Multimodal Spectroscopic Analysis of the Fe-S Clusters of the as-Isolated Escherichia coli SufBC2D Complex.

Iron-sulfur (Fe-S) clusters are essential inorganic cofactors dedicated to a wide range of biological functions, including electron transfer and catalysis. Specialized multiprotein machineries present in all types of organisms support their biosynthesis. These machineries encompass a scaffold protein, on which Fe-S clusters are assembled before being transferred to cellular targets. Here, we describe the first characterization of the native Fe-S cluster of the anaerobically purified SufBC2D scaffold from Escherichia coli by XAS and Mössbauer, UV-visible absorption, and EPR spectroscopies. Interestingly, we propose that SufBC2D harbors two iron-sulfur-containing species, a [2Fe-2S] cluster and an as-yet unidentified species. Mutagenesis and biochemistry were used to propose amino acid ligands for the [2Fe-2S] cluster, supporting the hypothesis that both SufB and SufD are involved in the Fe-S cluster ligation. The [2Fe-2S] cluster can be transferred to ferredoxin in agreement with the SufBC2D scaffold function. These results are discussed in the context of Fe-S cluster biogenesis.

Regulations of mitoNEET by the key redox homeostasis molecule glutathione.

Human mitoNEET (mNT) and CISD2 are two NEET proteins characterized by an atypical [2Fe-2S] cluster coordination involving three cysteines and one histidine. They act as redox switches with an active state linked to the oxidation of their cluster. In the present study, we show that reduced glutathione but also free thiol-containing molecules such as ß-mercaptoethanol can induce a loss of the mNT cluster under aerobic conditions, while CISD2 cluster appears more resistant. This disassembly occurs through a radical-based mechanism as previously observed with the bacterial SoxR. Interestingly, adding cysteine prevents glutathione-induced cluster loss. At low pH, glutathione can bind mNT in the vicinity of the cluster. These results suggest a potential new regulation mechanism of mNT activity by glutathione, an essential actor of the intracellular redox state.

Evidence for [2Fe-2S]2+ and Linear [3Fe-4S]1+ Clusters in a Unique Family of Glycine/Cysteine-Rich Fe-S Proteins from Megavirinae Giant Viruses.

We have discovered a protein with an amino acid composition exceptionally rich in glycine and cysteine residues in the giant virus mimivirus. This small 6 kDa protein is among the most abundant proteins in the icosahedral 0.75 µm viral particles; it has no predicted function but is probably essential for infection. The aerobically purified red-brownish protein overproduced inEscherichia coli contained both iron and inorganic sulfide. UV/vis, EPR, and Mössbauer studies revealed that the viral protein, coined GciS, accommodated two distinct Fe-S clusters: a diamagnetic S = 0 [2Fe-2S]2+ cluster and a paramagnetic S = 5/2 linear [3Fe-4S]1+ cluster, a geometry rarely stabilized in native proteins. Orthologs of mimivirus GciS were identified within all clades of Megavirinae, a Mimiviridae subfamily infecting Acanthamoeba, including the distantly related tupanviruses, and displayed the same spectroscopic features. Thus, these glycine/cysteine-rich proteins form a new family of viral Fe-S proteins sharing unique Fe-S cluster binding properties.

A complete biomimetic iron-sulfur cubane redox series.

Synthetic iron-sulfur cubanes are models for biological cofactors, which are essential to delineate oxidation states in the more complex enzymatic systems. However, a complete series of [Fe4S4]n complexes spanning all redox states accessible by 1-electron transformations of the individual iron atoms (n = 0-4+) has never been prepared, deterring the methodical comparison of structure and spectroscopic signature. Here, we demonstrate that the use of a bulky arylthiolate ligand promoting the encapsulation of alkali-metal cations in the vicinity of the cubane enables the synthesis of such a series. Characterization by EPR, 57Fe Mössbauer spectroscopy, UV-visible electronic absorption, variable-temperature X-ray diffraction analysis, and cyclic voltammetry reveals key trends for the geometry of the Fe4S4 core as well as for the Mössbauer isomer shift, which both correlate systematically with oxidation state. Furthermore, we confirm the S = 4 electronic ground state of the most reduced member of the series, [Fe4S4]0, and provide electrochemical evidence that it is accessible within 0.82 V from the [Fe4S4]2+ state, highlighting its relevance as a mimic of the nitrogenase iron protein cluster.

The Ruthenium Nitrosyl Moiety in Clusters: Trinuclear Linear µ-Hydroxido Magnesium(II)-Diruthenium(II), µ3-Oxido Trinuclear Diiron(III)-Ruthenium(II), and Tetranuclear µ4-Oxido Trigallium(III)-Ruthenium(II) Complexes.

The ruthenium nitrosyl moiety, {RuNO}6, is important as a potential releasing agent of nitric oxide and is of inherent interest in coordination chemistry. Typically, {RuNO}6 is found in mononuclear complexes. Herein we describe the synthesis and characterization of several multimetal cluster complexes that contain this unit. Specifically, the heterotrinuclear µ3-oxido clusters [Fe2RuCl4(µ3-O)(µ-OMe)(µ-pz)2(NO)(Hpz)2] (6) and [Fe2RuCl3(µ3-O)(µ-OMe)(µ-pz)3(MeOH)(NO)(Hpz)][Fe2RuCl3(µ3-O)(µ-OMe)(µ-pz)3(DMF)(NO)(Hpz)] (7·MeOH·2H2O) and the heterotetranuclear µ4-oxido complex [Ga3RuCl3(µ4-O)(µ-OMe)3(µ-pz)4(NO)] (8) were prepared from trans-[Ru(OH)(NO)(Hpz)4]Cl2 (5), which itself was prepared via acidic hydrolysis of the linear heterotrinuclear complex {[Ru(µ-OH)(µ-pz)2(pz)(NO)(Hpz)]2Mg} (4). Complex 4 was synthesized from the mononuclear Ru complexes (H2pz)[trans-RuCl4(Hpz)2] (1), trans-[RuCl2(Hpz)4]Cl (2), and trans-[RuCl2(Hpz)4] (3). The new compounds 4-8 were all characterized by elemental analysis, ESI mass spectrometry, IR, UV-vis, and 1H NMR spectroscopy, and single-crystal X-ray diffraction, with complexes 6 and 7 being characterized also by temperature-dependent magnetic susceptibility measurements and Mössbauer spectroscopy. Magnetometry indicated a strong antiferromagnetic interaction between paramagnetic centers in 6 and 7. The ability of 4 and 6-8 to form linkage isomers and release NO upon irradiation in the solid state was investigated by IR spectroscopy. A theoretical investigation of the electronic structure of 6 by DFT and ab initio CASSCF/NEVPT2 calculations indicated a redox-noninnocent behavior of the NO ancillary ligand in 6, which was also manifested in TD-DFT calculations of its electronic absorption spectrum. The electronic structure of 6 was also studied by an X-ray charge density analysis.

Mixed Valence (µ-Phenoxido) FeII FeIII and FeIII FeIV Compounds: Electron and Proton Transfers.

Mixed-valence non-heme diiron centers are present at the active sites of a few enzymes and confer them interesting reactivities with the two ions acting in concert. Related (µ-phenoxido)diiron complexes have been developed as enzyme mimics. They exhibit very rich spectroscopic properties enabling independent monitoring of each individual ion, which proved useful for mechanistic studies of catalytic hydrolysis and oxidation reactions. In our studies of such complexes, we observed that these compounds give rise to a wide variety of electron transfers (intervalence charge transfer), proton transfers (tautomerism), coupled electron and proton transfers (H. abstraction and PCET). In this minireview, we present and analyze the main results illustrating the latter aspects.

Intercepting a transient non-hemic pyridine N-oxide Fe(III) species involved in OAT reactions.

In the context of bioinspired OAT catalysis, we developed a tetradentate dipyrrinpyridine ligand, a hybrid of hemic and non-hemic models. The catalytic activity of the iron(III) derivative was investigated in the presence of iodosylbenzene. Unexpectedly, MS, EPR, Mössbauer, UV-visible and FTIR spectroscopic signatures supported by DFT calculations provide convincing evidence for the involvement of a relevant FeIII-O-NPy active intermediate.

Mössbauer Spectroscopic and Computational Investigation of An Iron Cyclopentadienone Complex.

(Cyclopentadienone)iron carbonyl complexes have recently received particular attention for their use as catalysts in hydrogenation or transfer hydrogenation reactions including the N-alkylation of amines with alcohols. This is due to their easy synthesis from simple and cheap materials, air and water stabilities, and the crucial metal-ligand cooperation giving rise to unique catalytic properties. Here, we report a Mössbauer spectroscopic and computational investigation of such a complex and its corresponding activated species for dehydrogenation and hydrogenation reactions. This study affords a deeper understanding of the species formed by the reaction with Me3NO and their distribution upon the added amount of an oxidant.

In†Cellulo Mössbauer and EPR Studies Bring New Evidence to the Long-Standing Debate on Iron-Sulfur Cluster Binding in Human Anamorsin.

Human anamorsin is an iron-sulfur (Fe-S)-cluster-binding protein acting as an electron donor in the early steps of cytosolic iron-sulfur protein biogenesis. Human anamorsin belongs to the eukaryotic CIAPIN1 protein family and contains two highly conserved cysteine-rich motifs, each binding an Fe-S cluster. In vitro works by various groups have provided rather controversial results for the type of Fe-S clusters bound to the CIAPIN1 proteins. In order to unravel the knot on this topic, we used an in cellulo approach combining Mössbauer and EPR spectroscopies to characterize the iron-sulfur-cluster-bound form of human anamorsin. We found that the protein binds two [2Fe-2S] clusters at both its cysteine-rich motifs.

Iron Oxidation in Escherichia coli Bacterioferritin Ferroxidase Centre, a Site Designed to React Rapidly with H2 O2 but Slowly with O2.

Both O2 and H2 O2 can oxidize iron at the ferroxidase center (FC) of Escherichia coli bacterioferritin (EcBfr) but mechanistic details of the two reactions need clarification. UV/Vis, EPR, and Mössbauer spectroscopies have been used to follow the reactions when apo-EcBfr, pre-loaded anaerobically with Fe2+ , was exposed to O2 or H2 O2 . We show that O2 binds di-Fe2+ FC reversibly, two Fe2+ ions are oxidized in concert and a H2 O2 molecule is formed and released to the solution. This peroxide molecule further oxidizes another di-Fe2+ FC, at a rate circa 1000 faster than O2 , ensuring an overall 1:4 stoichiometry of iron oxidation by O2 . Initially formed Fe3+ can further react with H2 O2 (producing protein bound radicals) but relaxes within seconds to an H2 O2 -unreactive di-Fe3+ form. The data obtained suggest that the primary role of EcBfr in vivo may be to detoxify H2 O2 rather than sequester iron.

Iron Oxidation in Escherichia coli Bacterioferritin Ferroxidase Centre, a Site Designed to React Rapidly with H2O2 but Slowly with O2.

Both O2 and H2O2 can oxidize iron at the ferroxidase center (FC) of Escherichia coli bacterioferritin (EcBfr) but mechanistic details of the two reactions need clarification. UV/Vis, EPR, and Mössbauer spectroscopies have been used to follow the reactions when apo-EcBfr, pre-loaded anaerobically with Fe2+, was exposed to O2 or H2O2. We show that O2 binds di-Fe2+ FC reversibly, two Fe2+ ions are oxidized in concert and a H2O2 molecule is formed and released to the solution. This peroxide molecule further oxidizes another di-Fe2+ FC, at a rate circa 1000 faster than O2, ensuring an overall 1:4 stoichiometry of iron oxidation by O2. Initially formed Fe3+ can further react with H2O2 (producing protein bound radicals) but relaxes within seconds to an H2O2-unreactive di-Fe3+ form. The data obtained suggest that the primary role of EcBfr in vivo may be to detoxify H2O2 rather than sequester iron.

Bioinspired symmetrical and unsymmetrical diiron complexes for selective oxidation catalysis with hydrogen peroxide.

Two new symmetrical and unsymmetrical diiron(iii) complexes were synthesized and characterized by X-ray diffraction analysis, mass spectrometry, UV-visible and Mössbauer spectroscopies. They proved to be good catalysts for alkene and alkane oxidation reactions by H2O2 in acetonitrile solution, and interesting effects of both the nature and the symmetry of the complexes were observed on catalysis in the presence of water.

Single-Ion Magnetic Behaviour in an Iron(III) Porphyrin Complex: A Dichotomy Between High Spin and 5/2-3/2 Spin Admixture.

A mononuclear iron(III) porphyrin compound exhibiting unexpectedly slow magnetic relaxation, which is a characteristic of single-ion magnet behaviour, is reported. This behaviour originates from the close proximity (≈550 cm-1 ) of the intermediate-spin S=3/2 excited states to the high-spin S=5/2 ground state. More quantitatively, although the ground state is mostly S=5/2, a spin-admixture model evidences a sizable contribution (≈15 %) of S=3/2 to the ground state, which as a consequence experiences large and positive axial anisotropy (D=+19.2 cm-1 ). Frequency-domain EPR spectroscopy allowed the mS = |±1/2⟩→|±3/2⟩ transitions to be directly accessed, and thus the very large zero-field splitting in this 3d5 system to be unambiguously measured. Other experimental results including magnetisation, Mössbauer, and field-domain EPR studies are consistent with this model, which is also supported by theoretical calculations.

Evolution of Ate-Organoiron(II) Species towards Lower Oxidation States: Role of the Steric and Electronic Factors.

Ate-iron(II) species such as [Ar3 FeII ]- (Ar=aryl) are key intermediates in Fe-catalyzed couplings between aryl nucleophiles and organic electrophiles. They can be active species in the catalytic cycle, or lead to Fe0 and FeI oxidation states, which can themselves be catalytically active or lead to unwished organic byproducts. Analysis of the reactivity of the intermediates obtained by step-by-step displacement of the mesityl groups in high-spin [Mes3 FeII ]- by less hindered phenyl ligands was performed, and uncovered the crucial role of both steric and electronic parameters in the formation of the Fe0 and FeI oxidation states. The formation of quaternized [Ar4 FeII MgBr(THF)]- intermediates allows the bielectronic reductive elimination energy required for the formation of Fe0 to be reduced. Similarly, the small steric pressure of the aryl groups in [Ar3 FeII ]- enables the formation of aryl-bridged [{FeII (Ar)2 }2 (µ-Ar)2 ]2- species, which afford the FeI oxidation state by bimetallic reductive elimination. These results are supported by 1 H NMR, EPR, and 57 Fe Mössbauer spectroscopies, as well as by DFT calculations.

Octahedral iron(iv)-tosylimido complexes exhibiting single electron-oxidation reactivity.

High valent iron species are very reactive molecules involved in oxidation reactions of relevance to biology and chemical synthesis. Herein we describe iron(iv)-tosylimido complexes [FeIV(NTs)(MePy2tacn)](OTf)2 (1(IV)[double bond, length as m-dash]NTs) and [FeIV(NTs)(Me2(CHPy2)tacn)](OTf)2 (2(IV)[double bond, length as m-dash]NTs), (MePy2tacn = N-methyl-N,N-bis(2-picolyl)-1,4,7-triazacyclononane, and Me2(CHPy2)tacn = 1-(di(2-pyridyl)methyl)-4,7-dimethyl-1,4,7-triazacyclononane, Ts = Tosyl). 1(IV)[double bond, length as m-dash]NTs and 2(IV)[double bond, length as m-dash]NTs are rare examples of octahedral iron(iv)-imido complexes and are isoelectronic analogues of the recently described iron(iv)-oxo complexes [FeIV(O)(L)]2+ (L = MePy2tacn and Me2(CHPy2)tacn, respectively). 1(IV)[double bond, length as m-dash]NTs and 2(IV)[double bond, length as m-dash]NTs are metastable and have been spectroscopically characterized by HR-MS, UV-vis, 1H-NMR, resonance Raman, Mössbauer, and X-ray absorption (XAS) spectroscopy as well as by DFT computational methods. Ferric complexes [FeIII(HNTs)(L)]2+, 1(III)-NHTs (L = MePy2tacn) and 2(III)-NHTs (L = Me2(CHPy2)tacn) have been isolated after the decay of 1(IV)[double bond, length as m-dash]NTs and 2(IV)[double bond, length as m-dash]NTs in solution, spectroscopically characterized, and the molecular structure of [FeIII(HNTs)(MePy2tacn)](SbF6)2 determined by single crystal X-ray diffraction. Reaction of 1(IV)[double bond, length as m-dash]NTs and 2(IV)[double bond, length as m-dash]NTs with different p-substituted thioanisoles results in the transfer of the tosylimido moiety to the sulphur atom producing sulfilimine products. In these reactions, 1(IV)[double bond, length as m-dash]NTs and 2(IV)[double bond, length as m-dash]NTs behave as single electron oxidants and Hammett analyses of reaction rates evidence that tosylimido transfer is more sensitive than oxo transfer to charge effects. In addition, reaction of 1(IV)[double bond, length as m-dash]NTs and 2(IV)[double bond, length as m-dash]NTs with hydrocarbons containing weak C-H bonds results in the formation of 1(III)-NHTs and 2(III)-NHTs respectively, along with the oxidized substrate. Kinetic analyses indicate that reactions proceed via a mechanistically unusual HAT reaction, where an association complex precedes hydrogen abstraction.

Selective C-H halogenation over hydroxylation by non-heme iron(iv)-oxo.

Non-heme iron based halogenase enzymes promote selective halogenation of the sp3-C-H bond through iron(iv)-oxo-halide active species. During halogenation, competitive hydroxylation can be prevented completely in enzymatic systems. However, synthetic iron(iv)-oxo-halide intermediates often result in a mixture of halogenation and hydroxylation products. In this report, we have developed a new synthetic strategy by employing non-heme iron based complexes for selective sp3-C-H halogenation by overriding hydroxylation. A room temperature stable, iron(iv)-oxo complex, [Fe(2PyN2Q)(O)]2+ was directed for hydrogen atom abstraction (HAA) from aliphatic substrates and the iron(ii)-halide [FeII(2PyN2Q)(X)]+ (X, halogen) was exploited in conjunction to deliver the halogen atom to the ensuing carbon centered radical. Despite iron(iv)-oxo being an effective promoter of hydroxylation of aliphatic substrates, the perfect interplay of HAA and halogen atom transfer in this work leads to the halogenation product selectively by diverting the hydroxylation pathway. Experimental studies outline the mechanistic details of the iron(iv)-oxo mediated halogenation reactions. A kinetic isotope study between PhCH3 and C6D5CD3 showed a value of 13.5 that supports the initial HAA step as the RDS during halogenation. Successful implementation of this new strategy led to the establishment of a functional mimic of non-heme halogenase enzymes with an excellent selectivity for halogenation over hydroxylation. Detailed theoretical studies based on density functional methods reveal how the small difference in the ligand design leads to a large difference in the electronic structure of the [Fe(2PyN2Q)(O)]2+ species. Both experimental and computational studies suggest that the halide rebound process of the cage escaped radical with iron(iii)-halide is energetically favorable compared to iron(iii)-hydroxide and it brings in selective formation of halogenation products over hydroxylation.

Correction to: Contribution of Mössbauer spectroscopy to the investigation of Fe/S biogenesis.

The article "Contribution of Mössbauer spectroscopy to the investigation of Fe/S biogenesis", written by Ricardo Garcia­Serres, Martin Clémancey, Jean­Marc Latour, Geneviève Blondin was originally published electronically on the publisher's internet portal (currently SpringerLink) without open access.

Development of a Rubredoxin-Type Center Embedded in a de Dovo-Designed Three-Helix Bundle.

Protein design is a powerful tool for interrogating the basic requirements for the function of a metal site in a way that allows for the selective incorporation of elements that are important for function. Rubredoxins are small electron transfer proteins with a reduction potential centered near 0 mV (vs normal hydrogen electrode). All previous attempts to design a rubredoxin site have focused on incorporating the canonical CXXC motifs in addition to reproducing the peptide fold or using flexible loop regions to define the morphology of the site. We have produced a rubredoxin site in an utterly different fold, a three-helix bundle. The spectra of this construct mimic the ultraviolet-visible, Mössbauer, electron paramagnetic resonance, and magnetic circular dichroism spectra of native rubredoxin. Furthermore, the measured reduction potential suggests that this rubredoxin analogue could function similarly. Thus, we have shown that an α-helical scaffold sustains a rubredoxin site that can cycle with the desired potential between the Fe(II) and Fe(III) states and reproduces the spectroscopic characteristics of this electron transport protein without requiring the classic rubredoxin protein fold.

Contribution of Mössbauer spectroscopy to the investigation of Fe/S biogenesis.

Fe/S cluster biogenesis involves a complex machinery comprising several mitochondrial and cytosolic proteins. Fe/S cluster biosynthesis is closely intertwined with iron trafficking in the cell. Defects in Fe/S cluster elaboration result in severe diseases such as Friedreich ataxia. Deciphering this machinery is a challenge for the scientific community. Because iron is a key player, 57Fe-Mössbauer spectroscopy is especially appropriate for the characterization of Fe species and monitoring the iron distribution. This minireview intends to illustrate how Mössbauer spectroscopy contributes to unravel steps in Fe/S cluster biogenesis. Studies were performed on isolated proteins that may be present in multiple protein complexes. Since a few decades, Mössbauer spectroscopy was also performed on whole cells or on isolated compartments such as mitochondria and vacuoles, affording an overview of the iron trafficking. This minireview aims at presenting selected applications of 57Fe-Mössbauer spectroscopy to Fe/S cluster biogenesis.

ISCA1 is essential for mitochondrial Fe4S4 biogenesis in vivo.

Mammalian A-type proteins, ISCA1 and ISCA2, are evolutionarily conserved proteins involved in iron-sulfur cluster (Fe-S) biogenesis. Recently, it was shown that ISCA1 and ISCA2 form a heterocomplex that is implicated in the maturation of mitochondrial Fe4S4 proteins. Here we report that mouse ISCA1 and ISCA2 are Fe2S2-containing proteins that combine all features of Fe-S carrier proteins. We use biochemical, spectroscopic and in vivo approaches to demonstrate that despite forming a complex, ISCA1 and ISCA2 establish discrete interactions with components of the late Fe-S machinery. Surprisingly, knockdown experiments in mouse skeletal muscle and in primary cultures of neurons suggest that ISCA1, but not ISCA2, is required for mitochondrial Fe4S4 proteins biogenesis. Collectively, our data suggest that cellular processes with different requirements for ISCA1, ISCA2 and ISCA1-ISCA2 complex seem to exist.