TtcA a new tRNA-thioltransferase with an Fe-S cluster

International audienceTtcA catalyzes the post-transcriptional thiolation of cytosine 32 in some tRNAs. The enzyme from Es-cherichia coli was homologously overexpressed in E. coli. The purified enzyme is a dimer containing an iron–sulfur cluster and displays activity in in vitro assays. The type and properties of the cluster were investigated using a combination of UV-visible absorption , EPR and M ¨ ossbauer spectroscopy, as well as by site-directed mutagenesis. These studies demonstrated that the TtcA enzyme contains a redox-active and oxygen-sensitive [4Fe-4S] cluster, chelated by only three cysteine residues and absolutely essential for activity. TtcA is unique tRNA-thiolating enzyme using an iron–sulfur cluster for catalyzing a non-redox reaction

Formation of high-valent iron-oxo species in superoxide reductase: characterization by resonance Raman spectroscopy.

International audienceSuperoxide reductase (SOR), a non-heme mononuclear iron protein that is involved in superoxide detoxification in microorganisms, can be used as an unprecedented model to study the mechanisms of O2 activation and of the formation of high-valent iron-oxo species in metalloenzymes. By using resonance Raman spectroscopy, it was shown that the mutation of two residues in the second coordination sphere of the SOR iron active site, K48 and I118, led to the formation of a high-valent iron-oxo species when the mutant proteins were reacted with H2O2. These data demonstrate that these residues in the second coordination sphere tightly control the evolution and the cleavage of the O-O bond of the ferric iron hydroperoxide intermediate that is formed in the SOR active site

Elucidating dramatic ligand effects on SET processes: iron hydride versus Iron borohydride catalyzed reductive radical cyclization of unsaturated organic halides

An iron(II) borohydride complex ([(η1-H3BH)FeCl(NCCH3)4]) is employed as the precatalyst in iron-catalyzed radical cyclizations of unsaturated organic halides in the presence of NaBH4. Mechanistic investigations have established that the ligand bound to the metal center (acetonitrile versus ethylenebis(diphenylphosphine) (dppe)) plays a crucial role in the structure and reactivity of the active anionic iron(I) hydride ([HFeCl(dppe)2]−) and borohydride ([(η1-H3BH)FeCl(NCCH3)4]−) with unsaturated haloacetals. This work provides new insights into iron(I) hydride and borohydride species and their potential implication in single-electron processes

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

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

The Role of CyaY in Iron Sulfur Cluster Assembly on the E. coli IscU Scaffold Protein

Progress in understanding the mechanism underlying the enzymatic formation of iron-sulfur clusters is difficult since it involves a complex reaction and a multi-component system. By exploiting different spectroscopies, we characterize the effect on the enzymatic kinetics of cluster formation of CyaY, the bacterial ortholog of frataxin, on cluster formation on the scaffold protein IscU. Frataxin/CyaY is a highly conserved protein implicated in an incurable ataxia in humans. Previous studies had suggested a role of CyaY as an inhibitor of iron sulfur cluster formation. Similar studies on the eukaryotic proteins have however suggested for frataxin a role as an activator. Our studies independently confirm that CyaY slows down the reaction and shed new light onto the mechanism by which CyaY works. We observe that the presence of CyaY does not alter the relative ratio between [2Fe2S]2+ and [4Fe4S]2+ but directly affects enzymatic activity

Étude RMN du système de spins quantiques CuHpCl

We present an NMR study of magnetic properties of Cu2(C5H12N2)2Cl4 (CuHpCl). This system of quantum spins ½ has been considered for a long time as an experimental realization of a spin ladder system. Its phase diagram under magnetic field is very rich, in particular a spin liquid ground state in zero field and two quantum phase transitions at Hc1=7.5 Tesla and Hc2 = 13 Tesla. The first goal of this study was a clarification of some specific parts of the phase diagram, in particular the low temperature regime between Hc1 and Hc2, where tridimensional magnetic order sets in. Through the study of a compound deuterated on specific hydrogen bonds, we observed the commensurate nature of this ordered phase. The second-order phase transition was studied as a function of H and T. The main result of this study highlights the apparition at low temperature of a staggered transverse magnetisation, due to antisymetric magnetic interactions, like observed in other spin liquid compounds like NENP or SrCu2(BO3)2. This « anomalous » magnetisation appears for fieds Hc1 and persist for H> Hc2. CuHpCl is the first compound where the coexistence of a staggered magnetisation with 3D magnetic order can be observed. We also observed by torque magnetometry anomalous features due to this staggered magnetisation. Last, in order to understand the magnetic exchange pathways in CuHpCl, we also undertook NMR and torque measurements of methylated variants of CuHpCl.Cette thèse est consacrée à l'étude par résonance magnétique nucléaire (RMN) des propriétés magnétiques du composé Cu2(C5H12N2)2Cl4 (CuHpCl). Ce système de spins quantiques 1/2 a longtemps été considéré comme une réalisation expérimentale d'un système d'échelles de spins. Son diagramme de phase en fonction du champ magnétique est très riche, avec en particulier un état fondamental liquide de spin en champ nul et deux transition de phase quantique aux champ Hc1=7.5 Tesla et Hc2 = 13 Tesla. Le but premier de cette étude est une clarification de certaines zones de ce diagramme de phases, en particulier la phase basse température comprise entre Hc1 et Hc2 qui présente un ordre magnétique tridimensionnel. A travers l'étude d'un composé spécifiquement deutéré, nous avons montré la nature commensurable de cette phase ordonnée et étudié la nature de la transition en fonction de H et de T. Mais l'apport principal de cette étude, est la mise en évidence de l'apparition à basse température d'une aimantation transverse alternée due à des interactions anti-symétriques, similaire à celle observée dans d'autres composés liquide de spins tels que NENP ou SrCu2(BO3)2. Cette aimantation apparaît pour des champs inférieurs à Hc1 et persiste pour H> Hc2. CuHpCl semble être le seul composé à présenter la coexistence de cette aimantation alternée et d'une phase magnétique 3D. Cette aimantation alternée est également observée dans les mesures d'aimantations par couple menées sur ce composé. Enfin, dans le but de comprendre les chemins d'échanges dans CuHpCl, nous avons entrepris un étude préliminaire (RMN et mesures d'aimantation) de composés dérivés (méthylés) de CuHpCl

Mössbauer spectroscopy.

International audienceGiven its ability to detect all iron centers, to identify their electronic structures, and to quantify the ratios of the different iron forms present in a sample, many researchers turn to Mössbauer spectroscopy when wanting to address structural and mechanistic questions involving iron proteins. Yet, this technique applied to biochemistry is provided by only a few dedicated teams in the world. Technical difficulties ranging from sample preparation to data analysis and interpretation make necessary the collaboration between biochemists and Mössbauer spectroscopists. This chapter will be confined to iron Mössbauer. It will focus on giving biologists and biochemists the keys to understand what essential information Mössbauer spectroscopy can yield, and how to engage in successful collaborations with spectroscopists. After introducing the basic principles of a Mössbauer experiment, we will describe first how to prepare a suitable Mössbauer sample, then how this technique is applied to the identification of different iron species inside proteins

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

International audienceFe/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, $^{57}$Fe-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

Structural modeling of iron halogenases: synthesis and reactivity of halide-iron(iv)-oxo compounds.

International audienceA structural synthetic model of the iron(iv)-oxo-halide active species of non-heme iron dependent halogenases is reported. Compounds with general formula [Fe(IV)(O)(X)(Pytacn)](+) (1-X, X = Cl, Br) have been prepared and characterized spectroscopically and chemically with regard to their oxidizing ability. 1-X performs hydrogen-atom abstraction of C-H bonds at reaction rates 2-3 times faster than the corresponding solvato dicationic species, thus modelling the first step in C-H functionalization taking place in natural halogenation