complex of Europium(III) with Tyrosine

A five-coordinate chloride ion is believed to template the assembly of a pentadecanuclear lanthanide complex of europium(III ). This cluster (see picture) has been prepared by coordination of europium(III ) perchlorate with tyrosine at about pH 6. Single crystal X-ray analysis established an unprecedented structure in which 15 constituent europium(III ) ions are organized into three parallel pentagonal layers.     

Coordination-driven self-assembly of cavity-cored multiple crown ether derivatives and poly[2]pseudorotaxanes

The synthesis of a new 120 degree diplatinum(II) acceptor unit and the self-assembly of a series of two-dimensional metallacyclic polypseudorotaxanes that utilize both metal-ligand and crown ether-dialkylammonium noncovalent interactions are described. Judiciously combining complementary diplatinum(II) acceptors with bispyridyl donor building blocks, with an acceptor and/or donor possessing a pendant dibenzo[24]crown-8 (DB24C8) moiety, allows for the formation of three new rhomboidal bis-DB24C8, one new hexagonal tris-DB24C8, and four new hexakis-DB24C8 metallacyclic polygons in quantitative yields. The size and shape of each assembly, as well as the location and stoichiometry of the DB24C8 macrocycle, can be precisely controlled. Each polygon is able to complex two, three, or six dibenzylammonium ions without disrupting the underlying metallacyclic polygons, thus producing eight different poly[2]pseudorotaxanes and demonstrating the utility and scope of this orthogonal self-assembly technique. The assemblies are characterized with one-dimensional multinuclear ((1)H and (31)P) and two-dimensional ((1)H-(1)H COSY and NOESY) NMR spectroscopy as well as mass spectrometry (ESI-MS). Further analysis of the size and shape of each assembly is obtained through molecular force-field simulations. (1)H NMR titration experiments are used to establish thermodynamic binding constants and poly[2]pseudorotaxane/dibenzylammonium stoichiometries. Factors influencing the efficiency of poly[2]pseudorotaxane formation are discussed.     

Self‐Assembly and Gelation of Poly(aryl ether) Dendrons Containing Hydrazide Units: Factors Controlling the Formation of Helical Structures

Self‐assembly of AB₂ and AB₃ type low molecular weight poly(aryl ether) dendrons that contain hydrazide units were used to investigate mechanistic aspects of helical structure formation during self‐assembly. The results suggest that there are three important aspects that control helical structure formation in such systems with acyl hydrazide/hydrazone linkage: i) J‐type aggregation, ii) the hydrogen‐bond donor/acceptor ability of the solvent, and iii) the dielectric constant of the solvent. The monomer units self‐assemble to form dimer structures through hydrogen‐bonding and further assembly of the hydrogen‐bonded dimers leads to macroscopic chirality in the present case. Dimer formation was confirmed by NMR spectroscopy and by mass spectrometry. The self‐assembly in the system was driven by hydrogen‐bonding and π–π stacking interactions. The morphology of the aggregates formed was examined by scanning electron microscopy, and the analysis suggests that aprotic solvent systems facilitate helical fibre formation, whereas introduction of protic solvents results in the formation of flat ribbons. This detailed mechanistic study suggests that the self‐assembly follows a nucleation–elongation model to form helical structures, rather than the isodesmic model.     

S K-edge XAS and DFT calculations on SAM dependent pyruvate formate-lyase activating enzyme: nature of interaction between the Fe4S4 cluster and SAM and its role in reactivity

S K-edge X-ray absorption spectroscopy on the resting oxidized and the S-adenosyl-l-methionine (SAM) bound forms of pyruvate formate-lyase activating enzyme are reported. The data show an increase in pre-edge intensity, which is due to additional contributions from sulfide and thiolate of the Fe(4)S(4) cluster into the C-S σ* orbital. This experimentally demonstrates that there is a backbonding interaction between the Fe(4)S(4) cluster and C-S σ* orbitals of SAM in this inner sphere complex. DFT calculations that reproduce the data indicate that this backbonding is enhanced in the reduced form and that this configurational interaction between the donor and acceptor orbitals facilitates the electron transfer from the cluster to the SAM, which otherwise has a large outer sphere electron transfer barrier. The energy of the reductive cleavage of the C-S bond is sensitive to the dielectric of the protein in the immediate vicinity of the site as a high dielectric stabilizes the more charge separated reactant increasing the reaction barrier. This may provide a mechanism for generation of the 5'-deoxyadenosyl radical upon substrate binding.