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Find video protocols related to scientific articles indexed in Pubmed.
Cis/trans isomerizations in diiron complexes involving aniline or anilide ligands.
Inorg Chem
PUBLISHED: 09-25-2014
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We have recently reported a deprotonation-induced valence inversion within a phenoxido-bridged mixed-valent diiron(II,III) complex. The initial aniline coordinated to the Fe(II) site reacts with triethylamine, and the resulting complex contains an anilide ligand coordinated to the Fe(III) ion. The behavior of these complexes in acetonitrile is indeed more intricate. Owing to the very distinctive spectroscopic signatures of the complexes, the conjunction of NMR, Mössbauer, and UV-visible absorption spectroscopies allows one to evidence two isomerization reactions, one involving the aniline linked to Fe(II) and the other the anilide on Fe(III). Theoretical calculations sustain this conclusion. Aniline in the cis position versus the bridging phenoxide is shown to be the most stable isomer while the anilide trans to the phenoxido bridge is favored. The trans isomer of the aniline complex is more acidic than the cis one by 1 pKa unit. Isomerization of the anilide complex is 10 times faster than the analogous isomerization of the aniline complex. Both reactions are proposed to proceed through a unique mechanism. This is the first time that such isomerization reactions are evidenced in dinuclear complexes.
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Structural modeling of iron halogenases: synthesis and reactivity of halide-iron(IV)-oxo compounds.
Chem. Commun. (Camb.)
PUBLISHED: 08-05-2014
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A 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.
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The Diabetes Drug Target MitoNEET Governs a Novel Trafficking Pathway to Rebuild an Fe-S Cluster into Cytosolic Aconitase/Iron Regulatory Protein 1.
J. Biol. Chem.
PUBLISHED: 07-10-2014
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In eukaryotes, mitochondrial iron-sulfur cluster (ISC), export and cytosolic iron-sulfur cluster assembly (CIA) machineries carry out biogenesis of iron-sulfur (Fe-S) clusters, which are critical for multiple essential cellular pathways. However, little is known about their export out of mitochondria. Here we show that Fe-S assembly of mitoNEET, the first identified Fe-S protein anchored in the mitochondrial outer membrane, strictly depends on ISC machineries and not on the CIA or CIAPIN1. We identify a dedicated ISC/export pathway in which augmenter of liver regeneration, a mitochondrial Mia40-dependent protein, is specific to mitoNEET maturation. When inserted, the Fe-S cluster confers mitoNEET folding and stability in vitro and in vivo. The holo-form of mitoNEET is resistant to NO and H2O2 and is capable of repairing oxidatively damaged Fe-S of iron regulatory protein 1 (IRP1), a master regulator of cellular iron that has recently been involved in the mitochondrial iron supply. Therefore, our findings point to IRP1 as the missing link to explain the function of mitoNEET in the control of mitochondrial iron homeostasis.
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TtcA a new tRNA-thioltransferase with an Fe-S cluster.
Nucleic Acids Res.
PUBLISHED: 06-09-2014
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TtcA catalyzes the post-transcriptional thiolation of cytosine 32 in some tRNAs. The enzyme from Escherichia 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 Mössbauer 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.
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Efficient oxidation and destabilization of Zn(Cys)? zinc fingers by singlet oxygen.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 05-16-2014
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Singlet oxygen ((1)O2) plays an important role in oxidative stress in all types of organisms, most of them being able to mount a defense against this oxidant. Recently, zinc finger proteins have been proposed to be involved in its cellular detection but the molecular basis of this process still remains unknown. We have studied the reactivity of a Zn(Cys)4 zinc finger with (1)O2 by combinations of spectroscopic and analytical techniques, focusing on the products formed and the kinetics of the reaction. We report that the cysteines of this zinc finger are oxidized to sulfinates by (1)O2. The reaction of the ZnS4 core with (1)O2 is very fast and efficient with almost no physical quenching of (1)O2. A drastic (ca. five orders of magnitude) decrease of the Zn(2+) binding constant was observed upon oxidation. This suggests that the Zn(Cys)4 zinc finger proteins would release their Zn(2+) ion and unfold upon reaction with (1)O2 under cellular conditions and that zinc finger sites are likely targets for (1)O2.
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Mössbauer spectroscopy.
Methods Mol. Biol.
PUBLISHED: 03-19-2014
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Given 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.
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Triggering the generation of an iron(IV)-oxo compound and its reactivity toward sulfides by Ru(II) photocatalysis.
J. Am. Chem. Soc.
PUBLISHED: 03-13-2014
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The preparation of [Fe(IV)(O)(MePy2tacn)](2+) (2, MePy2tacn = N-methyl-N,N-bis(2-picolyl)-1,4,7-triazacyclononane) by reaction of [Fe(II)(MePy2tacn)(solvent)](2+) (1) and PhIO in CH3CN and its full characterization are described. This compound can also be prepared photochemically from its iron(II) precursor by irradiation at 447 nm in the presence of catalytic amounts of [Ru(II)(bpy)3](2+) as photosensitizer and a sacrificial electron acceptor (Na2S2O8). Remarkably, the rate of the reaction of the photochemically prepared compound 2 toward sulfides increases 150-fold under irradiation, and 2 is partially regenerated after the sulfide has been consumed; hence, the process can be repeated several times. The origin of this rate enhancement has been established by studying the reaction of chemically generated compound 2 with sulfides under different conditions, which demonstrated that both light and [Ru(II)(bpy)3](2+) are necessary for the observed increase in the reaction rate. A combination of nanosecond time-resolved absorption spectroscopy with laser pulse excitation and other mechanistic studies has led to the conclusion that an electron transfer mechanism is the most plausible explanation for the observed rate enhancement. According to this mechanism, the in-situ-generated [Ru(III)(bpy)3](3+) oxidizes the sulfide to form the corresponding radical cation, which is eventually oxidized by 2 to the corresponding sulfoxide.
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Highly reactive nonheme iron(III) iodosylarene complexes in alkane hydroxylation and sulfoxidation reactions.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 02-18-2014
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High-spin iron(III) iodosylarene complexes bearing an N-methylated cyclam ligand are synthesized and characterized using various spectroscopic methods. The nonheme high-spin iron(III) iodosylarene intermediates are highly reactive oxidants capable of activating strong C-H bonds of alkanes; the reactivity of the iron(III) iodosylarene intermediates is much greater than that of the corresponding iron(IV) oxo complex. The electrophilic character of the iron(III) iodosylarene complexes is demonstrated in sulfoxidation reactions.
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Peptide-based FeS4 complexes: the zinc ribbon fold is unsurpassed to stabilize both the FeII and FeIII states.
Dalton Trans
PUBLISHED: 01-21-2014
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Whereas Zn(Cys)4 zinc fingers exist with different protein folds, only the zinc ribbon fold is found in rubredoxin Fe(Cys)4 sites. To assess the significance of this observation, we have investigated the binding and stability of Fe(2+) and Fe(3+) ions by a set of four peptides designed to model Zn(Cys)4 zinc fingers with various folds, i.e. zinc ribbon, treble clef and a loosened zinc ribbon fold. All peptides were shown by means of UV-Vis and CD spectroscopies to form stable 1?:?1 Fe(II)/peptide complexes with binding constants higher than 10(7) M(-1) at pH 7. Their oxidation into Fe(III) complexes and the stability of the latter were compared. The UV-Vis absorption and CD spectroscopic properties of the Fe(II) and Fe(III) complexes were analysed with respect to the structures of the zinc analogues in order to get insight into the local arrangement of the Fe(Cys)4 core around the metal ion. The chemical stability of these complexes was rationalized according to the shielding from the solvent provided by the various peptide folds to the FeS4 core. In addition, we showed that whereas UV-visible spectra inform only on the FeS4, the information derived from the corresponding CD spectra extend to the C? orientation and the peptide fold. The results presented here demonstrate that while the zinc ribbon fold is not strictly required to obtain a Fe(Cys)4 site, it affords a drastically superior protection of the site toward external redox agents. This finding brings new clues to engineer stable and redox-active Fe(Cys)4 sites in de novo proteins.
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A diiron(III,IV) imido species very active in nitrene-transfer reactions.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 01-13-2014
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Metal-catalyzed nitrene transfer reactions arouse intense interest as clean and efficient procedures for amine synthesis. Efficient Rh- and Ru-based catalysts exist but Fe alternatives are actively pursued. However, reactive iron imido species can be very short-lived and getting evidence of their occurrence in efficient nitrene-transfer reactions is an important challenge. We recently reported that a diiron(III,II) complex is a very efficient nitrene-transfer catalyst to various substrates. We describe herein how, by combining desorption electrospray ionization mass spectrometry, quantitative chemical quench experiments, and DFT calculations, we obtained conclusive evidence for the occurrence of an {Fe(III) Fe(IV) ?NTosyl} intermediate that is very active in H-abstraction and nitrene-transfer reactions. DFT calculations revealed a strong radical character of the tosyl nitrogen atom in very low-lying electronic configurations of the Fe(IV) ion which are likely to confer its high reactivity.
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Escherichia coli RIC is able to donate iron to iron-sulfur clusters.
PLoS ONE
PUBLISHED: 01-01-2014
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Escherichia coli RIC (Repair of Iron Centers) is a diiron protein previously reported to be involved in the repair of iron-sulfur proteins damaged by oxidative or nitrosative stresses, and proposed to act as an iron donor. This possible role of RIC was now examined specifically by evaluating its ability to donate iron ions to apo-iron-sulfur proteins, determining the iron binding constants and assessing the lability of its iron ions. We show, by UV-visible, EPR and resonance Raman spectroscopies that RIC may participate in the synthesis of an iron-sulfur cluster in the apo-forms of the spinach ferredoxin and IscU when in the presence of the sulfide donating system IscS and L-cysteine. Iron binding assays allowed determining the as-isolated and fully reduced RIC dissociation constants for the ferric and ferrous iron of 10-27 M and 10-13 M, respectively. Mössbauer studies revealed that the RIC iron ions are labile, namely when the center is in the mixed-valence redox form as compared with the (?-oxo) diferric one. Altogether, these results suggest that RIC is capable of delivering iron for the formation of iron-sulfur clusters.
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An EPR/HYSCORE, Mössbauer, and resonance Raman study of the hydrogenase maturation enzyme HydF: a model for N-coordination to [4Fe-4S] clusters.
J. Biol. Inorg. Chem.
PUBLISHED: 08-20-2013
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The biosynthesis of the organometallic H cluster of [Fe-Fe] hydrogenase requires three accessory proteins, two of which (HydE and HydG) belong to the radical S-adenosylmethionine enzyme superfamily. The third, HydF, is an Fe-S protein with GTPase activity. The [4Fe-4S] cluster of HydF is bound to the polypeptide chain through only the three, conserved, cysteine residues present in the binding sequence motif CysXHisX(46-53)HisCysXXCys. However, the involvement of the two highly conserved histidines as a fourth ligand for the cluster coordination is controversial. In this study, we set out to characterize further the [4Fe-4S] cluster of HydF using Mössbauer, EPR, hyperfine sublevel correlation (HYSCORE), and resonance Raman spectroscopy in order to investigate the influence of nitrogen ligands on the spectroscopic properties of [4Fe-4S](2+/+) clusters. Our results show that Mössbauer, resonance Raman, and EPR spectroscopy are not able to readily discriminate between the imidazole-coordinated [4Fe-4S] cluster and the non-imidazole-bound [4Fe-4S] cluster with an exchangeable fourth ligand that is present in wild-type HydF. HYSCORE spectroscopy, on the other hand, detects the presence of an imidazole/histidine ligand on the cluster on the basis of the appearance of a specific spectral pattern in the strongly coupled region, with a coupling constant of approximately 6 MHz. We also discovered that a His-tagged version of HydF, with a hexahistidine tag at the N-terminus, has a [4Fe-4S] cluster coordinated by one histidine from the tag. This observation strongly indicates that care has to be taken in the analysis of data obtained on tagged forms of metalloproteins.
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A cyclic peptide-based redox-active model of rubredoxin.
Chem. Commun. (Camb.)
PUBLISHED: 03-06-2013
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A model of rubredoxin based on a cyclic peptide with a linear tail is presented. This model reproduces almost perfectly the fold, the spectroscopic characterizations and the redox activity of rubredoxins.
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On the design of zinc-finger models with cyclic peptides bearing a linear tail.
Chemistry
PUBLISHED: 02-21-2013
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Cyclic peptides with a linear tail (CPLT) have been successfully used to model two zinc fingers (ZFs) adopting the treble-clef- and loosened zinc-ribbon folds. In this article, we examine the factors that may influence the design of such ZF models: mutations in the sequence, size of the cycle, and size of the tail. For this purpose, several peptides derived from the CPLT-based models of the treble-clef- and loosened zinc-ribbon ZF were synthesized and studied. CPLT-based models appear to be robust toward mutations, accommodate various cycle sizes, and are sensible to the size of the linking region of the tail located between the cycle and the coordinating amino acids. Based on these criteria, we describe the design of a new CPLT-based model for the zinc-ribbon ZFs, LZR , and compare it to a linear analogue, LZR(lin) . The model complex Zn?LZR is able to fold correctly around the metal ion contrary to Zn?LZR(lin) , suggesting that CPLT-based models are more likely to yield structurally meaningful models of ZF sites than linear peptide models. Finally, we draw some rules that could allow the design of new CPLT-based metallopeptides with a controlled fold.
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Mammalian frataxin controls sulfur production and iron entry during de novo Fe4S4 cluster assembly.
J. Am. Chem. Soc.
PUBLISHED: 01-07-2013
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Iron-sulfur (Fe-S) cluster-containing proteins are essential components of cells. In eukaryotes, Fe-S clusters are synthesized by the mitochondrial iron-sulfur cluster (ISC) machinery and the cytosolic iron-sulfur assembly (CIA) system. In the mammalian ISC machinery, preassembly of the Fe-S cluster on the scaffold protein (ISCU) involves a cysteine desulfurase complex (NFS1/ISD11) and frataxin (FXN), the protein deficient in Friedreichs ataxia. Here, by comparing the biochemical and spectroscopic properties of quaternary (ISCU/NFS1/ISD11/FXN) and ternary (ISCU/NFS1/ISD11) complexes, we show that FXN stabilizes the quaternary complex and controls iron entry to the complex through activation of cysteine desulfurization. Furthermore, we show for the first time that in the presence of iron and L-cysteine, an [Fe(4)S(4)] cluster is formed within the quaternary complex that can be transferred to mammalian aconitase (mACO2) to generate an active enzyme. In the absence of FXN, although the ternary complex can assemble an Fe-S cluster, the cluster is inefficiently transferred to ACO2. Taken together, these data help to unravel further the Fe-S cluster assembly process and the molecular basis of Friedreichs ataxia.
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Oxidation of Zn(Cys)4 zinc finger peptides by O2 and H2O2: products, mechanism and kinetics.
Chemistry
PUBLISHED: 06-21-2011
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The reactivity of a series of Zn(Cys)(4) zinc finger model peptides towards H(2)O(2) and O(2) has been investigated. The oxidation products were identified by HPLC and ESI-MS analysis. At pH<7.5, the zinc complexes and the free peptides are oxidised to bis-disulfide-containing peptides. Above pH 7.5, the oxidation of the zinc complexes by H(2)O(2) also yields sulfinate- and sulfonate-containing overoxidised peptides. At pH 7.0, monitoring of the reactions between the zinc complexes and H(2)O(2) by HPLC revealed the sequential formation of two disulfides. Several techniques for the determination of the rate constant for the first oxidation step corresponding to the attack of H(2)O(2) by the Zn(Cys)(4) site have been compared. This rate constant can be reliably determined by monitoring the oxidation by HPLC, fluorescence, circular dichroism or absorption spectroscopy in the presence of excess ethyleneglycol bis(2-aminoethyl ether)tetraacetic acid. In contrast, monitoring of the release of zinc with 4-(2-pyridylazo)resorcinol or of the thiol content with 5,5-dithiobis(2-nitrobenzoate) did not yield reliable values of this rate constant for the case in which the formation of the second disulfide is slower than the formation of the first. The kinetic measurements clearly evidence a protective effect of zinc on the oxidation of the cysteines by both H(2)O(2) and O(2), which points to the fact that zinc binding diminishes the nucleophilicity of the thiolates. In addition, the reaction between the zinc finger and H(2)O(2) is too slow to consider zinc fingers as potential sensors for H(2)O(2) in cells.
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Anticancer drugs in surface waters: what can we say about the occurrence and environmental significance of cytotoxic, cytostatic and endocrine therapy drugs?
Environ Int
PUBLISHED: 06-20-2011
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This study considers the implications and research needs arising from anticancer (also referred to as antineoplastic) drugs being released into the aquatic environment, for the entire therapeutic classes used: cytotoxic, cytostatic and endocrine therapy drugs. A categorization approach, based on French consumption amounts, allowed to highlight parent molecules and several metabolites on which further occurrence and ecotoxicological studies should be conducted. Investigations of consumption trends at a national and a local scale show an increase in the use of anticancer drugs between 2004 and 2008, thus leading to increased levels released in the environment. It therefore appears necessary to continue surveying their presence in surface waters and in wastewater treatment plant (WWTP) effluents. Furthermore, due to the rise of anticancer home treatments, most of the prescribed molecules are now available in town pharmacies. Consequently, hospital effluents are no longer the main expected entry route of anticancer drugs into the aquatic environment. Concerning ecotoxicological risks, current knowledge remains insufficient to support a definitive conclusion. Risk posed by cytotoxic molecules is still not well documented and it is not possible to conclude on their long-term effects on non-target organisms. To date, ecotoxicological effects have been assessed using standardized or in vitro assays. Such tests however may not be suitable for anticancer drugs, and further work should focus on full-life cycle or even multigenerational tests. Environmental significance (i.e. occurrence and effects) of cytostatics (protein kinases inhibitors and monoclonal antibodies), if any, is not documented. Protein kinases inhibitors, in particular, deserve further investigation due to their universal mode of action. Finally, concerning endocrine therapy drugs, molecules such as antiestrogen Tamoxifen and its active metabolites, could be of concern. Overall, to accurately assess the ecotoxicological risk of anticancer drugs, we discuss the need to break away from tests on isolated molecules and to test effects of mixtures at the low ng.l(-1) range.
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Reversible (de)protonation-induced valence inversion in mixed-valent diiron(II,III) complexes.
Inorg Chem
PUBLISHED: 06-14-2011
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The coupling of electron and proton transfers is currently under intense scrutiny. This Communication reports a new kind of proton-coupled electron transfer within a homodinuclear first-row transition-metal complex. The triply-bridged complex [Fe(III)(?-OPh)(?(2)-mpdp)Fe(II)(NH(2)Bn)] (1; mpdp(2-) = m-phenylenedipropionate) bearing a terminal aminobenzyl ligand can be reversibly deprotonated to the anilinate complex 2 whose core [Fe(II)(?-OPh)(?(2)-mpdp)Fe(III)(NHBn)] features an inversion of the iron valences. This observation is supported by a combination of UV-visible, (1)H NMR, and Mössbauer spectroscopic studies.
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The role of CyaY in iron sulfur cluster assembly on the E. coli IscU scaffold protein.
PLoS ONE
PUBLISHED: 05-04-2011
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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.
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Coordination properties of zinc finger peptides revisited: ligand competition studies reveal higher affinities for zinc and cobalt.
J. Am. Chem. Soc.
PUBLISHED: 11-24-2010
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Zinc fingers are ubiquitous small protein domains which have a Zn(Cys)(4-x)(His)(x) site. They possess great diversity in their structure and amino acid composition. Using a family of six peptides, it was possible to assess the influence of hydrophobic amino acids on the metal-peptide affinities and on the rates of metal association and dissociation. A model of a treble-clef zinc finger, a model of the zinc finger site of a redox-switch protein, and four variants of the classical ??? zinc finger were used. They differ in their coordination set, their sequence length, and their hydrophobic amino acid content. The speciation, metal binding constants, and structure of these peptides have been investigated as a function of pH. The zinc binding constants of peptides, which adopt a well-defined structure, were found to be around 10(15) at pH 7.0. The rates of zinc exchange between EDTA and the peptides were also assessed. We evidenced that the packing of hydrophobic amino acids into a well-defined hydrophobic core can have a drastic influence on both the binding constant and the kinetics of metal exchange. Notably, well-packed hydrophobic amino acids can increase the stability constant by 4 orders of magnitude. The half-life of zinc exchange was also seen to vary significantly depending on the sequence of the zinc finger. The possible causes for this behavior are discussed. This work will help in understanding the dynamics of zinc exchange in zinc-containing proteins.
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Heart rate reduction by ivabradine reduces diastolic dysfunction and cardiac fibrosis.
Cardiology
PUBLISHED: 11-19-2010
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To determine if heart rate (HR) reduction with ivabradine (IVA), a selective inhibitor of the pacemaker I(f) current, prevents cardiac dysfunction associated with dyslipidemia.
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Chemotherapy for metastatic breast cancer. Comparison of clinical practice and cost of drugs in two cohorts of patients: 1994-1998 and 2003-2006.
Breast Cancer Res. Treat.
PUBLISHED: 09-14-2010
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Although new chemotherapeutic drugs for metastatic breast cancer (MBC) have been approved over the past decade, it is unclear whether this has changed the overall outcome of patients. This study assessed the clinical and economic impacts of these drugs. We retrospectively studied MBC patients receiving chemotherapy in our institution over two time periods, 1994-1998 and 2003-2006. Patient characteristics and outcomes, and treatment characteristics and costs (€, 2008) were compared. Three hundred and one patients were identified, 149 patients in the first cohort and 152 in second one. The median number of lines of chemotherapy was similar in the two cohorts (three lines). The median costs of chemotherapy per patient nearly doubled over time, from 6,272 € in the 1994-1998 cohort to 13,035 € in the 2003-2006 cohort (P < 0.001). No survival difference was observed between the two groups, with a 3-year survival rate estimated to 41% in the 1994-1998 cohort and 44% in the 2003-2006 cohort (P = 0.52). In multivariate analysis, prognostic factors associated with longer overall survival were single metastatic site (HR 0.48; P < 10?³), bone metastases (HR = 0.67; P = 0.007) and positive hormone receptors (HR 0.56; P = 0.0002). New chemotherapeutic agents induced a significant cost increase over time. The limited size and heterogeneity of our cohort do not allow any conclusion concerning their impact on survival.
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Iron-sulfur (Fe-S) cluster assembly: the SufBCD complex is a new type of Fe-S scaffold with a flavin redox cofactor.
J. Biol. Chem.
PUBLISHED: 05-11-2010
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Assembly of iron-sulfur (Fe-S) clusters and maturation of Fe-S proteins in vivo require complex machineries. In Escherichia coli, under adverse stress conditions, this process is achieved by the SUF system that contains six proteins as follows: SufA, SufB, SufC, SufD, SufS, and SufE. Here, we provide a detailed characterization of the SufBCD complex whose function was so far unknown. Using biochemical and spectroscopic analyses, we demonstrate the following: (i) the complex as isolated exists mainly in a 1:2:1 (B:C:D) stoichiometry; (ii) the complex can assemble a [4Fe-4S] cluster in vitro and transfer it to target proteins; and (iii) the complex binds one molecule of flavin adenine nucleotide per SufBC(2)D complex, only in its reduced form (FADH(2)), which has the ability to reduce ferric iron. These results suggest that the SufBC(2)D complex functions as a novel type of scaffold protein that assembles an Fe-S cluster through the mobilization of sulfur from the SufSE cysteine desulfurase and the FADH(2)-dependent reductive mobilization of iron.
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Evidence that yeast frataxin is not an iron storage protein in vivo.
Biochim. Biophys. Acta
PUBLISHED: 03-14-2010
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Yeast cells deficient in the yeast frataxin homolog (Yfh1p) accumulate iron in their mitochondria. Whether this iron is toxic, however, remains unclear. We showed that large excesses of iron in the growth medium did not inhibit growth and did not decrease cell viability. Increasing the ratio of mitochondrial iron-to-Yfh1p by decreasing the steady-state level of Yfh1p to less than 100 molecules per cell had very few deleterious effects on cell physiology, even though the mitochondrial iron concentration greatly exceeded the iron-binding capacity of Yfh1p in these conditions. Mössbauer spectroscopy and FPLC analyses of whole mitochondria or of isolated mitochondrial matrices showed that the chemical and biochemical forms of the accumulated iron in mitochondria of mutant yeast strains (Deltayfh1, Deltaggc1 and Deltassq1) displayed a nearly identical distribution. This was also the case for Deltaggc1 cells, in which Yfh1p was overproduced. In these mitochondria, most of the iron was insoluble, and the ratio of soluble-to-insoluble iron did not change when the amount of Yfh1p was increased up to 4500 molecules per cell. Our results do not privilege the hypothesis of Yfh1p being an iron storage protein in vivo.
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Discrete tetrairon(III) cluster exhibiting a square-planar Fe4(mu4-O) core: structural and magnetic properties.
Inorg Chem
PUBLISHED: 02-04-2010
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The aerobic reaction of the Schiff-base ligand N-(benzimidazol-2-yl)salicylaldimine (Hbisi) with iron(II) perchlorate in methanol leads to the formation of the remarkable coordination compound [Fe(4)(mu(4)-O)(mu-MeO)(4)(bisi)(4)](ClO(4))(2) x 4 MeOH (1), whose single-crystal X-ray structure reveals the presence of a discrete Fe(III)(4)(mu(4)-O) core. Magnetic and Mossbauer studies both show that the exchange interaction within the square tetranuclear iron(III) unit is dominated by the central bridging mu(4)-oxido ligand, the involvement of the mu-methoxido bridges being negligible.
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Post-translational modification of ribosomal proteins: structural and functional characterization of RimO from Thermotoga maritima, a radical S-adenosylmethionine methylthiotransferase.
J. Biol. Chem.
PUBLISHED: 12-09-2009
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Post-translational modifications of ribosomal proteins are important for the accuracy of the decoding machinery. A recent in vivo study has shown that the rimO gene is involved in generation of the 3-methylthio derivative of residue Asp-89 in ribosomal protein S12 (Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 1826-1831). This reaction is formally identical to that catalyzed by MiaB on the C2 of adenosine 37 near the anticodon of several tRNAs. We present spectroscopic evidence that Thermotoga maritima RimO, like MiaB, contains two [4Fe-4S] centers, one presumably bound to three invariant cysteines in the central radical S-adenosylmethionine (AdoMet) domain and the other to three invariant cysteines in the N-terminal UPF0004 domain. We demonstrate that holo-RimO can specifically methylthiolate the aspartate residue of a 20-mer peptide derived from S12, yielding a mixture of mono- and bismethylthio derivatives. Finally, we present the 2.0 A crystal structure of the central radical AdoMet and the C-terminal TRAM (tRNA methyltransferase 2 and MiaB) domains in apo-RimO. Although the core of the open triose-phosphate isomerase (TIM) barrel of the radical AdoMet domain was conserved, RimO showed differences in domain organization compared with other radical AdoMet enzymes. The unusually acidic TRAM domain, likely to bind the basic S12 protein, is located at the distal edge of the radical AdoMet domain. The basic S12 protein substrate is likely to bind RimO through interactions with both the TRAM domain and the concave surface of the incomplete TIM barrel. These biophysical results provide a foundation for understanding the mechanism of methylthioation by radical AdoMet enzymes in the MiaB/RimO family.
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PerR vs OhrR: selective peroxide sensing in Bacillus subtilis.
Mol Biosyst
PUBLISHED: 09-18-2009
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Bacteria adapt to elevated levels of reactive oxygen species (ROS) by increasing the expression of detoxification enzymes and repair proteins. These defences are regulated by transcription factors that activate specific genes in response to ROS. In Bacillus subtilis, the adaptive response to peroxide stress is mainly under control of three proteins: sigma(B), PerR and OhrR. sigma(B) is a general stress response transcription factor. PerR is a dimeric zinc protein with a regulatory site that coordinates either a Fe(2+) or a Mn(2+) metal ion. In the presence of iron, PerR mediates strong induction of the perR regulon in response to hydrogen peroxide (H(2)O(2)). In contrast to PerR, the OhrR protein is weakly activated by H(2)O(2) but it shows a much higher reactivity for organic hydroperoxides. OhrR controls the expression of a thiol-dependent peroxidase that reduces organic hydroperoxides into their corresponding alcohols. In this review we emphasis peroxide sensing mechanisms for both proteins, focusing on recent biochemical and structural data.
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Cooperative metal binding and helical folding in model peptides of treble-clef zinc fingers.
Chemistry
PUBLISHED: 04-24-2009
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Two peptides, L(TC) and L(TC)(T) have been synthesised to model the treble-clef zinc fingers encountered in many Zn(Cys)(4)-site-containing proteins. Both are cyclic peptides with a linear tail grafted on a glutamate side chain of the cycle. They differ by the length of this tail, which lacks five amino acids in L(TC)(T) compared to L(TC). Both peptides bind Zn(2+) and Co(2+) in 1:1 metal/peptide ratio and the structure of these complexes have been characterised by NMR, UV/Vis and CD spectroscopy. Both peptides fold the same way around the metal ion and they fully reproduce the classical fold of treble-clef zinc fingers and display an extended hydrogen-bond network around the coordinating sulfur atoms. The structures of the ML(TC) complexes reveal that the linear tail forms a short two-turn alpha-helix, present in the metallated form only. The formation of this helix constitutes a rare example of metal-induced folding. The second turn of this helix is composed of the five amino acids that are absent in L(TC)(T). The study of the pH-dependence of the Zn(2+) binding constants shows that the metal ion is bound by four cysteinates above pH 5.2 and the binding constants are the highest reported so far. Interestingly, the binding constant of Zn x L(TC) is about tenfold higher than that of Zn x L(TC)(T). This difference clearly indicates that the helix, present in Zn x L(TC) only, stabilises the Zn(2+) complex by about 1.2 kcal mol(-1). The origin of this stabilisation is ascribed to an electrostatic interaction between the [ZnS(4)](2-) centre and the helix. This reveals a cooperative effect: zinc binding allows the folding of the tail into a helix which, in turn, strengthens the zinc complex.
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Structural and functional characterization of 2-oxo-histidine in oxidized PerR protein.
Nat. Chem. Biol.
PUBLISHED: 01-16-2009
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In Bacillus subtilis, PerR is a metal-dependent sensor of hydrogen peroxide. PerR is a dimeric zinc protein with a regulatory site that coordinates either Fe(2+) (PerR-Zn-Fe) or Mn(2+) (PerR-Zn-Mn). Though most of the peroxide sensors use cysteines to detect H(2)O(2), it has been shown that reaction of PerR-Zn-Fe with H(2)O(2) leads to the oxidation of one histidine residue. Oxidation of PerR leads to the incorporation of one oxygen atom into His37 or His91. This study presents the crystal structure of the oxidized PerR protein (PerR-Zn-ox), which clearly shows a 2-oxo-histidine residue in position 37. Formation of 2-oxo-histidine is demonstrated and quantified by HPLC-MS/MS. EPR experiments indicate that PerR-Zn-H37ox retains a significant affinity for the regulatory metal, whereas PerR-Zn-H91ox shows a considerably reduced affinity for the metal ion. In spite of these major differences in terms of metal binding affinity, oxidation of His37 and/or His91 in PerR prevents DNA binding.
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An N-bridged high-valent diiron-oxo species on a porphyrin platform that can oxidize methane.
Nat Chem
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High-valent oxo-metal complexes are involved in key biochemical processes of selective oxidation and removal of xenobiotics. The catalytic properties of cytochrome P-450 and soluble methane monooxygenase enzymes are associated with oxo species on mononuclear iron haem and diiron non-haem platforms, respectively. Bio-inspired chemical systems that can reproduce the fascinating ability of these enzymes to oxidize the strongest C-H bonds are the focus of intense scrutiny. In this context, the development of highly oxidizing diiron macrocyclic catalysts requires a structural determination of the elusive active species and elucidation of the reaction mechanism. Here we report the preparation of an Fe(IV)(µ-nitrido)Fe(IV)?=?O tetraphenylporphyrin cation radical species at -90 °C, characterized by ultraviolet-visible, electron paramagnetic resonance and Mössbauer spectroscopies and by electrospray ionization mass spectrometry. This species exhibits a very high activity for oxygen-atom transfer towards alkanes, including methane. These findings provide a foundation on which to develop efficient and clean oxidation processes, in particular transformations of the strongest C-H bonds.
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4-Demethylwyosine synthase from Pyrococcus abyssi is a radical-S-adenosyl-L-methionine enzyme with an additional [4Fe-4S](+2) cluster that interacts with the pyruvate co-substrate.
J. Biol. Chem.
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Wybutosine and its derivatives are found in position 37 of tRNA encoding Phe in eukaryotes and archaea. They are believed to play a key role in the decoding function of the ribosome. The second step in the biosynthesis of wybutosine is catalyzed by TYW1 protein, which is a member of the well established class of metalloenzymes called "Radical-SAM." These enzymes use a [4Fe-4S] cluster, chelated by three cysteines in a CX(3)CX(2)C motif, and S-adenosyl-L-methionine (SAM) to generate a 5-deoxyadenosyl radical that initiates various chemically challenging reactions. Sequence analysis of TYW1 proteins revealed, in the N-terminal half of the enzyme beside the Radical-SAM cysteine triad, an additional highly conserved cysteine motif. In this study we show by combining analytical and spectroscopic methods including UV-visible absorption, Mössbauer, EPR, and HYSCORE spectroscopies that these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster displaying a free coordination site that interacts with pyruvate, the second substrate of the reaction. The presence of two distinct iron-sulfur clusters on TYW1 is reminiscent of MiaB, another tRNA-modifying metalloenzyme whose active form was shown to bind two iron-sulfur clusters. A possible role for the second [4Fe-4S] cluster in the enzyme activity is discussed.
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Molecular organization, biochemical function, cellular role and evolution of NfuA, an atypical Fe-S carrier.
Mol. Microbiol.
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Biosynthesis of iron-sulphur (Fe-S) proteins is catalysed by multi-protein systems, ISC and SUF. However, non-ISC, non-SUF Fe-S biosynthesis factors have been described, both in prokaryotes and eukaryotes. Here we report in vitro and in vivo investigations of such a non-ISC, non SUF component, the Nfu proteins. Phylogenomic analysis allowed us to define four subfamilies. Escherichia coli NfuA is within subfamily II. Most members of this subfamily have a Nfu domain fused to a degenerate A-type carrier domain (ATC*) lacking Fe-S cluster co-ordinating Cys ligands. The Nfu domain binds a [4Fe-4S] cluster while the ATC* domain interacts with NuoG (a complex I subunit) and aconitase B (AcnB). In vitro, holo-NfuA promotes maturation of AcnB. In vivo, NfuA is necessary for full activity of complex I under aerobic growth conditions, and of AcnB in the presence of superoxide. NfuA receives Fe-S clusters from IscU/HscBA and SufBCD scaffolds and eventually transfers them to the ATCs IscA and SufA. This study provides significant information on one of the Fe-S biogenesis factors that has been often used as a building block by ISC and/or SUF synthesizing organisms, including bacteria, plants and animals.
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Human mitochondrial ferritin improves respiratory function in yeast mutants deficient in iron-sulfur cluster biogenesis, but is not a functional homologue of yeast frataxin.
Microbiologyopen
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We overexpressed human mitochondrial ferritin in frataxin-deficient yeast cells (?yfh1), but also in another mutant affected in [Fe-S] assembly (?ggc1). Ferritin was correctly processed and expressed in the mitochondria of these cells, but the fraction of total mitochondrial iron bound to ferritin was very low, and most of the iron remained in the form of insoluble particles of ferric phosphate in these mitochondria, as evidenced by gel filtration analysis of the mitochondrial matrix (fast protein liquid chromatography [FPLC]) and by Mössbauer spectroscopy. Mutant cells in which ferritin was overexpressed still accumulated iron in the mitochondria and remained deficient in [Fe-S] assembly, suggesting that human mitochondrial ferritin is not a functional homologue of yeast frataxin. However, the respiratory function was improved in these mutants, which correlates with an improvement of cytochrome and heme synthesis. Overexpression of mitochondrial ferritin in [Fe-S] mutants resulted in the appearance of a small pool of high-spin ferrous iron in the mitochondria, which was probably responsible for the improvement of heme synthesis and of the respiratory function in these mutants.
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Studies of inhibitor binding to the [4Fe-4S] cluster of quinolinate synthase.
Angew. Chem. Int. Ed. Engl.
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Stop for NadA! A [4Fe-4S] enzyme, NadA, catalyzes the formation of quinolinic acid in de?novo nicotinamide adenine dinucleotide (NAD) biosynthesis. A structural analogue of an intermediate, 4,5-dithiohydroxyphthalic acid (DTHPA), has an in?vivo NAD biosynthesis inhibiting activity in E. coli. The inhibitory effect can be explained by the coordination of DTHPA thiolate groups to a unique Fe site of the NadA [4Fe-4S] cluster.
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[Fe(IV)?O(TBC)(CH3CN)]2+: comparative reactivity of iron(IV)-oxo species with constrained equatorial cyclam ligation.
J. Am. Chem. Soc.
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[Fe(IV)?O(TBC)(CH(3)CN)](2+) (TBC = 1,4,8,11-tetrabenzyl-1,4,8,11-tetraazacyclotetradecane) is characterized, and its reactivity differences relative to [Fe(IV)?O(TMC)(CH(3)CN)](2+) (TMC = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane) are evaluated in hydrogen atom (H-atom) abstraction and oxo-transfer reactions. Structural differences are defined using X-ray absorption spectroscopy and correlated to reactivities using density functional theory. The S = 1 ground states are highly similar and result in large activation barriers (~25 kcal/mol) due to steric interactions between the cyclam chelate and the substrate (e.g., ethylbenzene) associated with the equatorial ?-attack required by this spin state. Conversely, H-atom abstraction reactivity on an S = 2 surface allows for a ?-attack with an axial substrate approach. This results in decreased steric interactions with the cyclam and a lower barrier (~9 kcal/mol). For [Fe(IV)?O(TBC)(CH(3)CN)](2+), the S = 2 excited state in the reactant is lower in energy and therefore more accessible at the transition state due to a weaker ligand field associated with the steric interactions of the benzyl substituents with the trans-axial ligand. This study is further extended to the oxo-transfer reaction, which is a two-electron process requiring both ?- and ?-electron transfer and thus a nonlinear transition state. In oxo-transfer, the S = 2 has a lower barrier due to sequential vs concerted (S = 1) two electron transfer which gives a high-spin ferric intermediate at the transition state. The [Fe(IV)?O(TBC)(CH(3)CN)](2+) complex is more distorted at the transition state, with the iron farther out of the equatorial plane due to the steric interaction of the benzyl groups with the trans-axial ligand. This allows for better orbital overlap with the substrate, a lower barrier, and an increased rate of oxo-transfer.
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Proton-coupled intervalence charge transfer: concerted processes.
J. Am. Chem. Soc.
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The kinetics of proton-induced intervalence charge transfer (IVCT) may be measured electrochemically by generating one of the members of the IVCT couple in situ and following its conversion by means of the electrochemical signature of the other member of the couple. In the case of the diiron complex taken as an example, the reaction kinetics analysis, including the H/D isotope effect, clearly points to the prevalence of the concerted proton-intervalence charge transfer pathway over the stepwise pathways. A route is thus open toward systematic kinetic studies of proton-induced IVCT aiming at uncovering the main reactivity parameters and the factors that control the occurrence of concerted versus stepwise pathways.
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