8èmes Journées Scientifiques

de l’Institut des Métaux

en Biologie de Grenoble

Autrans - 30 et 31 Mai 2011

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Le mot du Directeur Bonjour et merci de votre participation aux 8èmes Journées de l’Institut des Métaux en Biologie de Grenoble. A un moment où les indicateurs de la recherche ne sont pas spécialement positifs, il est très réconfortant de voir que notre Institut par son dynamisme a pu être reconduit sous la forme d’une structure fédérative de recherche, grâce au soutien fort de l’université Joseph Fourier. Notre mission restera de favoriser les échanges de chercheurs et d’enseignants-chercheurs dont le point commun est un projet scientifique : la Chimie et la Biologie des métaux dans les systèmes vivants et leurs applications dans le domaine de la catalyse, de l’environnement et de la santé. Après une longue période liée aux évaluations de la recherche, il est aussi encourageant de noter que l’essence même de notre Institut, l’interdisciplinarité, est montrée comme exemple au sein de l’université de Grenoble et ailleurs. Ainsi, notre structure restera pour les années à venir un lieu de rencontre de la communauté grenobloise, et ceci grâce à vous tous qui avez contribué à faire vivre et nourrir notre thématique. Pour illustrer ce dynamisme, l’année dernière s’est tenu le 4ème congrès international de l’IMBG qui s’est singularisé par la mise en place pour la première fois d’une école thématique autour du rôle des métaux dans les bioénergies. Cette manifestation a été une grande réussite puisque plus de 40 personnes étaient présentes pendant ces deux jours, le congrès quant à lui ayant réunis 90 participants de 10 nations. Le futur de l’Institut va consister à consolider cette école thématique et favoriser son rayonnement à l’échelle nationale et internationale. Nous continuerons encore à nourrir l’interdisciplinarité de la science. Nous poursuivrons évidemment toutes les autres actions comme les propositions de bourses pour soutenir la participation d’étudiants et post-doctorants à des congrès internationaux et les journées thématiques. L’année 2012 sera le temps d’un nouveau congrès international. Un point aussi très important va être l’organisation en Juillet 2013 du congrès ICBIC XVI à Alpexpo, dont le comité d’organisation sera présidé par Marc Fontecave. De nouveau, la visibilité de l’Institut a influencé le choix de Grenoble comme ville organisatrice. L’implication de certains d’entre vous sera aussi un gage de réussite pour le congrès phare de la chimie bioinorganique. Enfin, l’Institut pour continuer à progresser a besoin de tous et toutes les propositions seront les bienvenues. Bonnes conférences et que les discussions scientifiques soient prolifiques. Pensez à visiter le site : http://imbg.ujf-grenoble.fr. Vous y retrouverez les missions de l’Institut. Grenoble le 6 Mai 2011.

Au nom du comité de pilotage Stéphane Ménage

Directeur de l’IMBG

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PROGRAMME

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Lundi 30 Mai 9h00 Accueil, enregistrement 9h15 Ouverture par Stéphane Ménage 9h20-12h20 Conférences Modérateur : Fabien PIERREL 9h20 Conférence invitée : Hélène PUCCIO Identifying the primary and essential function of mammalian frataxin in iron-sulfur cluster biogenesis 10h10 Catherine PHAM Arsenic uptake and speciation in the green marine alga Ulva lactuca: development of a coastal aquatic

bioindicator 10h30 Olivier HAMELIN Photocatalytic sulfide oxygenation using water as the unique oxygen atom source. Sun, water, what else? 10h50 Pause 11h20 Elodie MARINONI Structure of IscS-IscU complex from Archaeoglobus fulgidus, an intermediate in biogenesis of FeS centers

in proteins 11h40 Hélène JAMET QM and QM/MM studies of the binding modes for inhibitors of Tyrosinase 12h00 Gaylord TALLEC Highly stable and soluble Gd, Nd, Yb complexes as potential bimodal MRI/NIR imaging agents 12h20 Déjeuner

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14h00-15h30 Posters 15h30-18h30 Conférences Modérateur : Yohann MOREAU 15h30 Conférence invitée : Xavier ASSFELD Electronic excited etates of macromolecules containing transition metal 16h20 Hugo LEBRETTE Nickel import in bacteria. Structural studies of the periplasmic nickel-binding protein NikA 16h40 Pause 17h10 Amélie KOCHEM Complexes de ligands Salen biomimétiques de la Galactose oxydase : mise en évidence d’un 3ème site

d’oxydation potentiel 17h30 Beate BERSCH Structural information on metal proteins and their binding sites obtained by NMR 17h50 Mohammad OZEIR Coenzyme Q biosynthesis: Coq6 catalyzes the C5-hydroxylation reaction and substrate analogues rescue

Coq6 deficiency 18h10 Juan FONTECILLA-CAMPS Structural bases for the assembly of biological Fe-S clusters 19h00 Apéritif 20h00 Dîner 21h30-23h00 Assemblée générale

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Mardi 31 Mai 8h30-12h10 Conférences Modérateur : Carole DUBOC 8h30 Conférence invitée : Stéphane PETOUD Near-infrared Luminescent Lanthanide Compounds for Applications in Biology: Polymetallic Dendrimers and

Metal-Organic Frameworks

9h20 Michael CARBONI A biomimetic approach to investigate the reactivity of iron-manganese oxygenases 9h40 Isabelle MICHAUD-SORET Secondary metal sites in Helicobacter pylori metalloregulators and cross regulation of nickel and iron

homeostasis 10h00 Florie SCHILD Analyse fonctionnelle de la protéine Selenium Binding Protein 1 (SBP1) et de ses capacités de liaison à

différents métaux/métalloides chez Arabidopsis thaliana 10h20 Pause 10h50 Daniel IMBERT Novel Gd (III) chelate appended quantum dots: dual – modal and multimeric MRI contrast agents 11h10 Jacques COVES Structural basis for metal sensing by CnrX 11h30 Johan ESTELLON Methodological improvement for large-scale metalloproteins identification in bacterial proteomes: the iron-

sulfur proteins case study 11h50 Thibault STOLL Molecular systems for phocatalytical reduction of protons 12h10 Déjeuner 14h00-15h50 Conférences Modérateur : Sandrine OLLAGNIER de CHOUDENS 14h00 Conférence invitée : Hilde de REUSE Transporter, sensor, storage proteins and chaperones for protection against and specific delivery of nickel in

the gastric pathogen Helicobacter pylori 14h50 Eugen ANDREIADIS Cobalt-catalyzed hydrogen evolution from water 15h10 Catherine GEREZ NfuA : a peculiar component in the E. coli iron-sulfur cluster biosynthesis 15h30 Pascale DELANGLE A series of tripodal cysteine derivatives as water-soluble chelators highly selective for Copper (I)

15h50 Clôture

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CONFERENCES

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COMMUNICATIONS

ORALES

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Identifying the primary and essential function of mammalian frataxin in iron-sulfur cluster biogenesis

H. Puccio

Department of Translational Medecine and Neurogenetics, Institut de Génétique et de Biologie Moculaire et Cellulaire (IGBMC), CNRS UMR7104, INSERM U964, University of Strasbourg, Illkirch, France.

e-mail : hpuccio@igbmc.fr

Friedreich’s ataxia (FA) is a rare neurodegenerative disorder frequently associated with a cardiomyopathy. The gene responsible for the disease (FXN) encodes a small mitochondrial protein called frataxin. The major mutation corresponds to a (GAA)n expansion within the first intron that triggers a drastic decrease of transcription, leading to a residual level of frataxin in patients. The main biochemical characteristics of the disease in patients are iron-sulfur cluster (Fe-S) enzymes deficit, mitochondrial iron deposits and signs of oxidative stress.

Despite the identification of the protein over 13 years ago, the function of frataxin is still unclear and a matter of debate. Indeed, the protein has been proposed to display many different cellular functions: iron donor in iron-sulfur cluster (Fe-S) and/or heme biosynthesis, inhibitor of Fe-S biosynthesis, regulator of Fe-S repair, mitochondrial iron storage protein, implicated in oxidative stress defense, and tumor suppressor gene. In the present work, using a combination of in vivo, in cellulo and in vitro approaches, we have collected the evidences for the essential role of frataxin in early Fe-S biogenesis.

Using a mouse model with a specific deletion of frataxin in the liver, we have been able to identify at 2 weeks of age, a clear strong Fe-S cluster deficit in all cellular compartments, in the absence of iron dysregulation, ultrastructural abnormalities, transcriptional deregulation, and oxidative stress. By 4 weeks, additional features were detected including mitochondrial iron accumulation and deposits, mitochondrial enlargement, signs of steatosis and cellular death. However, no cellular damage caused by oxidative stress could be observed. These results clearly indicate that frataxin primary role is in Fe-S biosynthesis, and that iron dysregulation occurs secondarily.

Using co-immunoprecipitation and GST pulldown experiment, we identify the main interactors of mature human frataxin in the early Fe-S biosynthesis machinery. Directed mutagenesis led to the identification of essential residues involved in the interaction, and disruption of the interactions in cellulo recapitulated biochemical features associated with FRDA. Furthermore, we show that mature frataxin is sufficient to rescue the features associated with total loss of function, further supporting the essential role of frataxin as a monomeric protein. Altogether, our data propose an essential function for frataxin in early Fe-S biogenesis and provide the working model capable to explain the different sometimes contradictory hypothesis presented in the recent years for the role of frataxin in Fe-S cluster biosynthesis.

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Arsenic Uptake & Speciation in the Green Marine Alga Ulva lactuca: Development of a Coastal Aquatic Bioindicator

C. Pham,a,b L. Charlet,b G. Spositoa

a) Division of Ecosystem Sciences, University of California, Berkeley, CA 94720-3114, USA

b) Institut des Sciences de la Terre, Université Joseph Fourier, BP 53, 38041, Grenoble, France

e-mail : pham@cal.berkeley.edu

Algae are ubiquitous in surface waters and are known to influence the biodynamics of the priority toxic metalloid arsenic (As) in polluted marine environments. In the present study, we investigated the bioavailability and chemical forms of As in algae sampled from contaminated coastal waters in France and California in order to understand As cycling in the marine environment using synchrotron-based spectroscopic techniques.

Given the alga’s unique morphology and cell size (15-50 µm cell diameter; 50-100 µm thallus thickness), STXM and µXRF were used for the first time to date to analyze Ulva lactuca to distinguish the different target organelles and map As speciation in situ. We observed shifts at the carbon 1s edge in response to As and phosphate gradients and are exploring the importance of arsenosugars.

Figure 1: Cross-section of Ulva lactuca perpendicular to the axis of the thallus using STXM at 288.2 eV.

We are characterizing the potential risks As may pose to both ecological and human health as it is transformed into various chemical forms and moves from algae up trophic levels, potentially to fish and humans. Our research may serve as a basis for the future use of algae in biomonitoring and phytoremediation scenarios related to metalloid contamination in aquatic ecosystems.

40um

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Photocatalytic Sulfide Oxygenation using Water as the Unique Oxygen Atom Source

Sun, Water, What else ?

O. Hamelin, P. Guillo, F. Loiseau, N. McClenaghan, S. Ménage

Laboratoire de Chimie et Biologie des Métaux, UMR 5249 UJF-CEA-CNRS ; 17 avenue des martyrs, 38054 Grenoble

e-mail : ohamelin@cea.fr

As a consequence of the inevitable end of fossil energy resources, tremendous efforts to develop photocatalytic systems, able to convert solar energy into chemical energy, have increased exponentially in the last decade.1 In our research program aiming to develop new polypyridyl ruthenium-based catalysts for oxidation, we were interested in the combination of a photosensitizer and a catalytic fragment within the same complex to achieve catalytic light-driven oxidation. Due to the lack in such a field, we developed a new eco-aware catalytic system able to use light to activate water molecule to perform the oxygenation of sulfides via an oxygen atom transfer from H2O to the substrate. This approach avoids the use of classical (and sometimes relatively toxic and/or hazardous) oxidants such as peroxides and peracids. Up to 200 turnover numbers were achieved upon irradiation thanks to a blue LEDs system of low wattage. In particular, we demonstrated a synergetic effect between both partners of the dyad with regard to the photocatalytic activity compared to the bimolecular system.2

In addition, based on literature reports and on electrochemical and photophysical studies, a mechanism involving the formation of an oxidant Ru(IV)=O species thanks to a proton coupled electron transfer emerged.

Ru Ru

468 nm

Electronacceptor

Electron transfer

R1S

R2 + H2O

R1S

R2

O

+ 2H+

phot catRu Ru

468 nm

Electronacceptor

Electron transfer

R1S

R2 + H2O

R1S

R2

O

+ 2H+

phot cat

1.S. Fukuzumi, T. Kishi, H. Kotani, Y.-M.; Lee, W. W. Nam, Nat. Chem. 2011, 3, 38; W. Chen, F. N. Rein, R. C. Rocha Angew. Chem. Int. Ed. 2009, 48, 9672-9675; T. P. Yoon, M. A. Ischay, J. Du, Nat. Chem. 2010, 2, 527 –532; H. W. Shih, M. N. V. Wal, R. L. Grange, D. W. C. MacMillan J. Am; Chem. Soc. 2010, 132, 13600; D. A. Nicewicz, D. W. C. MacMillan, Science 2008, 322, 77; J. M. R. Narayanam, J. W. Tucker, C. R. J. Stephenson, J. Am. Chem. Soc. 2009, 131, 8756; A. G. Condie, J. C. Gonzalez-Gomez, C. R. J. Stephenson J. Am. Chem. Soc. 2010, 132, 1464; P. Melchiorre Angew. Chem. Int. Ed 2009, 48, 1360; C Dai, J M R Narayanam, C. R. J. Stephenson, Nat. Chem., 2011, DOI: 10.1038/nchem.949 2. O. Hamelin, P. Guillo, F. Loiseau, M. F. Boissonnet, S. Ménage ; Submited ; P. Guillo, O. Hamelin, P. Batat, G. Jonusauskas, N. D. McClenaghan, S. Ménage, Submitted.

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Structure of IscS-IscU complex from Archaeoglobus fulgidus, an intermediate in biogenesis of FeS centers in proteins

E. Marinonia, J. Simoes de Oliveirab, Y. Nicoleta, L. Martina, E. C. Raulfsb, D. R. Deanb, J. C. Fontecilla-Campsa

a) Métalloprotéines, Institut de Biologie Structurale J.P. Ebel, CEA, CNRS, Université Joseph Fourier, 41 Rue Jules Horowitz, 38027 Grenoble Cedex 1, France

b) Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA

e-mail : elodie.marinoni@ibs.fr

Iron sulfur centers are one of the most common prosthetic groups in proteins. They are found in the three kingdoms, archea, bacteria and eukarya. Their roles include electron transfer, substrate binding and activation, gene expression regulation, sulfur donor, etc. (1). They are biosynthesized by specific machineries: one of which is the ISC (iron-sulfur cluster) bacterial system. ISC is composed of IscS, IscU, IscA, HscA, HscB and ferredoxin. IscS, a cysteine desulfurase and IscU, a scaffold protein, are the core of this machinery. IscS provides sulfur to IscU. Using iron provided by another protein, IscU assembles an iron-sulfur center and transfers it to its client apoproteins. We are currently studying the interaction between IscS and IscU, in order to better understand how iron-sulfur clusters are assembled in IscU.

During my oral presentation I will present the structure of the IscS-IscU complex from Archaeoglobus fulgidus, obtained by in vivo trapping in Escherichia coli (2).

1. D.C. Johnson, D.R. Dean, A.D. Smith, M.K. Johnson, Annual Review of Biochemistry, 2005, 74, 247-281.

2. Raulfs E. C, O'Carroll I. P, Dos Santos P. C, Unciuleac M. C, Dean D. R, Proc Natl Acad Sci U S A, 2008, 105, 8591–8596.

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QM and QM/MM Studies of the Binding Modes for Inhibitors of Tyrosinase

H. Jamet, a E. Favre, b P-A. Carrupt, b M. Orio, a Y. Moreau, c C. Dubois, d R. Hardré,d M. Réglier, d C. Bochot, a C. Belle a

a) Département Chimie Moléculaire, UMR-CNRS 5250, BP 53, 38041 Grenoble 9, France b) Section des sciences pharmaceutiques, Université de Genève, Université de Lausanne, 30, Quai E.-Ansermet, CH 1211 Genève

4, Suisse c) iRTSV/CBM/MCT CEA Grenoble, 17 avenue des martyrs, 38054 Grenoble 9, France.

d) Aix-Marseille Université, Institut des Sciences Moléculaires de Marseille, équipe BiosCiences, iSm2, UMR-CNRS 6263, 13397 Marseille Cedex 20, France. e-mail : hjamet@ujf-grenoble.fr

Tyrosinases (Ty) are copper-containing metalloenzymes, which catalyze the oxidation of phenolic compounds into catechols (phenolase activity) and catechol into o-quinone (catecholase activity) successively. After further chemical transformations the formed quinones lead to the formation of melanin pigments. Tyrosinase inhibition is a well-known strategy to control the over production and accumulation of melanins. In this context, theoretical chemistry can give molecular–levels insights into inhibition mechanisms through the determination of the binding mode for the inhibitors on the dicopper catalytic centers of the enzyme. In this work we consider two inhibitors, the well-known acid kojic1 and the 2-hydroxypyridine-N-oxide reported in a previous study2 as a new and efficient competitive inhibitor for mushroom tyrosinase. Two different binding modes (see scheme 1) were investigated. Calculations were done on biomimetic model complexes but also on the met form of one bacterial Ty3 using the LSCF/MM scheme at ab-initio level.4Because these systems feature two unpaired electrons carried by the copper centers, magnetic properties were studied. To our knowledge it is the first QM/MM calculations of magnetic properties of such systems. Our results show a good agreement with experimental data.5

Scheme 1 1. G. Battaini, E. Monzani, L. Casella, L. Santagostini, R. Pagliarin, Inhibition of the catecholase activity of biomimetic

dinuclear copper complexes by acid kojic (2000), J. Biol. Inorg. Chem., 5:262-268. 2. E. Peyroux, W. Ghattas, R. Hardré, M. Giorgi, B. Faure, A. J. Simaan, C. Belle, M. Réglier, binding of 2-

hydroxypyridine-N-oxide on dicopper(II) centers : Insights into Tyrosinase inhibition mechanism by transition-state analogs (2009), Inorg. Chem., 48:10874-10876.

3. Y. Matoba, T. Kumagai, A. Yamamoto, H. Yoshitsu, M. Sugiyama, Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis (2006) J. Biol. Chem. 281: 8981-90.

4. a) Assfeld, X. and Rivail, J.-L. Quantum chemical computations on parts of large molecules : the ab initio local self consistent field method, (1996), Chem. Phys. Lett, 263:100-106 b) Ferré, N.; Rivail, J.-L. and Assfeld, X., Specific force field parameters dtermination for the hybrid ab initio QM/MM LSCF method (2002), J. Comp. Chem. 23:610-624.

5. L. Bubacco, E. Vijgenboom, C. Gobin, A. W.J.W Tepper, J. Salgado, G. W. Canters, Kinetic and paramagnetic NMR investigations of the inhibition of Streptomyces antibioticus tyrosinase. (2000) J. Mol. Catalysis B : Enzymatic, 8: 27-35.

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Highly Stable and Soluble Gd, Nd, Yb Complexes as Potential Bimodal MRI/NIR Imaging Agents

G. Tallec, D. Imbert, P. H. Fries, M. Mazzanti

Laboratoire de Reconnaissance Ionique, Service de Chimie Inorganique et biologique

CEA/DSM/INAC 17, rue des Martyrs, F-38 054 Grenoble, Cedex 09, France

e-mail: gaylord.tallec@cea.fr

Magnetic resonance imaging is a commonly used diagnostic method in medicinal practice as well as in biological and preclinical research. However, the relaxivity of commercial CAs is only a few percent of the theoretically predicted relaxivity while the "new generation" target specific CAs require higher relaxivity. The simultaneous optimization of the molecular parameters determining the relaxivity (number of coordinated water molecules, water-exchange rate, rotation dynamics of the whole complex, electronic relaxation, ion-nuclear distance, solvation) is essential to prepare more efficient contrast agents.

Multimodal luminescent /magnetic contrast agents are particularly attractive because they can be used to couple high sensitivity optical imaging to high resolution MRI. Recent studies have suggested that 8-hydroxyquinolinate based lanthanide complexes are good candidates for the design of near infra-red (NIR) emitting luminescent probes for biomedical application due to their good stability, low cytotoxicity, sizeable quantum yields in water, long excitation wavelength, ability to interact with proteins.[1-4] . Because of these properties, we developed tripodal ligands based on the 8-hydroxyquinolinate binding unit yielding soluble and highly stable Gd3+ complexes in water, showing high relaxivity with an enhancement upon albumin binding. Furthermore, Nd3+, Yb3+ analogues show sizeable NIR emission upon excitation at 370 nm providing a new architecture for the development of bimodal agents.

We will present the synthesis, the thermodynamic, the photophysical and the relaxometric properties of this new Ln(III) complexes based on hydroxyquinolinate group.

1. D. Imbert, S. Comby, A. S. Chauvin and J. C. G. Bunzli, Chem. Commun., 2005, 1432-1434.

2. S. Comby, D. Imbert, A. S. Chauvin and J. C. G. Bunzli, Inorg. Chem., 2006, 45, 732-743.

3. S. Comby, D. Imbert, C. Vandevyver and J. C. G. Bunzli, Chem. Eur. J., 2007, 13, 936-944.

4. A. Nonat, D. Imbert, J. Pecaut, M. Giraud and M. Mazzanti, Inorg. Chem., 2009, 48, 4207-4218.

5. G. Tallec, D. Imbert, P.H. Fries and M. Mazzanti, Dalton Trans., 2010, 39, 9490-9492

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Electronic Excited States of Macromolecules containing Transition Metal

X. Assfeld, T. Very, A. Monari

Equipe de Chimie et Biochimie Théorique, UMR CNRS UHP 7565, Nancy Université, 54506 Vandoeuvre.

e-mail : Xavier.Assfeld@cbt.uhp-nancy.fr

Several challenges are still tickling the theoretical chemistry community. Among them, we decide to tackle the description of electronic excited states in macromolecular systems, and especially those containing transition metals. This type of systems presents several challenges for the theory. Excited states, albeit the nowadays wide spread of Time Dependent Density Functional Theory (TD-DFT), are still difficult to obtain. The large size of these systems is another dilemma for quantum chemistry, and one generally is obliged to use hybrid methods allying Quantum Mechanics and Molecular Mechanics (QM/MM). The electronic response of the surroundings1 of the chromophore, during the photon’s absorption, is also difficult to take into account. However, transition metals are quite often encountered when one deals with biomolecules. They can be complexed by the protein itself (think of zinc finger proteins, blue copper proteins, hemoglobin …), or the complexes can interact with biomolecules (for example the famous cis-platin drug against cancer).

This lecture will be divided in two parts. The first one will focus on the interactions between Ruthenium complexes2 and DNA (these complexes are envisaged as an alternative to cis-platin), while the second will deal with the absorption spectra of Cupper containing proteins (plastocyanines). These studies illustrate the potentiality of the method we have developed and shed some light on the complicated electronic phenomenon under study.

1. A. D. Laurent, X. Assfeld, Interdisp. Sci. Comput. Life Sci. 2010, 2, 38-47.

2. A. Monari, X. Assfeld, M. Beley, P. Gros, J. Phys. Chem. A. 2011, in press

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Nickel import in bacteria

Structural studies of the periplasmic nickel-binding protein NikA

H. Lebrettea, P. Amaraa, M.V. Cherrierb, E. Lafflya, L. Martina, J.C. Fontecilla-Camps,a

C. Cavazzaa

a) Institut de Biologie Structurale Jean-Pierre Ebel, Metalloproteins group, UMR 5075, CEA, CNRS, Université Joseph Fourier-Grenoble 1, 41 rue Horowitz, 38027 Grenoble Cedex 1, France.

b) BM16-CRG Consorci Laboratori de Llum de Sincrotro (LLS) c/o ESRF, 38043 Grenoble, France.

e-mail : hugo.lebrette@ibs.fr

Although nickel is generally present in the environment in nanomolar concentrations1, it is both toxic and an essential cofactor of at least 8 enzymes involved in diverse cellular processes such as energy metabolism and virulence2. For example, E. coli requires nickel for the synthesis of [NiFe] hydrogenases under anaerobic growth conditions3. Highly specific and controlled nickel transport and trafficking systems are therefore necessary in bacteria. Nickel import through the inner membrane proceeds either via a Ni/Co permease or via a unique ABC-transporter. In E. coli, the NikABCDE ABC-transporter is suggested to be hydrogenase-specific4. The crystal structure of the periplasmic binding protein NikA revealed the existence of a nickel-chelator, that we called ‘nickelophore’. Based on the electron density map, it was modelled as butane-1,2,4-tricarboxylate, the three carboxylate functions interacting with Ni(II). His416 completes a square planar coordination for the metal ion and is the only direct metal-protein contact5. Recently, we have studied the physiological consequences of mutating His416 into Ile. In agreement with the role suggested previously by His416, its mutation provoked 1) the absence of the metal ion in the mutated NikA crystal structure, 2) a low concentration of intracellular nickel and 3) a residual hydrogenase activity6. All these observations confirm the central role that plays His416. We are also investigating the chemical structure of the nickelophore using X-ray crystallography, molecular docking, mass spectrometry and in vivo experiments. Among NikABCDE transporters, the ones from Brucella suis7 and Staphylococcus aureus8 have been studied. In the two bacteria, nickel is required for urease maturation and inactivation of the nik operons severely affected urease activity. NikA from S. aureus and B. suis will be purified, characterized and crystallized for the determination of their crystal structures. This would give information about the potential use of a nickelophore by these bacteria.

1. T. Eitinger and M.A. Mandrand-Berthelot, Arch. Microbiol. 2000, 173, pp. 1–9. 2. Y.J. Li and D.B. Zamble, Chem. Rev. 2009, 109, pp. 4617–43. 3. S.P. Ballantine and D.H. Boxer, J. Bacteriol. 1985, 163, pp. 454–59. 4. J.L. Rowe, G.L. Starnes and P.T. Chivers, J. Bacteriol. 2005, 187, pp. 6317–23. 5. M.V. Cherrier, C. Cavazza, C. Bochot, D. Lemaire and J.C. Fontecilla-Camps, Biochemistry 2008, 47, pp. 9937–43. 6. C. Cavazza, L. Martin, E. Laffly, H. Lebrette, M.V. Cherrier, L. Zeppieri, P. Richaud, M. Carrière and J.C. Fontecilla-

Camps, FEBS Lett. 2011, 585, pp. 711-15. 7. V. Jubier-Maurin, A. Rodrigue, S. Ouahrani-Bettache, M. Layssac, M.A. Mandrand-Berthelot, S. Köhler and J.P.

Liautard, J. Bacteriol. 2001, 183, pp. 426-34. 8. A. Hiron, B. Posteraro, M. Carrière, L. Remy, C. Delporte, M. La Sorda, M. Sanguinetti, V. Juillard and E. Borezée-

Durant, Mol. Microbiol. 2010, 77, pp. 1246-60.

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Complexes de ligands Salen biomimétiques de la Galactose oxydase : mise en évidence d’un 3ème site d’oxydation potentiel

A. Kochem,a O. Jarjayes,a B. Baptiste,b F. Thomas a

a) Département de Chimie Moléculaire(b), équipe de Chimie Inorganique Rédox,

301 rue de la chimie, 38041 Grenoble Cedex 9.

e-mail : amelie.kochem@hotmail.fr

L’association métal-radical est présente dans certaines métalloprotéines, notamment la Galactose oxydase, qui catalyse des oxydations aérobies. Son site actif renferme un ion cuivrique en géométrie pyramide à base carrée coordiné à 4 acides aminés, le cinquième ligand étant le substrat. Avec pour but d’étudier les propriétés spectroscopiques de cette métalloenzyme nous développons des complexes modèles. Dans ce contexte, les ligands tétradentates de type « Salen » se sont avérés être d’excellents systèmes, les radicaux étant suffisamment stables pour être isolés à l’état solide, mais également assez réactifs vis-à-vis des alcools pour effectuer rapidement un très grand nombre de cycles catalytiques. De nombreuses études ont été effectuées dans le but de déterminer l’influence des groupements en ortho et para des phénols1-2 ainsi que de la nature du métal3 sur les propriétés physico-chimiques, magnétiques ainsi que sur la réactivité des espèces oxydées correspondantes. Jusqu’ici, ces études ont mis en évidence la présence de deux sites d’oxydation potentiels au niveau de ces complexes : le site métallique d’une part (conduisant à des espèces monooxydées où le métal est à haut degré d’oxydation)2, et le ligand d’autre part (le phenolate s’oxydant en radical phenoxyl coordiné au métal)3. Pour la première fois, nous démontrerons dans ce travail que le pont reliant les deux unités salicylidène peut être considéré comme un troisième site d’oxydation potentiel. Notre stratégie est basée sur l’enrichissement électronique (par des groupements méthoxy) du pont phénylènediamine afin de privilégier ce site d’oxydation. La comparaison avec son homologue méthoxylé en position para des phénols sera abordée.

Complexes de Salen discutés au cours de cet exposé, et site actif de la Galactose oxydase.

1. R. C. Pratt, T. Daniel P. Stack, J. Am. Chem. Soc. 2003, 125, 8716-8717

2. T. Storr, P. Verma, R. C. Pratt, E. C. Wasinger, Y. Shimazaki, T. Daniel. P. Stack, J. Am. Chem. Soc. 2008, 130, 15448-15459

3. M. Orio, O. Jarjayes, H. Kanso, C. Philouze, F. Neese, F. Thomas, Angew. Chem. Int. Ed. 2010, 49, 4989-4992

22

Structural information on metal proteins and their binding sites obtained by NMR

Beate Berscha

a) Groupe de RMN Biomoléculaire, Institut de Biologie Structurale, UMR5075, 41 rue Jules Horowitz, F-38027 Grenoble Cedex 1

e-mail : beate.bersch@ibs.fr

Biomolecular NMR is widely used in order to obtain detailed information on the three-dimensional structure and on the dynamics of proteins. However, due to the sensitivity of the chemical shift to changes in the electronic environment of a given nucleus, NMR is a powerful technique for the study of molecular complexes. The chosen examples will illustrate identification of Cu+, Ag+, and Zn2+ binding sites in proteins using mainly NMR data. In addition, other spectroscopic techniques as for example X-ray absorption or Electron Paramagnetic Resonance spectroscopy can provide geometric information on the ligand sphere. NMR also provides some insight into the conformational changes a protein undergoes upon metal binding.

1. Bersch, B., Derfoufi, K.-M., De Angelis, F., Auquier, V., Ngonlong Ekendé, E., Mergeay, M., Ruysschaert, J.-M., Vandenbussche, G. Biochemistry 2011, 50, 2194-2204.

2. Sarret G., Favier A., Covès J., Hazemann J.-L., Mergeay M., Bersch B. J. Am. Chem. Soc. 2010, 132, 3770-3777.

3. Bersch B., Favier A., Schanda P., van Aelst S., Vallaeys T., Covès J., Mergeay M., Wattiez R. J. Mol. Biol. 2008, 380, 386-403.

23

Coenzyme Q biosynthesis: Coq6 catalyzes the C5-hydroxylation reaction and substrate analogues rescue Coq6 deficiency

M. Ozeir,a U. Muhlenhoff,b H. Webert,b, R. Lill,b M. Fontecave,a, c F. Pierrela

a) Laboratoire de Chimie et Biologie des Métaux; UMR5249 CNRS-CEA-UJF, CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France

b)Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Koch-Strasse 6, 35032 Marburg, Germany

c) Collège de France, 11 place Marcellin-Berthelot, 75005 Paris, France

e-mail : fabien.pierrel@cea.fr

Coenzyme Q (Q) is an essential component of eukaryotic cells and its biosynthesis requires several enzymes to modify the aromatic ring of 4-hydroxybenzoic acid. Mutations in six of the corresponding genes have been described to cause clinically heterogeneous diseases that can sometimes be treated by oral Q supplementation (1). Attribution of specific function to particular biosynthesis proteins of the Q pathway, mainly by studies in the yeast Saccharomyces cerevisiae, has been difficult because Q biosynthetic intermediates seldom accumulate in mutants. For example, the function of Coq6, a predicted flavin-dependent monooxygenase (2), has remained uncertain. Coq6 has been proposed to catalyze either the C1 or the C5-hydroxylation reactions or even both (3). In this work, we show that under certain conditions, a yeast coq6 mutant strain accumulates Q biosynthetic intermediates which prove that Coq6 catalyzes exclusively the C5-hydroxylation reaction. We also propose that, in an unusual way, the ferredoxin Yah1 and the ferredoxin reductase Arh1 may be the in vivo source of electrons for Coq6. In addition, we show that C5-hydroxylated analogues of 4-hydroxybenzoic acid, such as vanillic acid or 3,4-dihydroxybenzoic acid, restore Q biosynthesis and respiration in a yeast coq6 mutant strain. Our results demonstrate that appropriate analogues of 4-hydroxybenzoic acid have the potential to bypass deficient Q biosynthetic activities which may represent an alternative to oral Q supplementation in the treatment of some primary Q deficiencies.

1. Quinzii CM & Hirano M (2010) Dev Disabil Res Rev 16, 183-188.

2. van Berkel WJH, Kamerbeek NM, & Fraaije MW (2006) J. Biotechnol. 124, 670-689.

3. Gin P, Hsu AY, Rothman SC, Jonassen T, Lee PT, Tzagoloff A, & Clarke CF (2003) J Biol Chem 278, 25308 25316.

24

Structural bases for the assembly of biological Fe-S clusters

E. N. Marinoni,a J. S. de Oliveira,b E. C. Raulfs,b Y. Nicolet,a D. R. Dean,b J. C. Fontecilla-Campsa

a) Métalloprotéines, Institut de Biologie Structurale J.P. Ebel, CEA, CNRS, Université Joseph Fourier, 41 rue Jules Horowitz, 38027 Grenoble, France; and b) Department of Biochemistry, Virginia Tech, Blacksburg, VA, 24061 USA

e-mail : juan.fontecilla@ibs.fr The Archaeoglobus fulgidus genome encodes proteins having a high degree of primary structure similarity when compared to the IscS and IscU proteins from other organisms. Recombinantly produced IscS protein was crystallized and the structure solved to 1.43Å resolution. A. fulgidus IscS was found to be an unusual member of the structurally related class of IscS proteins because it does not have an active site lysine residue capable of forming a Schiff’s base with pyridoxal phosphate. Nevertheless, the crystal structure contains this cofactor and is highly homologous to other IscS proteins. Although the as-isolated form of IscS lacks detectable L-cysteine desulfurase activity, the 2.74 Å resolution structure of a recombinantly produced (IscU-Ala35-IscS)2 complex contains an [Fe-S] species within IscU. The oxidized complex structure reveals that the IscS Cys321-containing loop is ordered and that the active cysteine thiol group approaches the nascent [Fe-S] species in IscU. Crystallographic refinement and comparisons with previously published IcsU structures indicate that this species is a [2Fe-S-S] center, which may represent an intermediate in the formation of a canonical [2Fe-2S] cluster or, alternatively, an oxidation product of the as-isolated complex. Conversely, the 2.55Å resolution structure obtained from DTT-treated crystals shows a standard [2Fe-2S] cluster with IscS Cys321 being one of the cluster ligand. Based on these observations and the relevant literature we propose a plausible [2Fe-2S] cluster assembly mechanism that involves direct S donation to the iron ions by the IscS active site persulfided cysteine. The crystal structure determination of the (IscU-Ala35-IscS)2 complexes, as well as their contribution to our understanding of the Fe-S assembly mechanism will be addressed by Elodie Marinoni at the meeting.

25

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Near-infrared Luminescent Lanthanide Compounds for Applications in Biology: Polymetallic Dendrimers and Metal-Organic Frameworks

Stéphane Petoud,a,b Kristy A. Gogick,b Alexandra Foucaut,a Sandrine Villette,a Hyounsoo Uhb

a) Centre de Biophysique Moléculaire, UPR4301, Rue Charles Sadron, 45000, Orléans, France

b) Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, 15217, USA

e-mail : stephane.petoud@cnrs-orleans.fr

Fluorescence and luminescence are detection techniques that possess important advantages for bioanalytical applications and biologic imaging: high sensitivity, versatility and low costs of instrumentation.

A common characteristic of biologic analytes is their presence in small quantities among complex matrices such as blood, cells, tissues and organs. These matrices emit significant background fluorescence (autofluorescence), limiting detection sensitivity.

The luminescence of lanthanide cations has several advantages to address these limitations : sharp emission bands for spectral discrimination, long luminescence lifetimes for temporal discrimination and strong resistance to photobleaching. Several lanthanides emit near-infrared (NIR) photons that induces improved detection sensitivity due to the absence of native NIR luminescence from tissues and cells. Such photons can cross deeply into tissues for non-invasive investigations.

The main requirement to obtain such NIR lanthanide emission is to sensitize them with an appropriate chromophore.

An innovative concept for such sensitization of lanthanide cations is proposed herein; the current limitation of low quantum yields experienced by most mononuclear lanthanide complexes is compensated for by using larger numbers of lanthanide cations number of emitted photons per unit of volume and the overall sensitivity of the measurement.

To apply this strategy, we are synthesizing, characterizing and testing several families We will present here results obtained with a) polymetallic dendrimers complexes1,2,3 b) lanthanide containing metal-organic frameworks (MOF).4,5,6 This presentation will also include the description of some examples

of applications of these compounds as reporters and sensors for biologic imaging in living cells and small animals.

1. J. P. Cross, M. Lauz, P. D. Badger and S. Petoud, J. Am. Chem. Soc. 2004, 126, 16278-16279. 2. D. R. Kauffman, C. M. Shade, H. Uh, S. Petoud and A. Star, Nature Chem. 2009, 1, 500-506. 3. M. A. Alcala, S. Ying Kwan, C. M. Shade, M. Lang, H. Uh, M. Wang, S. G. Weber, D. L. Bartlett, S. Petoud, Y. J. Lee,

Nanomedicine, 2010, advance article (doi:10.1016/j.nano.2010.09.002) 4. K. A. White, D. A. Chengelis, M. Zeller, S. J. Geib, J. Szakos, S. Petoud and N. L Rosi, Chem. Commun. 2009, 4506. 5. K. A. White, D. A. Chengelis, K. A. Gogick, J. Stehman, N. L. Rosi and S. Petoud, J. Am. Chem. Soc. 2009, 131, 18069. 6. J. An, C. M. Shade, D. A. Chengelis-Czegan, S. Petoud and N. L. Rosi, J. Am. Chem. Soc. 2011, 133, 1220-1223

Metal Organic Framework Dendrimer Complex

26

A biomimetic approach to investigate the reactivity of iron-manganese oxygenases

M. Carboni,a M. Clémencey,a F. Molton,c J. Pécaut,b C. Lebrun,b L. Dubois,b G. Blondin,a J.-M. Latoura

a) Laboratoire de Chimie et Biologie des Métaux - Equipe de Physico-chimie des Métaux en Biologie - UMR 5249 CEA-CNRS-UJF CEA - Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France

b) Service de Chimie Inorganique et Biologique - Reconnaissance Ionique et Chimie de Coordination - UMR-E 3 5249 CEA–UJF CEA - Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France

c) Université Joseph Fourier Grenoble 1 / CNRS, Département de Chimie Moléculaire, UMR 5250, Laboratoire de Chimie Inorganique Redox, Institut de Chimie Moléculaire de Grenoble FR-CNRS-2607, BP-53, 38041 Grenoble Cedex 9, France

e-mail : Michael.Carboni@cea.fr

Enzymes possessing a diiron nonheme active site are performing many essential functions such as ribonucleotide reduction (RNR) and methane oxygenation (MMO). These species have been spectroscopically identified as reaction intermediates for the dioxygen activating diiron proteins. Recently, new members of this protein family were isolated from bacteria and found to be quite different by the presence of a heterodinuclear Fe-Mn in the active site. The chemical potential of the heterodinuclear metal site is just starting to be characterized, but available data suggest that it may have capabilities for similarly versatile chemistry as the extensively studied diiron-carboxylate cofactor1.

In recent year, the study of models based on simple dinuclear metal complexes has became an important tool for gaining insight into the biological functions of such bimetallic cores. The design of binucleating ligands capable of providing asymmetric dinuclear complexes is a subject of great interest. We propose to synthesize dinuclear Fe-Mn complexes to investigate the reactivity of oxygenation reactions. By combining spectroscopic and electronical studies we hope to gain a better understanding on the reactivity of this new enzymatic system.

1. M. Carboni and J.-M. Latour, Coordination Chemistry Review, 2011, 255, 186.

27

Secondary metal sites in Helicobacter pylori metalloregulators and cross regulation of nickel and iron homeostasis

C. Bahlawanea, C. Dianb , S.Vitalea , C. Fauquanta, G. A. Leonardb, D. Leducc, K. Schauerc,

C. Mullerc, H. de Reusec , L.Terradot b and I.Michaud-Soreta

a) Laboratoire de Chimie et Biologie des Métaux; UMR 5249 CNRS/CEA/UJF, iRTSV, 17 avenue des Martyrs, Grenoble

F-38054 cedex 9, France ; b)The European Synchrotron Radiation Facility, BP 220 F-38043 Grenoble cedex 9, France ;

c) Unité de Pathogenèse de Helicobacter, Département de Microbiologie, Institut Pasteur, 28 rue Docteur Roux, 75724 Paris Cedex 15, France ;

e-mail : imichaud@cea.fr

In Helicobacter pylori, a human pathogen classified as a class 1 carcinogen, nickel and iron are needed to colonize the acidic gastric niche persistently as cofactors for essential metalloproteins but are potentially toxic. Therefore nickel and iron homeostasis must be tightly controlled.

Two transcriptional regulators were found to be central in the response of H. pylori to metal: the nickel responsive regulator HpNikR and the ferric uptake regulator HpFUR. An original overlap between the nickel, iron and acid-regulated genes has been revealed, including autoregulation of the genes encoding the two metalloregulators HpFur and HpNikR. This intricate regulation network is proposed to optimize the response of H. pylori to the variable gastric environment in terms of metal ions concentration and pH (1). Binding of divalent metal ions to these metalloregulators triggers a conformational change that activates the protein for binding to specific DNA on promoter regions of the regulated genes.

In order to decipher the molecular mechanisms underlying these regulations, functional and structural characterizations of HpNikR (2) and HpFUR (3) have been performed. These studies unravel key structural features for both metalloregulators from which we derive a model for their activations where: (i) main regulatory sites are required for DNA binding and (ii) metallation of secondary site modulates DNA binding.

This model could explain how these metalloregulators modulate the expression of various genes as a function of the metal ions. In the case of HpNikR, this regulation mechanism, specific to H. pylori, may allow for a gradual response to large variability in metal concentrations encountered in the gastric environment as a function of diet and/or of metal exposure.

1. A. Danielli, V. Scarlato. FEMS Microbiol Rev. 2010, 34(5):738-52. 2. C. Bahlawane, C. Dian, C. Muller, A. Round, C. Fauquant, C. Schauer, H. de Reuse, L. Terradot and I. Michaud-Soret,

Nucleic Acids Res. 2010;38(9):3106-18 3. C. Dian, S. Vitale, G.A. Leonard, C. Bahlawane, C. Fauquant, D. Leduc, C. Muller, H. de Reuse, I. Michaud-Soret, L.

Terradot, Mol Microbiol. 2011,79(5),1260-75

28

Analyse fonctionnelle de la protéine Selenium Binding Protein 1 (SBP1) et de ses capacités de liaison à différents métaux/métalloides chez

Arabidopsis thaliana

F. Schild, Agnès Jourdain, Renaud Dumas, Jacques Bourguignon et Véronique V. Hugouvieux

Laboratoire de Physiologie Cellulaire Végétale, iRTSV, CEA Grenoble ,17 rue des Martyrs, 38054 Grenoble cedex

e-mail : florie.schild@cea.fr et veronique.hugouvieux@cea.fr

Nous cherchons à identifier et à caractériser de nouveaux systèmes de détoxication que la plante met en place pour lutter contre le stress métallique. Les analyses protéomiques différentielles réalisées dans ce but sur des cellules d’Arabidopsis thaliana traitées ou non au cadmium (Cd) (1) ont révélé l’accumulation d’une protéine homologue à la protéine humaine liant le sélénium (Se), nommée Selenium Binding Protein (SBP1). Chez Arabidopsis thaliana, nous avons montré que SBP1 était impliquée dans les processus de détoxication du Se et du Cd (2, 3). La protéine SBP1 possède plusieurs sites de liaisons potentiels aux métaux et pour mieux comprendre sa fonction chez la plante, nous avons recherché si SBP1 pouvait interagir tout d’abord avec Se puis avec d’autres métaux. Pour se faire, la protéine recombinante GST-SBP1 a été purifiée dans un premier temps par affinité sur colonne de GSH Sépharose puis débarrassée de son étiquette GST par clivage avec le facteur Xa. La protéine SBP1 ainsi libérée a été ensuite purifiée à homogénéité par passage sur une colonne de filtration sur gel (Superdex S200). Son identité ainsi que son degré de pureté ont été analysés par western blot et spectrométrie de masse (coll. avec le laboratoire EDyP, BGE, CEA Grenoble). Nous avons analysé l’impact de Se et des différents métaux sur (i) la stabilité de la protéine en fonction de la température suivie par TSA (Thermal Shift Assay) et sur (ii) les variations d’émission de fluorescence des tryptophanes de SBP1. En parallèle, nous avons quantifié par analyse ICP MS (Inductively Coupled Plasma Mass Spectrometry) la quantité de Se et de métal liée à la protéine, après séparation sur colonne de filtration sur gel (G25) du métal lié à la protéine et du métal libre. Nous présenterons nos résultats qui concernent la capacité de liaison et de stabilité de SBP1 en présence de Se et des différents métaux testés Par ailleurs, nous avons générés au laboratoire des lignées d’Arabidopsis thaliana qui sur expriment SBP1 et qui sous expriment la famille entière des SBPs. La sensibilité de ces lignées aux différents ligands potentiels de la protéine sera ensuite analysée ainsi que son impact sur la quantité de métal dans la plante.

1. Sarry et. al, Proteomics 2006, 6, 2180-2198 2. Dutilleul et al., Plant Physiology 2008, 147, 239-251. 3. Hugouvieuxet al., Plant Physiology 2009, 151, 768-781.

29

Novel Gd (III) Chelate Appended Quantum Dots: Dual – Modal and Multimeric MRI Contrast Agents

G. J. Stasiuk,a S. Tamang,a M. Giardiello,a C. Poillot,b D. Imbert,a C. Gateau,a P. H. Fries,a P. Reiss,a M. DeWaard,b Marinella Mazzantia

a) Laboratoire de Reconnaissance Ionique, Service de Chimie Inorganique et Biologique CEA/DSM/INAC 17, rue des Martyrs, F-38 054 Grenoble, Cedex 09, France b) Grenoble Institut des Neurosciences Université Joseph Fourier 38042 Grenoble

e-mail : daniel.imbert@cea.fr

Magnetic Resonance (MR) imaging compared with other imaging modalities has excellent anatomical resolution; however, it suffers at the molecular scale due to its intrinsic low sensitivity. To produce a detectable change in water signal intensity, a relatively high concentration of contrast agent (0.01 - 0.1 mM) is required.1 This creates problems when imaging at the molecular level, as the most interesting targets are present at much lower concentrations, typically in the nano- or picomolar range. In order to overcome the inherent sensitivity problem of the NMR phenomenon,2 dual modal contrast agents is an area of research that shows great promise. This is the attachment of a secondary imaging agent for e.g. PET, SPECT or fluorescence imaging to an MRI contrast agent. The goal is to overlay images from different techniques, giving better image resolution and co-validation of the accumulation of targeted MR contrast agents at a specific site. Recent studies have shown that picolinate based lanthanide complexes,3 bearing thiol functionalities can be grafted to InP/ZnS Quantum Dots (QD). The QDs when excited with UV light, show strong emission in the visible region. Upon grafting the Gd (III) complexes this creates an excellent candidate for a dual modal MR/Fluorescent contrast agent. We have obtained an unprecedented high relaxivity up to 2000 s-1 mM-1 per QD, with a tissue retention time of over 4 hours, compared to commercial contrast agents which show r1 = 4 s-1 mM-1.

SS

SS

SS

SS

SS

SS

SS

= Gd(III) chela te = Quantum Dot

1. P. J. Endres, K. W. MacRenaris, S. Vogt, and T. J. Meade, Biocon. Chem. 2008, 19, 2049. 2. P. Caravan, Chem. Soc. Rev., 2006, 35, 512. 3. A. Nonat, C. Gateau, P. H. Fries and M. Mazzanti, Chem. Eur. J., 2006, 12, 7133

30

Structural basis for metal sensing by CnrX

J. Trepreau, E. Girard, A. P. Maillard, E. de Rosny, I. Petit-Haertlein, R. Kahn, J. Covès

Institut de Biologie Structurale-Jean-Pierre Ebel, UMR 5075 CNRS-CEA-UJF-Grenoble-1, 41, rue Jules Horowitz, 38027 Grenoble Cedex, France

e-mail : jacques.coves@ibs.fr

CnrX is the membrane-anchored periplasmic sensor of the CnrYXH complex that contributes to regulate the expression of the genes involved in Co and Ni resistance in Cupriavidus metallidurans CH34. The resistance is induced by the specific release of the ExtraCytoplasmic Function (ECF) sigma factor CnrH from the CnrYX complex upon sensing of Co or Ni in the environment. This suggests that the sensor domain is specific for these metal ions. We had previously determined the 3D-structure of a soluble form of CnrX spanning residues 31-148 that we referred to as CnrXs (Pompidor at al., 2008, FEBS Lett. 582, 3954-3958). The same crystal displayed both copper-bound CnrXs and apo-CnrXs and the structural differences were not sufficient to establish a link between conformational changes and signal onset.

Schematic representation of the CnrYXH complex. In the resting state, CnrY sequesters CnrH on the cytoplamsic side of the inner membrane. CnrX is represented as a dimer. The physical interaction of CnrY and CnrX in the periplasm or CnrY and CnrH in the cytoplasm are inferred from their functional interaction. The stoichiometry of the complex is still unknown.

We have now revisited the metal-dependent activation of CnrX and we present the high-resolution structures of CnrXs under the Ni-, Co-, and Zn-bound forms. Both Ni and Co ions elicit a biological response while Zn-bound CnrX represents an inactive form of the complex. A dramatic change of the geometry of the metal-binding site (trigonal bipyramidal in the presence of Zn vs octahedric in the presence of Ni or Co) is observed as a function of the bound metal ion. While the Zn ion is pentacoordinated in a 3N2O sphere, Ni or Co ions recruit the thioether sulfur of the only methionine (Met123) residue of the sequence as a sixth ligand. We propose that the Met123 side chain recruitment is the qualitative change that switches on the sensing mechanism by remodeling the four-helix bundle that accommodates the metal-binding site. The structures presented here also revealed how the protein in its active state can further discriminate between the strong Ni inducer and the poor Co inducer by a gradient of short-range to long-range effects that fine-tune the metal-sensor. The CnrXs dimer can be described as "contracted" in the presence of Co and "relaxed" in the presence of Ni, suggesting that the discrimination between Ni or Co sensing is coupled to a metal-dependent breathe of the protein. On the basis of preliminary results obtained with the homologous protein NccX, we provide spectroscopic evidences that CnrXs is a good structural model for the full-length membrane anchored protein. This led us to speculate in the discussion section on the propagation of the signal to the cytoplasm after metal sensing in the periplasm by the whole complex. To our knowledge these results represent the first complete structural study of a periplasmic metal sensor involved in transmembrane signal transduction for the activation of an ECF-type sigma factor. 1. Trepreau et al, J. Mol. Biol., 2011, in press

31

Methodological improvement for large-scale metalloproteins identification in bacterial proteomes: the iron-sulfur proteins case

study

J. Estellon,a S. Ollagnier de Choudens,b A. Viari,c , M. Fontecave,b Y. Vandenbroucka

a) Laboratoire de Biologie à Grande Echelle, CEA, IRTSV; INSERM U1038 ; Université Joseph Fourier, 38054 Grenoble Cedex 09, France

b) Laboratoire de Chimie et Biologie des Métaux, CEA, IRTSV ; CNRS, UMR5249 ; Université Joseph Fourier, 38054 Grenoble Cedex 09, France

c) Baobab team, INRIA Rhône-Alpes 38330 Montbonnot-Saint Martin, France e-mail: {johan.estellon, yves.vandenbrouck}@cea.fr

Metalloproteins are of major importance within the three domains of life. However, current methods dedicated to identify members of this large family within bacterial proteomes are either not suitable for large-scale screening purpose or present relatively limited performances when no 3D structural templates are available [1]. Within this context we developed an approach based on a linear model which combines the results of different sequence analysis tools. These tools are based on the screening of proteins descriptors (e.g. patterns, conserved domains, structural protein domains) or the building of protein profiles for remote homologs detection, each with different scoring function. We assessed their respective predictive power towards the identification of a subset of metalloproteins, the iron-sulfur proteins (FeS), either separately or in combination. The linear model is trained on a dataset composed of protein sequences from the PDB70 (protein structures databank). Each protein is represented by a binary vector that indicates the presence or the absence of each 83 FeS descriptors we considered in this study. Five predictive models were built: four correspond to each class of descriptors and a mixed one concatenating the whole set of descriptors. Each linear model was estimated by a logistic regression procedure which allowed to select and to weight the descriptors that proved to be the more efficient towards the prediction of genuine FeS proteins. We observed that descriptors based on distant homologies profiles are more sensible and less specific than those commonly used (patterns, domains), and that their use increases the global quality of the prediction when considered in combination in the predictive model. Then, we tested performances of each linear model on the complete genome of Escherichia coli K12 and noticed that the mixed model outperformed each approach considered separately (Table 1). The methodological framework, results applied to the identification of FeS proteins and the perspectives of this work will be presented and discussed.

Table 1. Performances of each predictive model: four models for each class of descriptors and the mixed model which comprises the overall set of FeS descriptors. All models were trained on the PDB70 without E.coli sequences and assessed on E.coli genome. True positives (TP) and negatives (TN), false positives (FP) and negatives (FN) were determined by comparison of the prediction with Hamap annotations and literature. Recall (Rec.), also called sensibility, is the fraction of correct predictions among all FeS proteins, while precision (Pre.) is the fraction of correct predictions among those that the algorithm believes to belong to the FeS proteins family. The F2 measure (F2) is the weighted harmonic mean of precision and recall; this latter metric reflects the efficiency of the model.

1. Cvetkovic, A., Menon, A. L., Thorgersen, M. P., Scott, J. W., Poole Ii, F. L., Jenney Jr, F. E., et al. (2010). Microbial metalloproteomes are largely uncharacterized. Nature, 466(7307), 779-782.

32

Molecular systems for phocatalytical reduction of protons

T. Stoll, I. Serrano, M. Gennari, J. Chauvin, J. Fortage, M.-N. Collomb, A. Deronzier

Université Joseph Fourrier Grenoble 1 - CNRS, Département de Chimie Moléculaire, UMR-5250, Laboratoire de Chimie Inorganique Redox, Institut de Chimie Moléculaire de Grenoble FR-CNRS-2607, BP 53, 38041 Grenoble Cedex 9, France

e-mail : Stoll.thibaut@gmail.com

In the next few decades, the development of renewable energy is one of the most important challenges for the researcher community. A very promising way to solve our energy problem is the conversion of solar light into electricity or other forms of energy[1]. An attractive approach would be the storage of solar energy into molecular hydrogen (H2) via light-driven water splitting. The process of water splitting can be divided into two reactions: (i) water oxidation, to evolve O2 and (ii) water reduction, to yield H2. In this context, this project aimed at using the photocatalytic properties of new molecular systems for hydrogen production and anchored them on semiconductors.

For this, we synthesized easily functionalisable rhodium complexes such as [RhIII(N^N)Cp*X]n+ and [RhIII(N^N)2Cl2]

+, due to their electrocatalytical reduction of protons to hydrogen property when they are immobilized on an electrode [2,3] Subsequently, these complexes were built in photocatalytic systems in homogeneous solution comprising a photosensitizer, [RuII(bpy)3]

2+, a proton source, and a sacrificial electron donor. Dyad type complexes were also synthesized by covalently grafting catalyst and photosensitizer and then their photocatalytical properties were studied.

[RuII(bpy)3]2+ [RhIII(N^N)Cp*X]n+ [RhIII(N^N)2Cl2]

+

Dyad system

1. Cook T.R., Dogutan D.K., Reece S.Y., Surendranath Y., Teets TS., Nocera D.G., Chem. Rev 2010, 110, 6474

2. Deronzier et coll., J. Chem. Soc, Chem. Commun. 1989, 1259

3. Lehn et coll., Helvetica Chemica Acta 1979, 62, 1361

NN

NN

RuIIN

N

2+ +

Cl

RhIIIN

N

R

R

+

NN

RhIII

ClCl

N

N

R

R

R

R

N

N

NN

N

NRuII

N

N

RhIII

3+

NN

ClCl

33

Transporter, sensor, storage proteins and chaperones for protection against and specific delivery of nickel in the gastric pathogen

Helicobacter pylori

Hilde DE REUSE

Institut Pasteur, Unité Pathogenèse de Helicobacter.

e-mail : hdereuse@pasteur.fr

Helicobacter pylori is a gram negative bacterium persistently colonizing the stomach of half of the human world population. Infection by this pathogen is associated with the development of chronic gastritis, peptic ulcers and adenocarcinoma. The transition metal nickel is essential for H. pylori, because it is required for two enzymes indispensable for in vivo colonization. These enzymes are the [NiFe] hydrogenase and the abundant urease, that contains 24 Ni2+ per complex and represents 10 % of the total protein content of H. pylori. Thus, H. pylori requires large amounts of nickel to survive in the stomach despite a low concentration of this ion in the human body (0.5 nM). We are studying the different aspects of nickel trafficking in H. pylori. We identified an original system for the acquisition of nickel comprising a TonB-dependent transporter in the outer membrane 1. H. pylori is equipped with several specific nickel-binding proteins that are thought to sustain nickel availability while providing protection from the metal’s harmful effects. Among these, H. pylori possesses a species-specific chaperone, HspA, homolog of the heat-shock protein GroES, that contains a unique His-rich C-terminal extension binding nickel in vitro. In H. pylori, HspA is involved in intracellular nickel sequestration and detoxification and plays a novel role as a specialized nickel chaperone involved in hydrogenase maturation 2. H. pylori abundantly synthesizes two short Histidine-rich proteins avidly binding nickel in vitro and for which the in vivo role is currently studied in our group. Finally, the expression of the genes involved in nickel trafficking is regulated by a nickel sensor, NikR, that depending on the target acts as an activator or a repressor 3. We recently demonstrated in vivo the existence of a chronological hierarchy in the NikR-dependent transcriptional response to nickel including positive and negative regulations that are coherent with the control of the homeostasis of this metal in H. pylori 4.

1. Schauer, K., Gouget, B., Carriere, M., Labigne, A. and De Reuse, H. Mol Microbiol (2007) 63, 1054-1068.

2. Schauer, K., Muller, C., Carriere, M., Labigne, A., Cavazza, C. and De Reuse, H. J Bacteriol. (2010) 192, 1231-1237.

3. Bahlawane, C., Dian, C., Muller, C., Round, A., Fauquant, C., Schauer, K., De Reuse, H., Terradot, L. and Michaud-

Soret, I. Nucleic Acids Res (2010) 38, 3106-3118.

4. Muller, C., Bahlawane, C., Aubert, S., Delay, C-M., Schauer, K., Michaud-Soret, I and De Reuse, H. Submitted.

34

Cobalt-catalyzed hydrogen evolution from water

E. S. Andreiadis ,a P.-A. Jacques, a M. Chavarot-Kerlidou, a M. Fontecave, a,b V. Artero a

a) Laboratoire de Chimie et Biologie des Métaux, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 9 (France)

b) Collège de France, 11 place Marcellin-Berthelot, 75005 Paris (France)

e-mail : eugen.andreiadis@cea.fr

Hydrogen production, through the reduction of water in electrolysers, is currently one of the most convenient ways to store energy durably, if the electrical energy is initially obtained from renewable resources. However, while electrolysis is a mature and robust technology, the most promising devices, based on proton exchange membranes, rely on the use of platinum as an electrocatalyst to accelerate both hydrogen evolution and water oxidation. However, this rare and expensive metal is not itself a renewable resource, so the viability of a hydrogen economy depends on the design of new efficient and robust, bio-inspired electrocatalytic materials based on earth-abundant elements.

Here, we report on a new family of cobalt diimine-dioxime complexes as efficient and stable electro-catalysts for hydrogen evolution from acidic nonaqueous solutions with slightly lower overvoltages and much larger stabilities towards hydrolysis as compared to previously reported cobaloxime catalysts [1,2]. The cobalt diimine-dioxime catalysts can be combined with iridium-based photosensitizers via a bimolecular approach to yield a photocatalytic system able to achieve the photochemical production of hydrogen with a TON of 307 (4h). Preliminary work towards the functionalization and covalent coupling of the cobalt catalyst to a photosensitizer, and the grafting of the supramolecular assembly onto a transparent electrode material will also be discussed.

2H+

e-

H2

Electrode

PhotosensitizerCobalt

catalyst

e-e- hv

1. A. Fihri, V. Artero, M. Razavet, C. Baffert, W. Leibl, and M. Fontecave, Angew. Chem. Int. Ed. 2008, 47, 564.

2. P.-A. Jacques, V. Artero, J. Pécaut, M. Fontecave, Proc. Natl. Acad. Sci. 2009, 106, 20627.

35

NfuA : a peculiar component in the E. coli iron-sulfur cluster biosynthesis

C. Gerez,a S. Ollagnier-de Choudens,a R. Garcia,a M. Fontecave,a

B. Pyb, D. Vinellab, L. Loiseau,b F. Barrasb

a) Laboratoire de Chimie et Biologie des Métaux, UMR5249, CEA 17 Ave. Des Martyrs, 38054 Grenoble

b) Laboratoire de Chimie Bactérienne, UPR9043, CNRS 31 Chemin Joseph Aiguier, 13402 Marseille

e-mail : cgerez@cea.fr

Iron-sulfur (Fe-S) proteins are crucial for all living organisms, carrying tasks related to numerous biological processes, such as electron transfer, gene expression regulation or enzymatic catalysis. Despite the chemical simplicity of Fe-S clusters, their biosynthesis and insertion into apoproteins within cells require complex protein machineries. Studies in Escherichia coli and a few other bacteria led to the discovery of two such systems referred to as ISC and SUF, the latter working under iron limitation and oxidative stress conditions. All of these machineries contain a cysteine desulfurase which provides sulfur from cysteine to form a Fe-S cluster within a scaffold, the second key player, that transfers it directly or indirectly via a so called [Fe-S] carrier to an apotarget protein. Three types of scaffold/[Fe-S] carrier proteins have been identified on the basis of their conserved cysteine motif: the A-Type proteins including the E. coli members IscA, SufA and the recently discovered ErpA, the U-Type represented by IscU in E. coli and the Nfu-Type including the C-terminal domain of Azobacter vinelandii NifU and its homologs. Recently, we identified and characterized the recalled protein NfuA that contains a Nfu-domain and a degenerated A-Type domain that had lost the conserved cysteine residues and named A*-domain. We showed that NfuA was a new factor required for maturing Fe-S proteins in E. coli under stress conditions which can bind a [4Fe-4S] cluster transferable to targets such apoaconitase (1). Using biochemical and genetic approaches, we now identified target proteins of NfuA : upon stress exposure, NfuA participates to the maturation of AcnB, NuoG and the essential Fe-S proteins IspG/H. To go further, we investigated by in vivo and in vitro experiments the roles of the Nfu- and A*-domains, both important for the NfuA protein function in vivo. Both domains could be obtained separately, characterized and functionally investigated. We could demonstrate that the Nfu-domain ensures [Fe/S] binding and transfer to the apotargets while the A*-domain is involved in the interaction of NfuA with targets. Finally, further investigations led us to establish relationships between NfuA and the A-Type [Fe-S] carriers and tentatively to place NfuA in the overall cellular architecture of the Fe-S cluster biogenesis network.

1 Angelini S., Gerez C., Ollagnier-de Choudens, Sanakis Y., Fontecave M., Barras F. and Py B. J. Biol. Chem. 2008, 283, 14084-91.

.

36

A series of tripodal cysteine derivatives as water-soluble chelators highly selective for Copper (I)

P. Delanglea, A. M. Pujol,a C. Gateau,a C. Lebrun,a M. Cuillel,b D. Cassio,c E. Mintzb

a) Laboratoire de Chimie Inorganique et Biologique (UMR_E 3 CEA UJF), INAC, CEA Grenoble

b) Laboratoire de Chimie et Biologie des Métaux (UMR 5249 CEA CNRS UJF), iRTSV, CEA Grenoble

c) INSERM, UMR-S757, Université Paris-Sud, Orsay

e-mail : pascale.delangle@cea.fr

Metallic elements play major roles in biochemistry. The essential transition metal ions are used by cells in structurally constrained binding sites in metalloproteins, where they can carry out structural, regulatory or catalytic roles. As these metal ions can also catalyze cytotoxic reactions, several families of proteins are present in cells to control their concentration and to confine them to vital roles. Whereas mercury (Hg) is a purely toxic metal which is present in vital organisms only in case of intoxication, copper (Cu) is an essential element which is used as cofactor in many redox proteins, involved in several vital processes. Free Cu can also promote Fenton-like reactions and would thus be very toxic even at low concentration. Therefore intracellular Cu concentration needs to be rigorously controlled so that it is only provided to the essential enzymes but does not accumulate to toxic levels.1 As the cytoplasm of most eukaryotic cells is a reducing environment, the predominant oxidation state of Cu in cells is Cu(I), which has a soft character as Hg(II), with a high affinity for soft donors like thiolates. This preference for soft sulfur ligands is exemplified in proteins involved in Cu homeostasis or in Hg detoxication, which mainly bind these latter ions with thiolates of cysteine side-chains.2,3

Figure 1. Cu(I) complexes of the ligand NTA(CysOEt)3.

To design efficient and selective soft metal ion chelators, we decided to take advantage of the high affinity of cysteine sulfur donors for Cu(I) or Hg(II) evidenced in proteins trafficking or sequestering these metals in cells. We will report here on a series of tripodal ligands derived from nitrilotriacetic acid extended by three converging metal-binding cysteine chains.4,5 These molecules chelate very efficiently Cu(I) as exemplified in Figure 1 and one derivative functionalized with ligands of receptors located at the surface of hepatocytes has recently been demonstrated to be an intracellular copper chelator.

1. Tottey, S.; Harvie, D. R.; Robinson, N. J. Acc. Chem. Res. 2005, 38, 775-783. 2. Sénèque, O.; Crouzy, S.; Boturyn, D.; Dumy, P.; Ferrand, M.; Delangle, P. Chem. Commun. 2004, 770-771. 3. Rousselot-Pailley, P.; Sénèque, O.; Lebrun, C.; Crouzy, S.; Boturyn, D.; Dumy, P.; Ferrand, M.; Delangle, P. Inorg. Chem.

2006, 45, 5510-5520. 4. Pujol, A. M.; Gateau, C.; Lebrun, C.; Delangle, P. J. Am. Chem. Soc. 2009, 131, 6928-6929. 5. Pujol, A. M.; Gateau, C.; Lebrun, C.; Delangle, P. Chem. Eur. J. 2011, 17, 4418-4428.

Cu(I)

NHNH HN

N

S

SS

O

OO

EtOOCEtOOC

COOEt

Cu6S9

Cu(I)

CuS3

logK1 = 19.2 logK2 = 20.7

NTA(CysOEt)3

37

POSTERS

38

39

Liste des posters

1 Laetitia Ancel Lanthanide-binding hexapeptides

2 Lucile Chiari Stereospecific isotopic labeling of methyl groups for NMR Studies of high molecular weight proteins

3 Aubérie Parent Mutational analysis of the metal and DNA binding sites of PerR

4 Clémantine Gibard Chiral-at-metal complexes as asymmetric photooxidation catalysts, using water as unique oxygen atom source

5 Yohann Moreau QM/MM study of the role of key residues in the reactivity of hppd

6 Emmanuel Tirel Preparation and characterization of {Cu2S} mixed-valent cores as potential minimalist active models of Nitrous Oxide Reductase

7 Simon Arragain The Radical-SAM family: the key for functionalization of unactivated C-H bonds

8 Chiekna Cissé Structure-function relationships of Ferric Uptake Regulator and their inhibitors: towards new antibacterial compounds

9 Carole Duboc Propriétés structurales, spectroscopiques et électrochimiques de complexes du Nickel à ligand(s) thiol(s) : une approche expérimentale et théorique

10 Aurélie Jacques Design and characterization of a new peptidic model of zinc ribbons

11 Agnieszka Niedzwiecka Synthetic lanthanide binding peptides for DNA recognition

12 Juliette Trepreau Structural Basis for Metal Sensing by CnrX

13 Gustav Berggren From maturation of [FeFe] hydrogenases to original artificial hydrogenases

14 Saioa Cobo H2 electro-evolution catalyzed by cobalt-oxide nanoparticles and their oxidative transformation into an O2-evolving catalytic film

15 Charlène Esmieu Design of artificial oxygenases for drug synthesis

16 Vincent Lebrun Reactivity of zinc finger sites toward singlet oxygen

17 Sandrine Ollagnier The presence of three Fe-S cluster assembly machineries in Blastocystis demonstrates an adaptation to anaerobic lifestyle

18 Constance Bochot The versatile binding mode of Tyrosinase inhibitors towards models dicopper(II) complexes

19 Florent Colin Characterisation of Frataxin function in the early step of mammalian iron-sulfur cluster biosynthesis

20 Caroline Fauquant Interference between nanoparticles and metal homeostasis

“ Nanoparticles dispersion & effect on E. coli viability ”

21 Lauréline Lecarme Structure électronique de complexes salen-Fe(III) oxydés à un électron

22 Elodie Ponce Synthesis of an antimicrobial agent directed against quinolinate synthase : 3-fluoro-aspartate

23 Romaric Bonnet Evaluation de complexes métalliques en tant que ligands de G-quadruplexes par étude SPR

40

24 Cédric Colomban Synthesis and characterisation of copper (II) complexes incorporating bulky aromatic substituents

25 Nicolas Gauthier Sensitization of luminescent lanthanides doped nanocrystals with organic ligands

26 Nicolas Leconte Synthesis of new transition metal complexes containing aniline moieties

27 Amandine Roux Zinc finger/DOTA-Tb conjugates : toward selective Zn2+ and Ag+ sensors

28 Marine Bacchi Toward to a hybrid photocatalyst for hydrogen production

29 Romaric Bonnet Evaluation de complexes métalliques en tant que ligands de G-quadruplexes par étude SPR

41

Lanthanide-binding hexapeptides

L. Ancel, A. Niedźwiecka, C. Lebrun, P. Delangle

CEA Grenoble, INAC / SCIB, UMR_E 3 CEA UJF, 17 avenue des martyrs, 38 054 Grenoble France

email: laetitia.ancel@cea.fr

Organic fluorophores are efficient tools to study supramolecular interactions involving biomolecules, but are sensitive to photobleaching. Therefore, it is attractive to use lanthanide ions as good optical tools to interpret interactions and folding of proteins. Indeed, these trivalent metal ions have particular physicochemical properties: a large magnetic moment, which makes them ideal contrast agents for Magnetic Resonance Imaging and nice emitting properties with long luminescence lifetimes and no photobleaching.1 In this context, we are designing lanthanide-binding peptides, which will be coupled to biomolecules or to relevant recognition units, to study supramolecular interactions in biological systems. Recently, we have demonstrated that hexapeptides containing two unnatural amino acids, bearing chelating aminodiacetate side chains (tridentate or tetradentate) provide LnIII-Peptide complexes with high-enough stability to avoid dissociation in water at physiological pH.2,3 We will present here preliminary results about higher denticity peptides which should induce a larger dehydration of the lanthanide ion. The luminescence properties of these novel Ln-Peptide complexes will be discussed.

Figure 1: Model of interaction between Lanthanide ion and an hexapeptide

1. S. Aime, M. Botta, E. Torreno, Adv. Inorg. Chem. 2005, 57, 173. 2. F. Cisnetti, C. Gateau, C. Lebrun, P. Delangle, Chem. Eur. J. 2009, 15, 7546. 3. F. Cisnetti, C. Lebrun, P. Delangle, Dalton Trans, 2010, 39, 3650.

GlyTrp NH2Ac

NH

LnIII

Spacer : 2 amino acids

Lanthanide sensitizer

unnatural amino acidsbearing chelating groups

42

Stereospecific Isotopic Labeling of Methyl Groups for NMR Studies of High Molecular Weight Proteins

L. Chiari,a O. Hamelin,a J. Boisbouvier,b P. Gans,b I. Ayalab.

a) Laboratoire de Chimie et Biologie des Métaux, UMR 5249CEA-UJF-CNRS, iRTSV, CEA-Grenoble

b) Institut de Biologie Structurale J.P. Ebel, 41, rue Jules Horowitz, F-38027 GRENOBLE Cedex 1

e-mail : lucile.chiari@cea.fr, ohamelin@cea.fr

Solution NMR spectroscopy is an extremely powerful technology for the structural determination of proteins and the study of their dynamic. Perhaps equally as important as the developed NMR technics (pulse schemes) has been the concomitant improvement in isotopic labeling technologies which have a significant impact on NMR studies of high-molecular-weight proteins. Methyl group are of particular interest in NMR studies of proteins because they occur frequently in the hydrophobic cores of these molecules and thus often serve as sensitive reporters of molecular structure and dynamics.

Whereas that production of methyl specifically labeled proteins can now be efficiently achieved using biosynthetic precursors for the incorporation of nearly any desired isotope labelling pattern into the side chains of Leucine, Isoleucine and Valine residues1,2 in proteins overexpressed in E. Coli, there is still a need for the labelling of other amino acids.

The goal of this project concerns the development of a strategy allowing the 13CH3 labelling of the threonine methyls into macroproteins. This requires the development of i) an efficient synthesis3,4, ii) a procedure to its in vivo incorporation without metabolic scrambling and iii) NMR applications to proteins of interest.

1. Gans, P. et al., Angew. Chem. Int. Ed. 2010, 49, 1958. 2. Plevin, M. J. et al., J. Biomol NMR 2011, 49, 61. 3. Belokon, Y. N et al. Tetrahedron : Asymmetry 1998, 9, 4249. 4. Soloshonok, V. A. et al. Tetrahedron : Asymmetry 1995, 6 , 1741.

43

Mutational analysis of the metal and DNA binding sites of PerR

A. Parent, C. Caux-Thang, C. Cicuttini, G. Blondin, V. Duarte, J.-M. Latour

Laboratoire Chimie et Biologie des Métaux, Equipe PMB, UMR5249 CEA-CNRS-UJF, iRTSV CEA-Grenoble, 17 avenue des Martyrs, 38054 Grenoble Cedex 9, France

e-mail : victor.duarte@cea.fr

An unavoidable consequence of aerobic life is the production of reactive oxygen species. To avoid the harmful effects of ROS, cells constitutively express enzymes to protect themselves and repair the damages. In addition, all organisms have adaptive and inducible responses to elevated levels of oxidative stress by the way of oxidant-specific sensors. In Bacillus subtilis, the H2O2 sensor PerR (BsPerR) which belongs to the Fur family is a zinc protein that binds DNA in the presence of a regulatory metal (Fe2+). The active site of BsPerR comprises 5 ligands (3 His and 2 Asp) arranged as a distorted square pyramid where His37, Asp85, His91 and Asp104 constitute the base of the pyramid and His93 occupies the apical position. This environment is quite adapted to an interaction with H2O2 on the position opposite to His93. Three mutants of BsPerR have been produced to evaluate the role of the two Asp residues in the reaction with H2O2. Asp (D) to Glu (E) mutations have been designed based on sequence alignments with other Fur proteins that are poorly sensitive to H2O2. The PerR-D85E, PerR-D104E and PerR-D85E-D104E proteins have been isolated with high purity. Interestingly, by using in vivo conditions where the wild type BsPerR is mainly oxidized (70 %), the three mutants show a significantly reduced susceptibility to oxidation. Preliminary in vitro experiments under anaerobic conditions in the presence of Fe2+ and H2O2 are in good agreement with the in vivo data. These results suggest that the two Asp residues play a crucial role in the detection of H2O2. The hypothesis where the D to E mutations change the geometry of the active site from square pyramidal to octahedral, in order to prevent the interaction of H2O2 in trans to His93, is evaluated by the use of several techniques including EPR and Mössbauer spectroscopies and X-Ray crystallography. In terms of DNA binding, sequence alignment of BsPerR with other Fur proteins in addition to site directed mutagenesis and EMSA experiments allowed us to identify a single residue that is absolutely essential for selective DNA recognition. While the Asn61 (N61) residue of the H4 DNA binding helice of BsPerR is highly conserved among the PerR proteins, it is important to note that this amino acid is largely replaced by an Arg (R) residue among most of the Fur proteins. Interestingly, the N61R mutation significantly decreases the affinity of BsPerR towards a perR-mrgA promoter and allows a highly specific interaction with a fur-box containing sequence (Kd = 1 nM). X-ray crystallography assays are currently performed to solve the structures of both the wild type and mutated PerR proteins with DNA.

44

Chiral-at-metal complexes as asymmetric photooxidation catalysts, using water as unique oxygen atom source

C. Gibard,a P. Guillo,a F. Loiseau, b S. Ménage. a O. Hamelin a

a) Laboratoire de Chimie et Biologie des Métaux, UMR CEA-UJF-CNRS n° 5249, iRTSV, CEA Grenoble

b) Département de Chimie Moléculaire, CIRE, Université Joseph Fourier (Grenoble I) e-mail: clementine.gibard@cea.fr; ohamelin@cea.fr

In the last decades, as a consequence of the inevitable end of fossil energy resources associated, attempts to develop “green solutions” have emerged all over the world. Tremendous efforts were made to take advantage of the exceptional photophysical properties of ruthenium polypyridyl complexes with the final objective to convert solar energy into chemical energy. We have made a combination of a photosensitizer and a catalytic fragment within the same complex to achieve catalytic light-driven oxidation. Here we have tackled a new eco-aware catalytic system able to perform the asymmetric photocatalytic oxidation of sulfides via an oxygen atom transfer from H2O to the substrate. This approach avoids the use of classical (and sometimes relatively toxic and/or hazardous) oxidants such as peroxides and peracids.

The originality of this project relies on four main points:

i) Water, as an abundant and non-toxic molecule, is used as the unique oxygene atom source, ii) Whereas all the metal-dependant asymmetric catalyses involve chiral organic ligands, in this project, the metal is one of the stereogenic center (Chirality ∆/Λ). iii) The photosensitizer, the chiral inducer and the catalytic center are associated within a unique catalyst. iv) Solar (light) or LED energy will be converted into chemical energy. This project involves the synthesis of chiral dyads in which one ruthenium center is the catalytic center and the second one is at the same time the photosensitizer and the chiral inducer. 1. O. Hamelin, P. Guillo, F. Loiseau, M.-F. Boissonnet, S. Ménage, submited

RuN

N N

N

N

NN

XN

N

N

Ru

Cl

4+

N

NElectron acceptor

Electron t ransfert

Substrat+

H2O

Enantioselective catalytic oxidation

45

QM/MM study of the role of key residues in the reactivity of hppd

Y. Moreau,a,b S. Crouzy,b M. Graindorge, a R. Dumas,c,d M. Matringe,d,e

a) Université Joseph Fourier, BP-53 F-38041 Grenoble cedex 9, France

b) iRTSV/CBM/MCT CEA Grenoble, 17 avenue des martyrs, F-38054 Grenoble cedex 9, France.

c) CNRS, UMR 5168, CEA Grenoble, 17 avenue des martyrs, F-38054 Grenoble cedex 9, France.

d) INRA, UMR 1200, CEA Grenoble, 17 avenue des martyrs, F-38054 Grenoble cedex 9, France.

e) iRTSV/PCV, CEA Grenoble, 17 avenue des martyrs, F-38054 Grenoble cedex 9, France.

e-mail : yohann.moreau@ujf-grenoble.fr

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) into homogentisate (HGA). The reaction mechanism of this key enzyme in both plants and animals has not yet been fully elucidated. In this poster, we present the theoretical part of a joint experimental/theoretical study aimed at understanding the whole enzymatic process. The theoretical work was mainly devoted to check the role of four specific residues (Q272, Q286, N261 and S246) surrounding the active site, known to be critical since their mutation have lead to a dramatic loss of activity of HPPD. In our study, we particularly focused on two key steps of the conversion of HPP into HGA using a QM/MM model including the active site (Iron-oxo and bound residues), substrate and surrounding critical residues. In a first part, we have focused on the structure of the binary complex between HPPD and HPP which shows a particular orientation of the substrate imposed by Q272 and Q286. The second part of our study concerns the critical step of hydroxylation of the aromatic ring of the substrate leading to the formation of HGA. Our calculations have proved that the hydroxide moiety of the substrate interacts strongly via a hydrogen-bond network with two polar residues: N261 and S246. These interactions are believed to keep the ring in a position that favors its hydroxylation over the easier arene-oxide formation in absence of the specific interactions mentioned, thus proving the essential role of the two residues despite their distance from the iron centre.

46

Preparation and characterization of {Cu2S} mixed-valent cores as

potential minimalist active models of Nitrous Oxide Reductase

E. Tirel, a M. Orio,b J. Pécaut,c H. Jamet,b L. Lepape,a S. Ménagea and S. Torellia

a) Laboratoire de Chimie et Biologie des Métaux - Université Joseph Fourier- CNRS, UMR 5249- CEA DSV/iRTSV/LCBM Bat K’ - 17, avenue des Martyrs, 38054 Grenoble Cedex 9 (France).

b) Equipe de Chimie Théorique – Département de Chimie Moléculaire - Université Joseph Fourier -CNRS, UMR 5250 - B.P. 53, 38041 Grenoble Cedex 9 (France).

c) Laboratoire de Reconnaissance Ionique et Chimie de Coordination – (UMR-E 3 CEA-UJF), SCIB/INAC, CEA Grenoble – 17, avenue des Martyrs, 38054 Grenoble Cedx 9 (France). e-mail : emmanuel.tirel@cea.fr; stephane.torelli@cea.fr

Due to its potent greenhouse and ozone-depleting effects, N2O remediation has emerged has a challenging initiative. Consequently, from an environmental point of view, compounds able of N2O reduction represent an adapted alternative. Nature is also able of N2O reduction during the last step of microbial denitrification using a unique metalloenzyme called nitrous oxide reductase, N2OR. Recent crystallographic,1 spectroscopic and theoretical investigations provide a clear depiction of the active site (called CuZ) as a µ-sulfido-tetracopper center and a provocative mechanism with a bent µ-1,3-binding coordination mode of N2O on two copper ions has been proposed.2 During the reaction, it is admitted that the {Cu4S} core alternates between (CuI)4, (CuII)2(CuI)2 and a CuII(CuI)3 partially delocalized MV resting state. To date, the unique example of reduced copper-containing compound that reacts with N2O has been isolated by Tolman’s group3 and possesses an original mixed-valent tricopper-disulfido cluster. An other example with an iron complex has been published by Chang and coll.4 The recent isolation of a mononuclear vanadium-N2O adduct5 also provided new possibilities on N2O coordination at metal center.

Inspired by the CuZ center of N2OR, and taking into account mechanistic considerations (that is, substrate fixation at a {Cu2S} edge), we investigated the preparation of dicopper species bearing a thiophenolate ligand. During our work, we isolated a series of disufide ligands that undergo reductive cleavage upon exposure to cupric ion. These new species have been characterized spectroscopically and contain a {Cu2S}

2+ mixed-valent motif, the delocalization depending on the organic skeleton. A striking Cu-Cu bonding interaction has been pointed out and confronted with calculations.6 The reactivity toward O2 allows the isolation of the oxidative products. Concerning N2O, the data obtained so far indicate a more that moderate activity.

1. Brown, K.; Tegoni, M.; Prudencio, M.; Pereira, A. S.; Besson, S.; Moura, J. J.; Moura, I.; Cambillau, C. Nat. Struct. Mol.

Biol. 2000, 7, 191. 2. Gorelsky, S. I.; Ghosh, S.; Solomon, E. I. J. Am. Chem. Soc. 2006, 128, 278. 3. Bar-Nahum, I.; Gupta, A. K.; Huber, S. M.; Ertem, M. Z.; Cramer, C. J.; Tolman, W. B. J. Am. Chem. Soc. 2009, 131,

2812. 4. Harman, W. H.; Chang, C. J. J. Am. Chem. Soc. 2007, 129, 15128. 5. Piro, N. A.; Lichterman, M. F.; Harman, W. H.; Chang, C. J. J. Am. Chem. Soc. 2011, 133, 2108. 6. Torelli, S.; Orio, M.; Pécaut, J.; Jamet, H.; Le Pape, L.; Ménage, S. Angew. Chem. Int. Ed. 2010, 49, 8249.

47

The Radical-SAM family: the key for functionalization of unactivated C-H bonds

S. Arragain,a E. Mulliez,a M. Fontecave,b and M. Attaa

a) LCBM, UMR 5249, CEA-CNRS-UJF, 17 Avenue des Martyrs, 38 054 CEA Grenoble, France.

b) Collège de France, 11 place Marcellin-Berthelot, 75005 Paris, France.

e-mail : mohamed.atta@cea.fr and etienne.mulliez@cea.fr

One of the most chemically difficult reactions in enzymology is the controlled cleavage of unreactive aliphatic C–H bonds. Such reactions invariably require specialized molecules such as hemes, di-iron clusters or vitamin B12 coenzymes. Recently, a class of enzymes has emerged that use S-adenosylmethionine (SAM) and a specialized [4Fe–4S]1+/2+ cluster to potentiate the cleavage of unreactive C–H bonds1. The reactions of the enzymes in this class designed as the Radical SAM superfamily are chemically extremely diverse and concern eventually any domain of the cellular metabolism. In this poster we will focus on methylthiotransferases (MTTases). These enzymes catalyse the insertion of a methylthio group in an unreactive C-H bond and are classified in three groups. The first group, the MiaB family, catalyses the formation of the 2-methylthio-N6-isopentenyladenosine 37 (ms2i6A-37) on the N6-isopentenyladenosine 37 (i6A-37) of several tRNAs2. The second group, the RimO family, catalyses the formation of the β-methylthio-aspartate 89 on the S12 ribosomal protein (β-ms-D89-S12)3-4-5. The third group, recently characterized as the MtaB family corresponds to the yqev and cdkal1 gene transcripts. This family catalyses the formation of 2-methylthio-N6-threoninecarbamoyl adenosine 37 (ms2t6A-37) on the N6-threoninecarbamoyladenosine 37 (t6A-37) of several tRNAs6-7.

Compared to other Radical SAM enzymes, there is, in the case of MTTases, an additional mechanistic step to be considered, namely the insertion of the thiomethyl group which is believed to be mediated by the N-terminal [4Fe–4S]1+/2+ cluster resulting in the formation of the reaction product. This is the central point of the MTTases mechanism that remains largely unknown. Our goal is to elucidate this mechanism which represents one of the most challenging problems in bioinorganic chemistry.

1. M. Atta, E. Mulliez, S. Arragain, F. Forouhar, JF. Hunt, M. Fontecave, Curr Opin Struct Biol 2010, 20, 1-9. 2. F. Pierrel, T. Douki, M. Fontecave, M. Atta, JBC 2004, 279, 47555-63. 3. BP. Anton, L. Saleh, JS. Benner, EA. Raleigh, S. Kasif, RJ. Roberts, PNAS 2008, 105, 1826-31. 4. KH. Lee, L. Saleh, BP. Anton, CL. Madinger, JS. Benner, DF. Iwig, RJ. Roberts, C. Krebs, SJ. Booker Biochemistry, 2009,

48, 10162-74. 5. S. Arragain, R. Garcia-Serres, G. Blondin, T. Douki, M. Clemancey, JM. Latour, F. Forouhar, H. Neely, GT.

Montelione, JF. Hunt, E. Mulliez, M. Fontecave, M. Atta JBC 2010, 285, 5792-801. 6. BP. Anton, SP. Russell, J. Vertrees, S. Kasif, EA. Raleigh, PA. Limbach, RJ. Roberts Nucleic Acids Res 2010, 38, 6195-

205. 7. S. Arragain, SK. Handelman, F. Forouhar, FY. Wei, K. Tomizawa, JF. Hunt, T. Douki, M. Fontecave, E. Mulliez, M.

Atta JBC 2010, 285, 28425-33.

48

Structure-function relationships of Ferric Uptake Regulator and their inhibitors: towards new antibacterial compounds

C. Cissé,a,b S. Vitalea, D. Boturync, P. Cattya, S. Crouzyb, I. Michaud-Soreta

a) LCBM/BioMet UMR5249 CEA/CNRS/UJF, iRTSV Grenoble

b) LCBM/MCT UMR5249 CEA/CNRS/UJF, iRTSV Grenoble

c) DCM, Grenoble University, France

e-mail : Cheickna.cisse@cea.fr; isabelle.michaud-soret@cea.fr; serge.crouzy@cea.fr

The overuse of antibiotics has induced the development of resistant pathogens. New antibacterial compounds and new targets must be found. The Ferric Uptake Regulator (FUR) is a global regulator in most bacteria which does not exist in eukaryotes. Since a link has been well established between iron bio-availability and pathogens virulence, FUR is a potential antibacterial target. Indeed, fur mutants exhibit decreased virulence. Once metallated with iron, Fe-FUR binds to DNA to regulate the transcription of genes involved in iron metabolism and in virulence.1 The aim of this work is to develop FUR inhibitors that were formerly identified by means of the peptide aptamer technology. These molecules specifically inhibit FUR and significantly decrease the virulence.2 Small peptides containing the active part of the anti-FUR peptide aptamers are able to inhibit FUR and are therefore very promising. Structural information on the inhibitor-target interactions using both theoretical and experimental approaches will set constraints for a virtual screening drug-design approach. This interdisciplinary work involves researchers from the LCBM/Grenoble: biochemists experts in metalloregulators such as FUR (Ferric Uptake Regulator) and theoretical chemists performing modelling and molecular dynamics simulations, and chemists from the DCM/Grenoble, having expertise in peptide chemistry.

1. A. Gonzalez de Peredo, C. Saint-Pierre, J.-M. Latour, I. Michaud-Soret, E. Forest, J. Mol. Biol. 2001, 1, 83.

2. N. Abed, M. Bickle, B. Mari, M. Schapira, R. Sanjuan-España, K. Robbe Sermesant, O. Moncorgé, S. Mouradian-Garcia, P. Barbry, B. B. Rudkin, M. O. Fauvarque, I. Michaud-Soret, P. Colas, Mol. Cell Prot. 2007, 6, 2110.

49

Propriétés structurales, spectroscopiques et électrochimiques de complexes du Nickel à ligand(s) thiol(s) : une approche expérimentale

et théorique

M. Gennari,a M. Orio,b M. Rétégan,a J. Pécaut,c F. Neese,b M.-N. Collomba, C. Duboca

a) Université Joseph Fourier Grenoble 1 / CNRS, Département de Chimie Moléculaire, UMR-5250, 38041 Grenoble Cedex 9 France

b) Institute for Physical and Theoretical Chemistry Universität Bonn, Wegelerstraße 12, D-53113 Bonn, Germany

c) Laboratoire de Reconnaissance Ionique et Chimie de Coordination, Service de Chimie Inorganique et Biologique, (UMR E-3 CEA/UJF, FRE3200 CNRS), CEA-Grenoble, INAC, 17 rue des Martyrs 38054 Grenoble cedex 9

e-mail : carole.duboc@ujf-grenoble.fr

Les ligands thiols ou thioethers sont présents dans le site actif de nombreuses métalloenzymes et confèrent à ces complexes soufrés des propriétés spectroscopiques et des réactivités particulières. Plus particulièrement, parmi les quatre enzymes à Ni connues, quatre font intervenir un complexe à ligands soufrés dans leur site actif. Dans ces systèmes, les sites à Ni présentent une très grande flexibilité à la fois dans la coordination du métal, et dans la chimie redox associée à la catalyse. Dans ce contexte, nous avons utilisé le ligand précédemment décrit par Artaud et al.1 pour synthétiser un complexe du NiII, [NiL] (Figure 1). Nous avons étudié ses propriétés spectroscopiques et électrochimiques révélant sa capacité à modéliser les changements structuraux liés au changement d’état redox du Ni pendant le cycle catalytique de la superoxide dismutase à Ni.2 L’étude de sa réactivité vis-à-vis de la S-méthylation, nous a permis d’isoler et de caractériser un complexe du NiII, [NiLCH3]

+, avec un environnement mixte thiol/thioether et d’étudier son influence sur la stabilité des complexes du NiI et NiIII correspondants.3 Nous avons également étudié cette réaction de S-méthylation et comparer la réactivité de complexes du Ni avec ceux du Zn correspondants.4 Une bonne compréhension de l’influence de la nucléophilie des fonctions thiols impliqués dans ces complexes de coordination en fonction de la nature de la sphère de coordination et/ou du métal est primordiale pour comprendre comment la Nature a modulé leur réactivité.

-1e-, + Im

-1e- +1e-

+1e-

CH3I

+1e-

-1e-

NiI NiII

NiII

NiIII

NiIII

RX

RX

EPR, DFT EPR, DFT

EPR, DFT

S

S

NN

SS

NN

S S

NNN

S S

NNN

S S

NN

-1e-, + Im

-1e- +1e-

+1e-

CH3I

+1e-

-1e-

NiI NiII

NiII

NiIII

NiIII

RX

RX

EPR, DFT EPR, DFT

EPR, DFT

S

S

NN

SS

NN

S S

NNN

S S

NNN

S S

NN

1. M. A. Kopf, D. Varech, J. P. Tuchagues, D. Mansuy and I. Artaud, J. Chem. Soc., Dalton Trans., 1998, 991-998.

2. M. Gennari, M. Orio, J. Pécaut, F. Neese, M.-N. Collomb and C. Duboc, Inorg. Chem., 2010, 49, 6399-6401.

3. M. Gennari, M. Orio, J. Pécaut, E. Bothe, F. Neese, M.-N. Collomb and C. Duboc, Inorg. Chem., 2011, 50, 3707-3716.

4. M. Gennari, M. Retegan, S. DeBeer, J. Pécaut, F. Neese, M. N. Collomb and C. Duboc, Inorg. Chem., 2011, submitted.

50

Design and characterization of a new peptidic model of zinc ribbons

A. Jacques, M. Clemancey, G. Blondin, J.-M. Latour, O. Sénèque

Laboratoire de Chimie et Biologie des Métaux, CEA/iRTSV/LCBM, UMR 5249 CNRS/Université Joseph Fourier/CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble, France.

e-mail : aurelie.jacques@cea.fr

Zinc fingers are small protein domain containing a Zn(Cys)4-x(His)x (x = 0,1 or 2) centre which were first found in transcription factors.[1] These sites were long considered as structural tools for the folding proteins and have been classified into seven groups according to their fold.[1] More recent studies have shown that some zinc fingers can act as active sites[2-3] or redox switches[4-5].

In order to better understand the role of zinc fingers, we developed a new design of peptidic models based on small cyclic peptides bearing a side-chain tail.[6-7] These models are efficient tools to

reproduce the folding around the metal and the reactivity toward H2O2 oxidation.[6]

Based on this design, we synthesized a model of zinc ribbons, Rib1. Zinc ribbons contain a Zn(Cys)4 centre composed of two β-hairpin each containing a CXXC motif.[1] Metal binding was investigated by UV-visible and CD titrimetry with Zn2+, Co2+, Cd2+ and Fe2+ showing complexes with 1:1 metal/Rib1 ratio only. The NMR structure showed that our model reproduced the loops around the metal (Figure). Finally, oxidation of Fe(II)-Rib1 into Fe(III)-Rib1 showed an interesting similarity with native rubredoxin.

Figure : Solution structure of ZnRib1

1. S. S. Krishna, I. Majumdar, N. V. Grishin, Nucleic Acids Res. 2003, 31, 532-550.

2. H. Takinowahi, Y. Matsuda, T. Yoshida, Y. Kobayashi, T. Ohkubo, Protein Sci. 2006, 15, 487-497.

3. J. Penner-Hahn, Curr.Opin. Chem. Biol. 2007, 11, 166-171.

4. C. Kumsta, U. Jakob, Biochemistry 2009, 48, 4666-4676.

5. A. T. Dinkova-Kostova, W. D. Holtzclaw, N. Wakabayashi, Biochemistry 2005, 44, 6889-6899.

6. O. Sénèque, E. Bourlès, V. Lebrun, E.Bonnet, P. Dumy, J.-M. Latour, Angew. Chem. Int. Ed. 2008, 47, 6888-6891.

7. O. Sénèque, E. Bonnet, F. L. Joumas, J.-M. Latour, Chem. Eur. J. 2009, 15, 4798-4810.

51

Synthetic lanthanide binding peptides for DNA recognition

A. Niedźwiecka, F. Cisnetti, C. Gateau, C. Lebrun, P. Delangle

CEA-Grenoble INAC/ Laboratoire de Reconnaissance Ionique et Chimie de Coordination (UMR_E 3 CEA/UJF, FRE 3200 CNRS), 17 avenue des martyrs, 38 054 Grenoble, France

e-mail : agnieszka.niedzwiecka@gmail.com

The application of a peptide scaffold to chelate lanthanide (LnIII) ions is very attractive as

Ln-peptide complexes combine biocompatibility, high water solubility as well as the amazing

spectroscopic and magnetic properties of 4f-elements. In particular, Lanthanide Binding Tags (LBTs)

tightly bind Ln ions and efficiently sensitize their luminescence by a tryptophan moiety incorporated in

a peptide sequence.1 Nevertheless LBTs, built only of natural amino acids, exhibit moderate affinity for

Ln ions in physiological conditions (max. 57 nM).

Here we report on the design of short peptide scaffolds bearing polydentate Ln coordinating units

anchored on unnatural amino acids, with increased, nanomolar and picomolar affinity.2,3 Complexation

properties were optimized by modifying denticity of chelating groups, length

of the coordinating arms and sequence of the spacer. The luminescent properties of the

Ln-peptide complexes where enhanced by decreasing the number of coordinated water molecules and

diminishing the distance between the sensitizer and the Ln ion.

Currently, the short Ln binding peptides are applied to sequence-specific artificial transcription factors

and their interactions with cancer-related sequences of DNA are investigated.

1. M. Nitz, B. Imperiali et al., Angew. Chem., Int. Ed., 2004, 43, 3682.

2. F. Cisnetti, C. Gateau, C. Lebrun and P. Delangle, Chem.–Eur. J., 2009, 15, 7456.

3. F. Cisnetti, C. Lebrun and P. Delangle, Dalton Trans., 2010, 39, 3560.

52

Structural Basis for Metal Sensing by CnrX

J. Trepreau, E. Girard, A. P. Maillard, E. de Rosny, I. Petit-Haertlein, R. Kahn, J. Covès

Institut de Biologie Structurale Jean-Pierre Ebel, UMR 5075, CNRS-CEA-UJF Grenoble 1, 41, rue Jules Horowitz,

38027 Grenoble Cedex, France

e-mail : jacques.coves@ibs.fr

CnrX is the metal sensor and signal modulator of the three-protein transmembrane signal transduction

complex CnrYXH of Cupriavidus metallidurans CH34 that is involved in the setup of cobalt and nickel

resistance. We have determined the atomic structure of the soluble domain of CnrX in its Ni-bound,

Co-bound, or Zn-bound form. Ni and Co ions elicit a biological response, while the Zn-bound form is

inactive. The structures presented here reveal the topology of intraprotomer and interprotomer

interactions and the ability of metal-binding sites to fine- tune the packing of CnrX dimer as a function

of the bound metal. These data suggest an allosteric mechanism to explain how the complex is

switched on and how the signal is modulated by Ni or Co binding. These results provide clues to

propose a model for signal propagation through the membrane in the complex.

J.Trepreau†, E. Girard†, A.P. Maillard, E. de Rosny, I. Petit-Haertlein, R. Kahn, and J. Covès, J Mol Biol, 2011, 408(4), 766–79.

† J.T. and E.G. contributed equally to this work.

53

From maturation of [FeFe] hydrogenases to original artificial hydrogenases

G. Berggrena, M. Attaa, V. Arteroa, M. Fontecavea,b

a) Laboratoire de Chimie et Biologie des Métaux - CEA Grenoble

17 rue des martyrs, 38054 Grenoble Cedex 9, France

b) Collège de France, 11 place Marcellin-Berthelot, 75005 Paris, France

e-mail : gustav.berggren@cea.fr

The development of an efficient method for harnessing solar energy and transforming it into a « solar fuel » is arguably the most important challenge facing the scientific community today. One possible fuel is H2, however the development of an hydrogen economy requires new technologies for both the production of H2 and its combustion (fuel cells). In nature these reactions are catalyzed by hydrogenase enzymes (e.g. the [FeFe] hydrogenases) which operate at high rates and with high efficiency. However, so far attempts at developing dinuclear iron catalysts mimicking the active site of the [FeFe] hydrogenases have been hampered by the low stability of synthetic dinuclear iron complexes and their requirement for large over-potentials during catalysis [1]. As part of our laboratory’s continues efforts towards the development of better catalysts we wish to construct hybrid systems in which synthetic organometallic complexes are incorporated into a protein (HydF) scaffold. This approach will allow us to study how a protein environment influences the coordination behavior of the complexes and their catalytic properties. The choice of scaffold protein is driven by the observation that HydF serves as a scaffold during the maturation of the [FeFe] hydrogenase enzyme [2]. Thus the HydF protein features a binding pocket suitable for this class of complexes.

This poster presents the project outline and our initial results.

Apo-HydF

S

Fe S

Fe

Fe S

S Fe

Y

S

Fe

S

Fe

Hybrid-HydF

X

X

X

X

X XS

Fe S

Fe

Fe S

S Fe

S S

Fe FeOC

OCCO

CO CO

CO

S S

Fe FeOC

OCNH2-n-pr

CO CO

CO

S S

Fe FeOC

OCCN

CO CO

CO

S S

Fe FeOC

OCCN

CN CO

CO

Apo-HydF

S

Fe S

Fe

Fe S

S Fe

Y

S

Fe

S

Fe

Hybrid-HydF

X

X

X

X

X XS

Fe S

Fe

Fe S

S Fe

S S

Fe FeOC

OCCO

CO CO

CO

S S

Fe FeOC

OCNH2-n-pr

CO CO

CO

S S

Fe FeOC

OCCN

CO CO

CO

S S

Fe FeOC

OCCN

CN CO

CO

1. Tard, C. and C. J. Pickett, Chemical Reviews 2009, 109, 2245-2274.

2. McGlynn, S. E., E. M. Shepard, et al., FEBS Letters 2008, 582, 2183-2187.

54

H2 electro-evolution catalyzed by cobalt-oxide nanoparticles and their oxidative transformation into an O2-evolving catalytic film

S. Cobo,a V. Fourmond,a L. Guetaz,b M. Fontecave,a, c V. Arteroa

a) Laboratoire de Chimie et Biologie des Métaux (CEA/Université Grenoble 1/CNRS), 17 rue des Martyrs, 38054 Grenoble cedex 09, France

b) CEA, LITEN, Département des Technologies de l’Hydrogène, Laboratoire des Composants PEM (LCPEM), 17, Rue des Martyrs, 38054 Grenoble, France

c) Collège de France, 11 place Marcelin-Berthelot, 75231 Paris cedex 05, France

e-mail : saioa.cobo@cea.fr

There has been a renewed interest in the past years for first-row transition metal oxide materials as catalysts for the oxidation of water and the evolution of O2 under neutral pH 7 conditions.1,2 Such catalysts, usually formed under oxidative conditions, thus hold promises for the substitution of noble metals (Pt, IrO2) at the anode of water electrolysers. We also found that cobalt oxide materials can also be deposited onto FTO or carbon electrodes under reductive conditions, though as nanoparticles in this case, and that such particles catalyze H2 evolution under neutral conditions. The structural characterization and electrocatalytic properties of these nanoparticles will be described. Actually, this material was found to be a bifunctional catalyst, also active for oxygen evolution, when the potential was switched to oxidative conditions. The catalytic switch is correlated with a reversible structural transformation of the cobalt deposit.

a) Charge passed through a FTO electrode (1 cm2) during bulk electrolysis at –1 V vs. Ag/AgCl initially in 0.5 M KPi, pH 7 containing 0.5mM Co(NO3)2 and after transfer to a cobalt-free 0.5 M KPi, pH 7 electrolyte, with potential switching between oxidative (blue, 1.16 V vs. Ag/AgCl) and reductive conditions (red, -1 V vs. Ag/AgCl); Hydrogen and oxygen evolution detected by gas chromatography c) SEM images of the cobalt nanoparticles formed at the electrode under reductive conditions.

1. Jiao, F.; Frei, H. Energy Environ. Sci. 2010, 3, 1018. 2. Kanan, M. W.; Nocera, D. G. Science 2008, 321, 1072.

2µm

-5

0

5

10

15

0 10000 20000 30000 40000

0,0

0,3

0,6

0,9

1,2

1,5

Q (

C)

µm

ol/c

m²

O2

H2

Time (s)

55

Design of artificial oxygenases for drug synthesis

C. Esmieu,a E. Girgenti,a A. Jorge-Robin,a C. Marchi-Delapierre,a S. Ménage,a M. Cherrier,b M. Iannello,c J. Fontecilla-Camps,c P. Amara,c C. Cavazzac

a) UJF/CEA Grenoble iRTSV/LCBM/CRBio, 17 rue des martyrs, 38054 Grenoble CEDEX 9, France.

b) Spanish CRG Beam Line BM16, ESRF, 6 rue Jules Horowitz, BP 220, 38043 Grenoble, France.

c) LCCP-IBS, 41 rue Jules Horowitz, 38027 Grenoble, France.

e-mail : caroline.marchi-delapierre@cea.fr

Exploring new reactions and designing efficient catalysts considering environmental and economic aspects remains a big challenge. Enzymes are indeed powerful biocatalysts which can provide enantiopure products under mild conditions; however they are very substrate-specific which limits their application. In this context, artificial metalloenzymes have appeared as a promising avenue to a new generation of “green” efficient enantioselective biocatalysts1. The combination of non polluant inorganic iron complexes with protein scaffolds leads to systems which could display both enzymatic and homogeneous inorganic catalysis advantages. In this context, the laboratory has focused on the design of artificial monooxygenases. Our strategy consists on the development of modular biocatalytic systems in which the folding of the protein will modulate the selectivity of the oxygen transfer reaction whereas the inorganic complexes will conduct the reactivity.

We decided to exploit the NikA technology2 in the synthesis of drugs based on a sulfoxide backbone as the anti-secretory gastric Omeprazole ® or the anthelmitic Fenbendazole ®.

N S

MeMeO

Me

HN

NOMe

O

Omeprazole

SNH

NFenbendazoleoxide N

HO

O

O

N S

MeMeO

Me

HN

NOMe

O

Omeprazole

SNH

NFenbendazoleoxide N

HO

O

O

N S

MeMeO

Me

HN

NOMe

O

Omeprazole

SNH

NFenbendazoleoxide N

HO

O

O

Based on docking experiments, we choose a new sulfide family which showed a good recognition with the catalytic pocket. First, three new model sulfides have been synthesized as well as the corresponding oxidation products sulfoxides and sulfones. The second part of the project has been devoid to the determination of the right experimental conditions due to the poor solubility and high molecular weight of the new substrates. We are now testing their oxidation pathway with NaOCl with a series of hybrids. The first catalysis results will be discussed.

1. Heinisch, T.; Ward, T. S; Curr. Opin. Chem. Biol. 2010, 14, 184-199. Ueno, T.; Abe, S.; Yokoi, N.; Watanabe Y., Coord. Chem. Rev. 2007, 251, 2717-2731. Lu, Y., Angew. Chem. Int. Ed. 2006, 45, 5588-5601.

2. Cavazza, C.; Bochot, C.; Rousselot-Pailley, P.; Carpentier, P.; Cherrier, M.V.; Martin, L.; Marchi-Delapierre, C.; Fontecilla-Camps, J.C.; Ménage, S. Nature Chem. 2010, 2(12), 1069-76.

56

Reactivity of zinc-finger sites toward singlet oxygen

V. Lebrun,a C. Lebrun,

b J.L. Ravanat,

c J.M. Latour,

a O. Sénèque

a

a) CEA, iRSTV, LCBM, Physico-chimie des Métaux en Biologie

b) CEA, INAC/SCIB, Reconnaissance Ionique et Chimie de Coordination c) CEA, INAC/SCIB, Lésions des Acides Nucléiques

e-mail : olivier.seneque@cea.fr

Singlet oxygen (1O2) is one of the most reactive oxygen species. Leaving aside photosynthetic organisms, its biology has long been neglected. Nevertheless, recently, several generation pathways of singlet oxygen in cells were identified, as well as some of its biological targets (amino acids, DNA, lipids, etc…) and the damages it causes. Finally, defence genes against singlet oxygen have been identified in all types of organisms (not only photosynthetic organisms), suggesting that singlet oxygen stress is ubiquitous in the living world. Among 1O2 targets, cysteines, especially in their deprotonated form, reacts rapidly with singlet oxygen, leading to various oxidized species: disulfides, thiosulfinates, sulfinates and sulfonates. Despite the importance of metal-bound cysteines in biology, their reactivity with 1O2 has scarcely been investigated, and it was limited to Ni, Co and Pt complexes. In addition, organic ligands used in these studies are much more rigid than proteins, possibly avoiding the formation of some oxidation products.

Zinc-finger zinc site are constituted by a zinc ion coordinated by four side chains with the general formula Zn(Cys)x(His)y-x (x=2,3 or 4). It has been estimated that ca 8% of proteins contain a zinc site. Because of this and because cysteine and histidine are well known 1O2 targets, the reactivity of zinc fingers need to be investigated. For such a study, we used biomimetic complexes already developed by the team: these peptide-based models mimic perfectly the structure of native protein’s site and they have similar reactivity. They are much more robust and can be more easily studied than native proteins.

We will report on the products formed by reaction of Zn(Cys4) and Zn(Cys2)(His2) sites with 1O2 and on some mechanistic aspects of these oxidations.

57

The presence of three Fe-S cluster assembly machineries in Blastocystis demonstrates an adaptation to anaerobic lifestyle

A. D. Tsaousis,a E. Gentekaki,a S. Long,b S. Ollagnier de Choudens,c D. Gaston,a A. Stechmann,a B. Py,d M. Fontecave,c F. Barras,d J. Lukes,b A. J. Rogera

a) Centre for Comparative Genomics and Evolutionary Bioinformatics, Dalhousie University, Department of Biochemistry and

Molecular Biology, Halifax, Canada, B3H 1X5 b) Biology Centre, Institute of Parasitology, Czech Academy of Sciences, and Faculty of Natural Sciences, University of South

Bohemia, Ceské Budejovice (Budweis), Czech Republic c) Laboratoire de Chimie et Biologie des Métaux, iRTSV/CEA-Grenoble, UMR5249, 17 Rue des Martyrs, 38054 Grenoble

Cedex-09, France

d) Laboratoire de Chimie Bacterienne, Institut Fédératif de Recherche 88 - Institut de Microbiologie de la Mediterranée, Centre National de la Recherche Scientifique, Marseille, France

e-mail : sollagnier@cea.fr

Proteins with iron-sulphur (Fe-S) clusters play key part in a variety of cellular processes, suggesting an important role in the function of each individual organism (1). In bacterial, archaeal and eukaryotic cells the assembly of Fe-S clusters can be achieved by several systems. Among these, the Iron-Sulphur Cluster (ISC) assembly, the SUlphur mobilisation (SUF) and the Cytosolic Iron-Sulfur Assembly (CIA) machineries are important among eukaryotes (2). The ISC machinery is localised inside mitochondria and is required for the generation of mitochondrial and cytosolic Fe-S proteins in eukaryotes, while the SUF machinery is found in plastids where it is typically used as a response to oxygen stress and/or iron limitation (3). In contrast, the CIA is a dedicated cytosolic machinery for the support of nuclear and cytosolic Fe-S proteins. The eukaryote Blastocystis, a unicellular anaerobic intestinal human parasite, contains mitochondrion-related organelles (MROs) that appear to lack classical aerobic mitochondrial pathways and function in complete absence of oxygen. In order to clarify functions of these organelles, expressed sequence tags (ESTs) were generated (4). Data from Blastocystis ESTs survey identified the core proteins for the mitochondrial ISC system, which are essential in other mitochondria, along with members of the CIA machinery. In addition, and unique among non-plastid bearing eukaryotes, a gene corresponding to the ancestral SufCB operon in bacteria was identified, suggesting the existence of a SUF system. We performed structural and phylogenetic analyses, cellular localization and functional investigations of members of the ISC machinery along with the SUF candidate protein of Blastocystis (SufCB). Our findings demonstrate the first non-plastid bearing organism with two different functional Fe-S cluster biosynthetic machineries to strategically overcome environmental stress conditions.

1. H. Beinert, P. Kiley, Curr. Opin. Chem. Biol. 1999, 3, 152-157

2. R. Lill, U. Muhlenhoff. Wwww, Trends Biochem. Sci. 2005, 30, 133-141

3. J. Balk, M. Pilon, Trends Plant Sci. 2011, 847, 1-9.

4. Stechmann et al. Curr. Biol. 2008, 18, 580-585

58

The versatile binding mode of Tyrosinase inhibitors towards models dicopper(II) complexes

C. Bochot,a M. Orio,a C. Dubois,b G. Gellon,a R. Hardré,b H. Jamet,a D. Luneau,c B. Baptiste,a C. Philouze,a M. Réglier,b G. Serratrice,a C. Bellea

a) Département de Chimie Moléculaire, Equipes CIRe et Chimie Théorique, Université J. Fourier, UMR 5250, ICMG FR-2607 BP 53, 38041 Grenoble

b) Institut des Sciences Moléculaires de Marseille, équipe BiosCiences, Aix-Marseille Université, UMR-CNRS 6263 Avenue Escadrille Normandie-Niemen, 13397 Marseille Cedex 20

c) Laboratoire des Multimatériaux et Interfaces, UMR-CNRS 5615, Université Claude Bernard, Campus de le Doua, 69622 Villeurbanne

e-mail : constance.bochot@ujf-grenoble.fr

Tyrosinase (Ty) and Catechol oxidase (CO) belongs to type-3 copper enzymes. With molecular dioxygen both enzymes catalyze the conversion of o-diphenols into the respective quinones and are involved in melanins (protective pigments) biosynthesis.1 Furthermore, Ty inhibition is a well-known approach against increased production and accumulation of melanins. The o-diphenols oxidation chemistry of type-3 copper enzymes has inspired efforts to design synthetic copper complexes that duplicate the structure and reactivity of such enzymes. In relation with Ty inhibition mechanism, the molecules targeted binuclear copper sites represent a relevant strategy to achieve Ty inhibition specificity. The latest developments include inhibition and binding studies through a series of substituted pyridine-N-oxide as ‘catechol like’ compounds. From kinetic studies, HOPNO (2-hydroxypyridine-N-oxide) appears as an efficient competitive inhibitor for mushroom Ty (Figure 1).2,3 A combination of structural data, DFT calculations and spectroscopic studies were also carried out to investigate small molecules binding modes to specific model complexes ([Cu2(BPMP)(µ-OH)](ClO4)2, [Cu2(BPEP)(µ-OH)](ClO4)2 and [Cu2(BPMEP)(µ-OH)](ClO4)2). Our goal is to determine the substrate binding modes of the molecules with their chemical nature and/or the size of the model complex. We also aim to find a potential correlation between the inhibitor activity of the molecules with their coordination mode to the binuclear copper site.

Figure 1.

1. Rolff, M. R.; Schottenheim, J.; Decker, H.; Tuczek, F. Chem. Soc. Rev. 2010

2. Peyroux, E.; Ghattas, W.; Hardre, R.; Giorgi, M.; Faure, B.; Simaan, A. J.; Belle, C.; Réglier, M. Inorg. Chem. 2009, 48, 10874.

3. Orio, M.; Bochot, C.; Dubois, C.; Gellon, G.; Hardré, R. ; Jamet, H. ; Luneau, D. ; Philouze, C.; Réglier, M.; Serratrice, G. ; Belle C. Chem. Eur. J. Under revision.

59

Characterisation of Frataxin Function in the early Step of mammalian Iron-Sulfur Cluster Biosynthesis

F. Colina, S. Schmukera, A. Martellia, C. Birckc, S. Ollagnier de Choudensb, H. Puccioa

a) Department of Translational Medicine and Neurogenetics, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France

b) Laboratoire de Chimie et Biologie des Métaux, iRTSV/CEA-Grenoble, UMR5249, 17 Rue des Martyrs, 38054 Grenoble Cedex-09, France

c) Plateforme de Biologie Structurale et Genomique, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France

e-mail : Florent.Colin@igbmc.fr

Friedreich ataxia is a rare neurodegenerative disease caused by a decreased expression of frataxin (FXN), a highly conserved mitochondrial protein essential in superior eukaryotes. The molecular consequences of this disease are a significant decrease of iron-sulfur (FeS) cluster enzymes activity, a dysregulation of iron homeostasis followed by intramitochondrial accumulation, and a hypersensitivity to oxidative stress. Frataxin is proposed to be involved in FeS clusters biosynthesis either as an iron donor or transporter, or as a regulator of iron incorporation and/or sulfur enzymatic production (Reviews (1) and (2)). In the mammalian system, FeS cluster biosynthesis and transfer to apoproteins requires more than twenty different proteins (3), but the initial formation of these clusters requires a more limited number of partners, with the core four different proteins, NFS1, ISD11, ISCU and FXN, recently shown to form a tight quaternary complexe (4). NFS1 is the cysteine desulfurase of the system, a homodimeric protein in complex with ISD11, a small protein required for its stability. ISCU is the scaffold protein on which the FeS cluster is proposed to be synthesized and subsequently transported toward apoproteins. This protein is able to generate a ternary complex with NFS1/ISD11 with which FXN can finally interact to form the quaternary complex NFS1/ISD11/ISCU/FXN. The role of frataxin within the quaternary complex has been recently proposed to be an allosteric activator of the cysteine desulfurase activity of NFS1, positively affected by Fe2+ under strict anaerobic conditions (5). Thus frataxin could be considered as an activator of FeS clusters biosynthesis. However, other reports suggest that frataxin, at least the bacterial homolog, is an inhibitor of FeS cluster biosynthesis (6). In order to resolve some of the controversies in the field, and to gain a deeper understanding of frataxin function, we have set out to biochemically characterise the mammalian ternary and quaternary complexes. We have confirmed that FXN is indeed an activator of NFS1 cysteine desulfurase activity. Furthermore, we are currently evaluating the Fe2+ and Fe3+ fixation abilities and their respective effects on cysteine desulfurase activities within both ternary and quaternary complexes. The final aim is to follow FeS cluster biosynthesis on specific iron pre-charged complexes and monitor the effect of FXN on it. Various mutations of FXN, ISCU and NFS1 will be used to further investigate the residues required for iron fixation and complexes stability, and their influence on the FeS cluster biosynthesis. Our results will enable to propose a general in vitro mechanism of FXN during the early step of FeS cluster biosynthesis.

1. S. Schmucker, H. Puccio, Hum Mol Genet. 2010, 19(R1), R103-110

2. M. Pandolfo, A. Pastore, J. Neurol. 2009, 256 Suppl1, 9-17

3. R. Lill, Nature. 2009, 460, 831-838.

4. S. Schmucker et al., Plos One. 2011, 6(1), e16199

5. C.L. Tsai, D.P. Barondeau, Biochemistry. 2010, 49(43), 9132-9139

6. S. Adinolfi et al., Nat Struc Mol Biol. 2009, 16(4), 390-396

60

Interference between nanoparticles and metal homeostasis “ Nanoparticles dispersion & effect on E. coli viability ”

C. Fauquant-Pecqueur,a A.-N. Petit,a A. Casanova,b N. Herlin-Boime,b R. Miras,a V. Nivière,a

S. Ollagnier de Choudens,a M Carrière,c I. Michaud-Soreta

a) Laboratoire de Chimie et Biologie des Métaux UMR 5249 CEA-CNRS-UJF, 17 rue des Martyrs, 38054 Grenoble Cedex

09, France b) Laboratoire Edifices Nanométriques URA 2453 CEA-CNRS-IRAMIS, 91191 Gif-sur-Yvette, France

c) Laboratoire Lésions des Acides Nucléiques UMR E3 CEA-UJF, 17 rue des Martyrs, 38054 Grenoble Cedex 09, France e-mail : Caroline.Pecqueur@cea.fr & imichaud@cea.fr

The TiO2 nanoparticles (NPs) are now produced abundantly and used widely in a variety of consumer products. Due to the important increase in the production of TiO2-NPs, potential widespread exposure of humans and environment may occur during both the manufacturing process and final use. Therefore, the potential toxicity of TiO2-NPs on human health and environment has attracted particular attention. Unfortunately, the results of the large number of studies on the toxicity of TiO2-NPs differ significantly, mainly due to an incomplete characterisation of the used nanomaterials in terms of size, shape and crystalline structure and to their unknown state of agglomeration/aggregation (1-3). First studies using TiO2-NPs with well-characterized physicochemical parameters have shown that TiO2-NPs caused cytotoxicity, were accumulated in cells and induced an elevation in the level of reactive oxygen species (ROS) in both prokaryotic and eukaryotic systems (1-2). From these results, the purpose of our project entitled NanoBioMet is to investigate further mechanisms leading to cellular effects of TiO2-NPs, using proteomic and molecular approaches on both prokaryotic (E. coli, B. subtilis) and eukaryotic cells (A549 human pneumocytes, macrophages, and hepatocytes). More particularly, a perturbation in metal homeostasis upon TiO2-NPs exposure which could generate ROS production will be evaluated. Preliminary results about nanoparticles dispersion and viability of E. coli MG1655 and mutants in presence of nanoparticles will be presented. 1. A. Simon-Deckers et al., Toxicology 2008, 253, 137–146. 2. A. Simon-Deckers et al., Environ. Sci. Technol 2009, 43, 8423–8429. 3. J. Boczkowski and P. Hoet, Nanotoxicology 2010, 4, 1–14.

61

Structure électronique de complexes salen-Fe(III) oxydés à un électron

L. Lecarme, B. Baptiste, O. Jarjayes, F. Thomas

Département de Chimie Moléculaire, Chimie Inorganique Redox Biomimétique (CIRE), UMR-5250, Université Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France.

e-mail : laureline.lecarme@gmail.com

Les ligands de type bis-salicylidene (« salen ») sont parmi les plus courants de la chimie de coordination. Une des applications de ces complexes est la catalyse d’oxydation, notamment par le catalyseur de Jacobsen représenté sur la figure ci-dessous. Au cours du cycle catalytique le manganèse oscille entre les degrés d’oxydation +III et +V, sans qu’aucune activité rédox ne puisse être observée au niveau du ligand. 1

NN

t-But-Bu

OOt-Bu t-BuMn

Cl

III

NN

t-But-Bu

OOt-Bu t-BuMn

Cl

V

NaOCl

NaClR2R1

R2R1

O

O

Nous avons récemment montré que l’incorporation de groupements fortement électro-donneurs en positions ortho et para des phénols pouvait induire une activité rédox centrée sur le ligand, comme en témoignent les structures par diffraction des RX de complexes salen radicalaires de cuivre et nickel. 2 Nous présentons dans ce poster les premiers résultats obtenus avec des complexes de fer (métal dont les préférences géométriques se rapprochent du manganèse) formés à partir du ligand de Jacobsen ainsi que ses dérivés dont les phénols sont enrichis en électrons. Les études par électrochimie, UV-Vis et RPE suggèrent que l’oxydation mono-électronique de complexes monomères et dimères phénolate-Fe(III) n’est pas centrée sur le métal, mais conduit à des radicaux phénoxyle coordinés à du Fer(III).

OFeIII - e-

+ e-O

FeIII

OFeIV

ou ?

1. W. Zhang, J. L. Loebach, S. R. Wilson, E. N. Jacobsen, J. Am. Chem. Soc. 1990, 112, 2801

2. M. Orio, O. Jarjayes, H. Kanso, C. Philouze, F. Neese, and F. Thomas, Angew. Chem. Int. Ed. 2010, 49, 4989

62

Synthesis of an antimicrobial agent directed against quinolinate synthase : 3-fluoro-aspartate

E. Ponce, O. Hamelin,a M. Fontecave,b S. Ollagnier de Choudensb

Laboratoire de Chimie et Biologie des Métaux, Equipe BioCE (a), Equipe Biocatalyse (b), CEA, iRTSV ; CNRS, UMR5249 ; Université Joseph Fourier, 38054 Grenoble Cedex 09, France

e-mail: sollagnier@cea.fr, ohamelin@cea.fr

Nicotinamide adenine dinucleotide (NAD) plays a crucial role as a cofactor in numerous

essential redox biological reactions1. It is possible to synthesize NAD by two different pathways which have a common intermediate, quinolinic acid (QA). The first pathway occurs in eukaryotes through the degradation of L-tryptophan. In pathogenic bacteria Mycobacterium tuberculosis, Mycobacterium leprae, Helicobater pylori, opportunistic bacteria Escherichia coli, Thermotoga maritima, … and in plant chloroplasts (Arabidopsis thaliana) it is synthesized through a second pathway from L-aspartate involving two enzymes, L-aspartate oxidase (NadB) that converts L-aspartate to Imino-aspartate and Quinolinate synthase (NadA), an oxygen sensitive Fe/S enzyme, that catalyzes the condensation between iminoaspartate and dihydroxyacetone phosphate to generate QA1. Whereas the eukaryotic pathway was well studied, the catalytic mechanism of quinolinate synthase NadA is poorly understood1. Besides the de novo synthesis of NAD, a salvage pathway may exist that enables NAD to be recycled (from nicotinic acid and nicotinamide. M. leprae and H. pylori were described as lacking such a salvage pathway and thus cannot recycle NAD. H. pylori colonizes various areas of the stomach and duodenum and is strongly linked to the development of duodenal and gastric ulcers and some stomach cancers. M. leprae is the causative agent of the disease leprosy that affected hundredth of thousand persons. The presence in most prokaryotes and eukaryotes of distinctly different pathways for the biosynthesis of quinolinic acid in addition to the absence of the salvage pathway for some microorganisms may reveal NadA as a key and novel target for the development of new specific antibacterial drugs. One objective of the NadA project is to shed light on the mechanism catalyzed by the quinolinate synthase at a molecular level, which so far has been a stumbling block in the elucidation of the de novo quinolinic acid biosynthesis pathway in bacteria. During my professional training at the CEA, I have synthesized the L-3-fluoro-aspartate, a substrate analogue, in order to inhibit NadA. This inhibitor could generate species unable to form quinolinate leading to an abortive mechanism. Synthesis of this analogue and its in vitro effect on quinolinate synthase activity are presented in the poster.

OP

O

HO H2N COOH

COOH

+

O

HO HN COOH

COOH HO

O HN COOH

COOHPiHO

N C OOH

C OOHHO

OH

NH

COOH

COOH H2 O

H2O

N COOH

COOH

OP

O

HOHN COOH

COOH

+

OP

HO

O H2N COOH

COOH

+

HO

N COOH

COOHH2O Pi

N COOH

COOH

OP

HO

H2O

Fe Fe

Fe Fe

(1)

(2)

Proposed mechanisms for quinolinic acid biosynthesis from DHAP and IminoAspartate. 1. Begley, T. P.; Kinsland, C.; Mehl, R. A.; Osterman, A.; Dorrestein, P. Vitam Horm 2001, 61, 103-19.

63

Synthesis and characterisation of copper (II) complexes incorporating bulky aromatic substituents

C. Colomban, N. Leconte, F. Thomas

Département de Chimie Moléculaire, Chimie Inorganique Redox Biomimétique (CIRE), UMR-5250, Université Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France

e-mail : cedrix.colomban@laposte.net

Galactose oxidase (GO) is a metalloenzyme of fungal origin that catalyzes the oxidation of a broad range of primary alcohols to aldehydes with concomitant reduction of molecular oxygen.1 The catalytic activity is essentially due to the presence in the GO active site of an organic radical (phenoxyl) located on the enzyme peptidic chain and coordinated to a copper ion.1

Scheme 1: Proposed catalytic mechanism for Galactose oxydase2 (left); studying complexes (right) Recently, our team has focused on the preparation of GO models with N3O donor ligands having a phenolic arm that is substituted in the ortho and para positions. Copper (II) complexes made from these tripodal ligands were found to be particularly interesting as they showed a stabilisation of the phenoxyl when electron-donating ortho/para substituents were used.2,3 To date, the effect of more sterically-demanding groups, i.e. aromatics, at the ortho position of the phenoxyl moiety has not been investigated. Bulky aromatics could bring not only stabilisation to the radical species but also fluorescence and intercalating properties to the copper complexes.

In this poster we present the synthesis of new family of copper complexes bearing bulky aromatic substituents as well as their characterization by the mean of X-Ray diffraction, electrochemistry, EPR and UV/Vis spectroscopies. 1. F. Michel, F. Thomas, S. Hamman, E. Saint-Aman, C. Bucher, J. -L.Pierre, Chem. Eur. J. 2004, 10, 4115. 2. F. Michel Thesis Association cuivre-radical phénoxyle: chimie en solution et modèles bio-inspirés de la Galatose Oxydase

2005,Université Joseph Fourier, Grenoble. 3. F. Thomas, Eur. J. Inorg. Chem. 2007, 2379.

64

Sensitization of Luminescent Lanthanides Doped Nanocrystals with Organic Ligands

N. Gauthier,a Daniel Imbert,a Olivier Raccurt,b Marinella Mazzantia

a) Laboratoire de Reconnaissance Ionique et Chimie de Coordination (RICC), Service de Chimie Inorganique et Biologique (SCIB), CEA, 17 rue des Martyrs, 38054 Grenoble, France

b) Département de Technologie des Nano-Matériaux, Laboratoire de nanoChimie et Sécurité des Nano-Matériaux, CEA, 17 rue des Martyrs, 38054 Grenoble, France

e-mail : nicolas.gauthier@cea.fr; daniel.imbert@cea.fr

Near-infrared (NIR) luminescent lanthanides exhibit useful properties that make them crucial components for applications such as photonic materials and optical telecommunication devices, as well as bioanalytical and biological imaging probes and sensors.1 Since f-f transitions are Laporte forbidden, free Ln3+ have low extinction coefficients, therefore it is necessary to sensitize these cations through a suitable chromophore/ligand with high molar absorption coefficient (“antenna effect”). However, this approach has intrinsic limitations because lanthanide luminescence is easily quenched through nonradiative routes when the cations are in close proximity to the vibrational overtones of -OH, -NH, and -CH groups present in the sensitizing ligand and/or solvent. This effect is particularly dramatic for NIR emitting Ln3+ because of relatively small energy gaps between ground and excited electronic states.

A strategy to overcome this drawback has been recently developed in sensitizing lanthanides doped nanocrystals with suitable organic chromophores.2 Indeed, the lanthanide cations are now well protected from the environment and is still sensitized by surrounding ligands at the surface. These materials exhibit intense fluorescent properties and could be useful for several applications.

In this context, we present here our contribution on the sensitization of visible and NIR emitting lanthanides doped nanocrystals (Tb or Yb for example) with functionalized bipyridine and terpyridine based ligands.3

1. S. V. Eliseeva, J.-C. G. Bünzli, Chem. Soc. Rev., 2010, 39, 189.

2. (a) J. Zhang, C. M. Shade, D. A. Chengelis, S. Petoud, J. Am. Chem. Soc. 2007, 129, 14834; (b) L. J. Charbonnière, J.-L. Rehspringer, R. Ziessel, Y. Zimmermann, New J. Chem., 2008, 32, 1055.

3. E. S. Andreiadis, R. Demadrille, D. Imbert, J. Pécaut, and M. Mazzanti, Chem. Eur. J. 2009, 15, 9458.

65

Synthesis of new transition metal complexes

containing aniline moieties

N. Leconte, A. Kochem, O. Jarjayes, F. Thomas

Département de Chimie Moléculaire, Equipe Chimie Inorganique REdox, UMR 5250 Université Joseph Fourier, BP 53, 38041 Grenoble Cedex 9

e-mail : nicolas.leconte@ujf-grenoble.fr

Galactose Oxidase (GO) belongs to a class of metalloenzymes that are able to generate an organic radical cofactor directly on their own peptidic chain to complete metal-driven electron-transfer. GO, whose active site includes a tyrosyl radical coordinated to a copper(II) ion, catalyzes the oxidation of a broad range of primary alcohols to aldehydes with concomitant reduction of molecular oxygen.1

Over the past 25 years many efforts have been devoted to the modeling of the copper(II)-phenoxyl entity of GO.2 Our team has focused on the preparation of GO models with special emphasis given to copper(II) complexes involving tripodal or salen ligands. Copper(II) complexes made from either N3O donor tripodal ligands having an phenolic arm, or salen-type ligands, exhibited a stabilisation of the phenoxyl radical when the phenol moieties were ortho/para disubstituted with electron-donating groups.3,4

To our knowledge no GO model built from aniline-based ligands has been studied. Replacing a phenol by an aniline would give original copper(II) complexes from which an anilinyl radical could be generated. In addition, employing such ligands could facilitate the preparation of copper(I) complexes owing to the azaphilic nature of copper(I) compared with copper(II).

In this poster we present the syntheses of original aniline-based ligands that have been used to prepare some new transition metal complexes (Scheme 1).

Scheme 1: Aniline-derived ligands studied.

1. J. A. Stubbe, W. Van Der Donk Chem. Rev. 1998, 98, 705.

2. F. Thomas Eur. J. Inorg. Chem. 2007, 2379.

3. F. Michel, F. Thomas, S. Hamman, E. Saint-Aman, C. Bucher, J.-L. Pierre Chem. Eur. J. 2004, 10, 4115.

4. M. Orio, O. Jarjayes, H. Kanso, C. Philouze, F. Neese, F. Thomas Angew. Chem. Int. Ed. 2010, 49, 4989.

NH2R

R

N

H2N R

R

N

NH2

R

R

N

N N

or Transition metals

66

Zinc finger/DOTA-Tb conjugates: toward selective Zn2+ and Ag+ sensors

A. Roux, J.-M. Latour, O. Sénèque

LCMB / PMB, UMR 5249 CNRS-CEA-UJF, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex

e-mail : olivier.seneque@cea.fr

Actuellement, la complexité du vivant nécessite des outils d’analyse de plus en plus pointus. Depuis quelques années, l’imagerie moléculaire, qui permet d’étudier sélectivement une molécule ou un ion, apparait comme une technique de choix pour étudier les phénomènes biologiques. C’est dans ce cadre là que s’inscrit notre sujet car nous essayons de développer des systèmes capables de détecter sélectivement certains cations tels que le zinc ou l’argent. En effet, l’homéostasie du zinc ou d’autres métaux physiologiques (Cu, Fe) est encore mal connue et sa dérégulation peut entrainer des maladies graves telles que la maladie d’Alzheimer. L’utilisation grandissante d’argent dans les nanotechnologies conduit aussi à la dispersion dans l’environnement de ce métal potentiellement toxique. Il convient donc de développer des sondes sélectives permettant la détection sélective de ces cations dans les milieux biologiques.

La conception de ces systèmes repose essentiellement sur deux critères majeurs : la complexation sélective du cation et la transformation de cette information en un signal détectable.

Dans notre projet, nous avons choisi de travailler sur des modèles peptidiques doigts de zinc. En effet, ceux-ci ont été particulièrement étudiés au laboratoire et montrent une forte sélectivité envers le zinc ainsi qu’un repliement du peptide lors de la complexation.

Pour la détection nous avons choisi d’exploiter la luminescence des lanthanides. Pour cela, nous avons incorporé dans nos peptides un tryptophane et un complexe DOTA-terbium. Le premier va jouer le rôle d’antenne (sensibilisation du lanthanide) tandis que le second sera l’émetteur permettant la détection. Les lanthanides sont particulièrement intéressants pour la luminescence car ils émettent dans le domaine du visible et possèdent un temps de demi-vie assez important qui permet de s’affranchir de la fluorescence du milieu biologique. De plus, l’émission du lanthanide va être sensible à la distance qui le sépare de l’antenne. Notre stratégie consiste à profiter du repliement du peptide en présence de zinc pour modifier la distance entre l’antenne et le lanthanide et donc les propriétés de luminescence.

Nous allons présenter ici nos premiers résultats concernant la synthèse de dérivés de doigt de zinc fonctionnalisés par un complexe DOTA-Tb et leurs propriétés de complexation et de luminescence.

67

Toward to a hybrid photocatalyst for hydrogen production

M . Bacchi,a M. Chavarot-Kerlidou,a M . Fontecave,a,b V. Arteroa

a) Laboratoire de Chimie et Biologie des Métaux, UMR CEA-CNRS-UJF-Grenoble 1, 17 rue des martyrs, Grenoble.

b) Collège de France, 11, place Marcelin Berthelot, 75231 Paris Cedex 05

e-mail : marine.bacchi@cea.fr

Molecular hydrogen is expected to be a major energy carrier in global human activity in the XXIth century. As this gas just exists in low quantity on Earth, the main issue is related to its production at low cost and in a sustainable manner.

In this context, sunlight-driven water-splitting appears as a very promising solution. Some microalgae, expressing hydrogenase enzymes coupled to photosystem I from the photosynthetic chain, are even capable to photolyze water and produce hydrogen.

Photosystem I is an optimized photosensitizer that can absorb ~45% of total solar radiation with a quantum yield that approaches unity. Upon light excitation, an electron with a low potential is transferred to a Fe/S cluster, called FB, that interact with a ferredoxin in the natural system. John H Golbeck has shown1 the possibility to modify FB in order to attach thiolate ligands further linked to platinum nanoparticles. In the presence of sodium ascorbate as a sacrificial electron donor, the PSI - nanoPt hydride system produces hydrogen under illumination.

We have developed in LCBM/Biocat group bio-inspired cobalt diimine-dioxime electrocatalysts based for hydrogen evolution2. We will report in this poster on the functionalization of such diimine-dioxime catalysts with thiolate groups for their ultimate attachment to the FB Fe/S cluster of a modified photosystem I.

1. XRebecca A. Grimme, Carolyn E. Lubner, Donald A. Bryant, and John H. Golbeck, JACS 2008, 130, 6308-6309.

2. Jacques, P.-A. ;Artero, V. ;Pécaut, J. ;Fontecave, M. , Proc. Natl. Acad. Sci. U.S.A., 2009, 106, 20627-20632.

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Evaluation de complexes métalliques en tant que ligands de G-quadruplexes par étude SPR

R. Bonneta, O. Jarjayesa, F. Thomasa, G. Pratvielb, P. Murata, N. Spinellia, A Van der Heydena,

P. Labbéa, P. Dumya and E. Defrancqa

a) Département de Chimie Moléculaire, UMR CNRS 5250, ICMG FR2607, Université Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France.

b) Laboratoire de Chimie de Coordination, CNRS, 205 route de Narbonne, 31077 Toulouse Cedex 4, France

e-mail : romaric.bonnet@ujf-grenoble.fr

De nombreux complexes métalliques sont développés actuellement pour interagir avec l’ADN notamment pour la conception d’anticancéreux. Parmi les stratégies utilisées, certaines visent les G-quadruplexes. En effet, ces structures secondaires d’ADN ou d’ARN formées via l’interaction de plusieurs guanines organisées sous forme de tétrades, sont actuellement de potentielles cibles pour des molécules anticancéreuses. Les applications les plus connues dans ce cadre se situent au niveau des télomères et des promoteurs d’oncogènes où de telles structures pourraient jouer un rôle d’ « interrupteurs biologiques ».

Dans ce cadre, de nombreuses équipes développent des ligands affins pour ces structures et divers complexes métalliques ont été testés tels que des porphyrines, des Salen, des Salphen, des terpyridines, etc. Comprendre où et comment des ligands interagissent avec ces structures peut permettre de mieux de les concevoir. Toutefois les G-quadruplexes sont des édifices complexes aux topologies variables qui diffèrent selon les boucles, l’orientation des brins et la taille des sillons. Cette diversité dépend des séquences utilisées et des conditions de formation. C’est pourquoi notre équipe travaille sur le développement de mimes de G-quadruplexes aux topologies contrôlées. La formation de ces mimes repose sur l’utilisation d’un gabarit peptidique qui, relié aux oligonucléotides via des liens chimiosélectifs, contraint la structure1. Actuellement cette stratégie est employée pour développer différents types de mimes de quadruplexes. L’utilisation de ces mimes a permis de développer un test de screening par SPR2 pour caractériser l’interaction des quadruplexes avec des ligands et d’étudier la sélectivité par rapport au duplexe.

Récemment, ces études se sont portées sur des ligands de type salen et porphyrines pour lesquels l’utilisation de métaux a permis d’obtenir un gain de sélectivité pour les G-quadruplexes. En effet, dans le cas des salen, le métal permet d’induire une géométrie particulière à la structure l’empêchant ainsi d’interagir avec le duplexe. Pour les porphyrines, l’utilisation de métaux présentant des ligands axiaux pourrait empêcher l’intercalation dans le duplexe obtenue pour d’autres ligands de ce type.

1. P. Murat, D. Cressend, N. Spinelli, A. Van der Heyden, P. Labbé, P. Dumy, E. Defrancq ChemBioChem 2008, 9, 2588-2591.

2. P. Murat, R. Bonnet, A. Van der Heyden, N. Spinelli, P. Labbé, D. Monchaud, M.-P. Teulade-Fichou, P. Dumy, E. Defrancq, Chem. Eur. J ., 2010, 16, , 6106-6114e.

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COORDONNEES

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Liste des participants

AMARA Patricia IBS / METALLO patricia.amara@ibs.fr

ANCEL Laetitia SCIB / RICC laetitia.ancel@cea.fr

ANDREIADIS Eugen LCBM / BIOCAT eugen.andreiadis@cea.fr

ARRAGAIN Simon LCBM / BIOCAT simon.arragain@cea.fr

ARTERO Vincent LCBM / BIOCAT vincent.artero@cea.fr

ASSFELD Xavier SRSMC / CBT xavier.assfeld@cbt.uhp-nancy.fr

ATTA Mohamed LCBM / BIOCAT mohamed.atta@cea.fr

BACCHI Marine LCBM / BIOCAT marine.bacchi@cea.fr

BARBIER Elodie IBS / GSY elodie.barbier@ibs.fr

BELLE Catherine DCM / CIRE catherine.belle@ujf-grenoble.fr

BERGGREN Gustav LCBM / BIOCAT gustav.berggren@cea.fr

BERSCH Beate IBS / RMN beate.bersch@ibs.fr

BOCHOT Constance DCM / CIRE constance.bochot@ujf-grenoble.fr

BONNET Romaric DCM / I2BM romaric.bonnet@ujf-grenoble.fr

CARBONI Michaël LCBM / PMB michael.carboni@cea.fr

CAUX-THANG Christelle LCBM / PMB christelle.caux-thang@cea.fr

CAVAZZA Christine IBS / METALLO christine.cavazza@ibs.fr

CHERRIER Mickaël IBS / METALLO mickael.cherrier@ibs.fr

CHIARI Lucile LCBM / BIOCE lucile.chiari@cea.fr

CISSE Cheikna LCBM / BIOMET cheickna.cisse@cea.fr

COBO Saioa LCBM / BIOCAT saioa.cobo@cea.fr

COLIN Florent LCBM / BIOCAT florent.colin@cea.fr

COLOMBAN Cédric DCM / CIRE cedrix.colomban@laposte.net

COVES Jacques IBS / METALLO jacques.coves@ibs.fr

DE REUSE Hilde IP / PHP hdereuse@pasteur.fr

DE ROSNY Eve IBS / METALLO eve.derosny@ibs.fr

DELANGLE Pascale SCIB / RICC pascale.delangle@cea.fr

D'HARDEMARE Amaury DCM / CIRE amaury.d-hardemare@ujf-grenoble.fr

DUARTE Victor LCBM / PMB victor.duarte@cea.fr

DUBOC Carole DCM / CIRE carole.duboc@ujf-grenoble.fr

ESMIEU Charlène LCBM / BIOCE charlene.esmieu@cea.fr

ESTELLON Johan BGE / EDyP johan.estellon@cea.fr

FAUQUANT-PECQUEUR Caroline LCBM / BIOMET caroline.pecqueur@cea.fr

FONTECAVE Marc LCBM / BIOCAT marc.fontecave@cea.fr

FONTECILLA-CAMPS Juan IBS / METALLO juan.fontecilla@ibs.fr

GAUTHIER Nicolas SCIB / RICC nicolas.gauthier@cea.fr

GEREZ Catherine LCBM / BIOCAT catherine.gerez@cea.fr

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GIBARD Clémentine LCBM / BIOCE clementine.gibard@cea.fr

GIRGENTI Elodie LCBM / BIOCE elodie.girgenti@cea.fr

HAMELIN Olivier LCBM / BIOCE olivier.hamelin@cea.fr

HUGOUVIEUX Véronique LPCV / RPSEML veronique.hugouvieux@cea.fr

IANNELLO Marina IBS / METALLO marina.iannello@ibs.fr

IMBERT Daniel SCIB / RICC daniel.imbert@cea.fr

JACQUES Aurélie LCBM / PMB aurelie.jacques@cea.fr

JAMET Hélène DCM / CT hjamet@ujf-grenoble.fr

JARJAYES Olivier DCM / CIRE olivier.jarjayes@ujf-grenoble.fr

JORGE-ROBIN Adeline LCBM / BIOCE adeline.jorge-robin@cea.fr

KOCHEM Amélie DCM / CIRE amelie.kochem@hotmail.fr

LEBRETTE Hugo IBS / METALLO hugo.lebrette@ibs.fr

LEBRUN Vincent LCBM / PMB vincent.lebrun@cea.fr

LECARME Lauréline DCM / CIRE laureline.lecarme@gmail.com

LECONTE Nicolas DCM / CIRE nicolas.leconte@ujf-grenoble.fr

MAILLARD Antoine IBS / METALLO antoine.maillard@ibs.fr

MARCHI-DELAPIERRE Caroline LCBM / BIOCE caroline.marchi-delapierre@cea.fr

MARINONI Elodie IBS / METALLO elodie.marinoni@ibs.fr

MARTIN Lydie IBS / METALLO lydie.martin@ibs.fr

MAZZANTI Marinella SCIB / RICC marinella.mazzanti@cea.fr

MENAGE Stéphane LCBM / BIOCE stephane.menage@cea.fr

MICHAUD-SORET Isabelle LCBM / BIOMET imichaud@cea.fr

MOLLE Thibaut LCBM / BIOCAT thibaut.molle@cea.fr

MOREAU Yohann LCBM / MCT yohann.moreau@cea.fr

NICOLET Yvain IBS / METALLO yvain.nicolet@ibs.fr

NIEDZWIECKA Agnieszka SCIB / RICC agnieszka.niedzwiecka@cea.fr

OLLAGNIER DE CHOUDENS Sandrine LCBM / BIOCAT sollagnier@cea.fr

OZEIR Mohammad LCBM / BIOCAT mohammad.ozeir@cea.fr

PAGNIER Adrien IBS / METALLO adrien.pagnier@ibs.fr

PARENT Aubérie LCBM / PMB auberie.parent@cea.fr

PETIT HARTLEIN Isabelle IBS / METALLO isabelle.petit-hartlein@ibs.fr

PETOUD Stéphane CBM / ISCV stephane.petoud@cnrs-orleans.fr

PHAM Catherine ISTerre catherine.pham@ujf-grenoble.fr

PIERREL Fabien LCBM / BIOCAT fabien.pierrel@cea.fr

PONCE Elodie LCBM / BIOCE elodie.ponce@cea.fr

PUCCIO Hélène IGBMC / PRA hpuccio@igbmc.u-strasbg.fr

ROUX Amandine LCBM / PMB amandine.roux@cea.fr

SCHILD Florie PCV / RPSEML florie.schild@cea.fr

SENEQUE Olivier LCBM / PMB olivier.seneque@cea.fr

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STOLL Thibaud DCM / CIRE stoll.thibaut@gmail.com

TALLEC Gaylord SCIB / RICC gaylord.tallec@cea.fr

THOMAS Fabrice DCM / CIRE fabrice.thomas@ujf-grenoble.fr

TIREL Emmanuel LCBM / BIOCE emmanuel.tirel@cea.fr

TORELLI Stéphane LCBM / CRBIO stephane.torelli@cea.fr

TREPREAU Juliette IBS / METALLO juliette.trepreau@ibs.fr

TRON Céline LCBM / BIOCAT cecile.tron@cea.fr

ZEPPIERI Laura IBS / METALLO laura.zeppieri@ibs.fr

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Liste des Laboratoires

SRSMC / CBT Groupe Chimie et Biochimie Théorique, Laboratoire Structure et Réactivité des Systèmes Moléculaires Complexes, UMR 7565 CNRS-UHP, Boulevard des Aiguillettes, BP 70239, 54506 Vandoeuvre-lès-Nancy Cedex

IP / PHP Unité Pathogenèse de Hélicobacter, Institut Pasteur, Département de Microbiologie, Bâtiment Fernbach, 25 Rue du Dr. Roux, 75724 Paris Cedex 15

CBM / ISCV Groupe Imagerie, Spectroscopie et Chimie du Vivant, Centre de Biophysique Moléculaire, UPR 4301 CNRS, Rue Charles Sadron, 45071 Cedex 2 Orléans

IGBMC / PRA Equipe Physiopathologie des Ataxies Récessives, Institut de Génétique et de Biologie Moléculaire et Cellulaire, B.P. 10142, 67404 Illkirsch Cedex

IBS / METALLO Groupe Métalloprotéines, Institut de Biologie Structurale J. P. Ebel, UMR 5075 CEA-CNRS-Université Joseph Fourier, 41 rue J. Horowitz, 38027 Grenoble Cedex 1

IBS / RMN Groupe RMN Biomoléculaire, Institut de Biologie Structurale J. P. Ebel, UMR 5075 CEA-CNRS-Université Joseph Fourier, 41 rue J. Horowitz, 38027 Grenoble Cedex 1

IBS / GSY Groupe Synchrotron, Institut de Biologie Structurale J. P. Ebel, UMR 5075 CEA-CNRS-Université Joseph Fourier, 41 rue J. Horowitz, 38027 Grenoble Cedex 1

ISTerre Institut des Sciences de la Terre, Université Joseph Fourier, BP 53, 38041, Grenoble

DCM / CIRE Equipe Chimie Inorganique Rédox, Département de Chimie Moléculaire, 301 rue de la Chimie, 38041 Grenoble Cedex 9

DCM / I2BM Equipe Ingénieurie et Intéractions Biomoléculaires, Département de Chimie Moléculaire, 301 rue de la Chimie, 38041 Grenoble Cedex 9

DCM / CT Equipe Chimie Théorique, Département de Chimie Moléculaire, 301 rue de la Chimie, 38041 Grenoble Cedex 9

SCIB / RICC Laboratoire de Reconnaissance Ionique et Chimie de Coordination, Service de Chimie Inorganique et Biologique, UMR_E 3 CEA UJF, FRE CNRS 3200, INAC, CEA-Grenoble, 17 avenue des Martyrs, 38054 Grenoble cedex 9

LCBM / BIOCAT Equipe Biocatalyse, Laboratoire de Chimie et Biologie des Métaux, UMR 5249 CNRS-CEA-UJF, iRTSV, 17 rue des Martyrs, 38054 Grenoble

LCBM / PMB Equipe Physico-chimie des Métaux en Biologie, Laboratoire de Chimie et Biologie des Métaux, UMR 5249 CNRS-CEA-UJF, iRTSV, 17 rue des Martyrs, 38054 Grenoble

LCBM / BIOCE Equipe Catalyse Bioinorganique et Environnementale, Laboratoire de Chimie et Biologie des Métaux, UMR 5249 CNRS-CEA-UJF, iRTSV, 17 rue des Martyrs, 38054 Grenoble

LCBM / BIOMET Equipe Biologie des Métaux, Laboratoire de Chimie et Biologie des Métaux, UMR 5249 CNRS-CEA-UJF, iRTSV, 17 rue des Martyrs, 38054 Grenoble

LCBM / MCT Equipe Modélisation et Chimie Théorique, Laboratoire de Chimie et Biologie des Métaux, UMR 5249 CNRS-CEA-UJF, iRTSV, 17 rue des Martyrs, 38054 Grenoble

BGE / EDyP Equipe Etude de la Dynamique des Protéomes, Laboratoire de Biologie à Grande Echelle, UMR_S 1038 CEA-INSERM-UJF, iRTSV, 17 rue des Martyrs, 38054 Grenoble

PCV / RPSEML Equipe Réponse de la Plante aux Stress Environnementaux et Métaux Lourds, Laboratoire Physiologie Cellulaire et Végétale, UMR 5168 CEA-CNRS-INRA-UJF, iRTSV, 17 rue des Martyrs, 38054 Grenoble