[en] Considering the environmental impact of the Fukushima nuclear accident, it is fundamental to study the mechanisms governing the effects of the released radionuclides on the biosphere and thus identify the molecular processes generating the transport and deposition of actinides, such as neptunium and uranium. However, the information about the microscopic aspect of the interaction between actinides and biological molecules (peptides, proteins...) is scarce. The data being mostly reported from a physiological point of view, the structure of the coordination sites remains largely unknown. These microscopic data are indeed essential for the understanding of the interdependency between structural aspect, function and affinity.The Calmodulin (CaM) (abbreviation for Calcium-Modulated protein), also known for its affinity towards actinides, acts as a metabolic regulator of calcium. This protein is a Ca carrier, which is present ubiquitously in the human body, may also bind other metals such as actinides. Thus, in case of a contamination, actinides that bind to CaM could avoid the protein to perform properly and lead to repercussions on a large range of vital functions.The complexation of Np and U was studied by EXAFS spectroscopy which showed that actinides were incorporated in a calcium coordination site. Once the thermodynamical and structural aspects studied, the impact of the coordination site distortion on the biological efficiency was analyzed. In order to evaluate these consequences, a calorimetric method based on enzyme kinetics was developed. This experiment, which was conducted with both uranium (50 - 500 nM) and neptunium (30 - 250 nM) showed a decrease of the heat produced by the enzymatic reaction with an increasing concentration of actinides in the medium. Our findings showed that the Calmodulin actinide complex works as an enzymatic inhibitor. Furthermore, at higher neptunium (250 nM) and uranium (500 nM) concentration the metals seem to have a poison-like behavior and 'kill' completely the enzymatic activity. (author)

[en] The determination of the binding constant between transferrin and thorium and the conformational changes of the protein upon metal complexation (thorium and plutonium) have been studied by both capillary electrophoresis (CE) and capillary isoelectric focusing (cIEF) coupled with inductively coupled plasma mass spectrometry (ICP-MS). This method allows the use of both the separation power of the cIEF and the low detection limit of ICP-MS which is critical when working with highly radioactive elements. To our knowledge, this is the first time a method coupling cIEF and ICP-MS is reported in the literature. Nitrilotriacetate was used to prevent from actinide hydrolysis and as a competitive ligand with transferrin. The binding constant for the complexation of transferrin and thorium, in the absence of bicarbonate at pH 7, was found to be log K = 18.65 ± 0.19. This value, close to that of transferrin with iron, evidenced the high affinity of the protein for thorium. The results obtained by the newly developed method, cIEF-ICPMS, showed no pI change after the addition of thorium or plutonium, whereas a pI shift (linked to conformational changes) occurred for the transferrin-indium complex. This suggests that, despite the high affinity towards the actinides, the protein does not undergo a significant structure change upon complexation. The important ionic radius of the cations Th⁴⁺ (1.05 angstrom, CN = 8) and Pu⁴⁺ (0.96 angstrom, CN = 8) with respect to Fe³⁺ (0.645 angstrom, CN = 6) and to a lesser extent to In³⁺ (0.800 angstrom, CN = 6) suggests that the transferrin lobe does not close completely after complexation. However, mixed indium-actinide complexes showed structural changes even at high concentrations of apotransferrin. The conformational change is not governed by the actinide but by the other metals present. (authors)

[en] After entering the blood, plutonium accumulates mainly in the liver and the bones. The mechanisms leading to its accumulation in bone are, however, completely unknown. We already know that another uptake pathway not involving the transferrin-mediated pathways is suspected to intervene in the case of the liver. Fetuin, a protein playing an important role in bone metabolism, is proposed as a potential transporter of Pu from serum to bone. For the first time, the binding constants of these two proteins (transferrin and fetuin) with tetravalent plutonium at physiological pH (pH 7.0) were determined by using capillary electrophoresis (CE) coupled with inductively coupled plasma mass spectrometry (ICP-MS). Their very close values (log(10) K-PuTf = 26.44 ± 0.28 and log(10) K-PuTf = 26.20 ± 0.24, respectively) suggest that transferrin and fetuin could compete to chelate plutonium, either in the blood or directly at bone surfaces in the case of Pu deposits. We performed competition reaction studies demonstrating that the relative distribution of Pu-protein complexes is fully explained by thermodynamics. Furthermore, considering the average concentrations of transferrin and fetuin in the blood, our calculation is consistent with the bio-distribution of Pu observed in humans. (authors)

[en] The threat of a dirty bomb which could cause internal contamination has been of major concern for the past decades. Because of their high chemical toxicity and their presence in the nuclear fuel cycle, uranium and neptunium are two actinides of high interest. Calmodulin (CaM) which is a ubiquitous protein present in all eukaryotic cells and is involved in calcium-dependent signaling pathways has a known affinity for uranyl and neptunyl ions. The impact of the complexation of these actinides on the physiological response of the protein remains, however, largely unknown. An isothermal titration calorimetry (ITC) was developed to monitor in vitro the enzymatic activity of the phosphodiesterase enzyme which is known to be activated by CaM and calcium. This approach showed that addition of actinyl ions (AnO₂ⁿ⁺), uranyl (UO₂²⁺) and neptunyl (NpO₂⁺), resulted in a decrease of the enzymatic activity, due to the formation of CaM-actinide complexes, which inhibit the enzyme and alter its interaction with the substrate by direct interaction. Results from dynamic light scattering rationalized this result by showing that the CaM-actinyl complexes adopted a specific conformation different from that of the CaM-Ca²⁺ complex. The effect of actinides could be reversed using a hydroxypyridonate actinide decorporation agent (5-LIO(Me-3,2-HOPO)) in the experimental medium demonstrating its capacity to efficiently bind the actinides and restore the calcium-dependent enzyme activation. (authors)

[en] Better understanding of uranyl-protein interactions is a prerequisite to predict uranium chemical toxicity in cells. The EF-hand motif of the calmodulin site I is about thousand times more affine for uranyl than for calcium, and threonine phosphorylation increases the uranyl affinity by two orders of magnitude at pH 7. In this study, we confront X-ray absorption spectroscopy with Fourier transform infrared (FTIR) spectroscopy, time-resolved laser-induced fluorescence spectroscopy (TRLFS), and structural models obtained by molecular dynamics simulations to analyze the uranyl coordination in the native and phosphorylated calmodulin site I. For the native site I, extended X-ray absorption fine structure (EXAFS) data evidence a short U-O${}_{eq}$ distance, in addition to distances compatible with mono- and bidentate coordination by carboxylate groups. Further analysis of uranyl speciation by TRLFS and thorough investigation of the fluorescence decay kinetics strongly support the presence of a hydroxide uranyl ligand. For a phosphorylated site I, the EXAFS and FTIR data support a monodentate uranyl coordination by the phosphoryl group and strong interaction with mono- and bidentate carboxylate ligands. This study confirms the important role of a phosphoryl ligand in the stability of uranyl-protein interactions. By evidencing a hydroxide uranyl ligand in calmodulin site I, this study also highlights the possible role of less studied ligands as water or hydroxide ions in the stability of protein-uranyl complexes. (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim)

[en] The first session of this colloquium concerned the nuclear fuel: elaboration of nanoparticles of actinide oxides, study of Na-U-O and Na-Np-O phase diagrams, relationships between precursors and sintering behaviour, study of uranium IV and thorium IV speciation. The second session addressed fuel processing: semi-quantitative analysis of metallic cations in organic phase, cathodic reduction of plutonium IV solutions, study of paramagnetism of actinide complexes, complexing of actinides III and IV, behaviour under alpha radiation of oximes and influence on redox properties of actinides. The third session addressed radionuclides and geosphere: study of the reactivity of surfaces of clayey phases, study of alpha-Al₂O₃/fulvic acid/Eu III interactions, determination of microscopic interactions between actinides and humic substances. Then, after a contribution of the evolution of the use of modelling tools of atmospheric dispersion and of the health and environmental impact in the support to the management of radiological emergencies, a fourth session addressed the relationship between radiochemistry and health: bio-accumulation and speciation of europium, mechanisms of interaction between actinides and a protein, speciation of (poly)molybdate and (poly)tungstate ions, study of astatine chemistry by means of quantum molecular modelling. The fifth session addressed storage safety: C¹⁴ diffusion in used fuel claddings, migration of deuterium in nuclear graphite, quantification of the production of radiolysis gas in radioactive waste parcels. The sixth session addressed tools for radiochemistry: advances on MARS (the beamline dedicated to radioactive materials at synchrotron SOLEIL), from radiolysis to radiochemistry (speciation of radio-elements under alpha and gamma radiation), presentation of γ3 (an electron/photon spectrometer with a high resolution and low background noise for the detection of ultra-traces of fission and activation products in the environment), analysis of wastes coming from the nuclear industry. Posters addressed the following issues: EF hand affins and uranium specific patterns engineering, Production of ⁶⁴Cu radionuclides, Plutonium electrochemical kinetics in nitric acid, Study of the self-radiolysis of tritiated water, Study of uranium complexing by phenylphosphonate, Synthesis, structure and solubility of Rhabophane as a monazite precursor, Determination of ⁹³Zr in radioactive effluents, Study of Tc redox in carbonate environment induced by radiolysis, Behaviour of rare gases in uranium-based nuclear fuels, Redox properties of Samarium in ionic liquids, Some chemical separations by chromatography developed for analytic purposes, Apatite composition effect (U-Th)/He thermo-chronometer, Coordination chemistry of uranium VI, In-situ Raman spectroscopy for the study of nuclear fuel salts, Dissolution of Thorium oxides, BDD/Si-PIN detector for direct spectrometry of actinides, Competition between chelating agents and identified target proteins of actinides, Nano-size metallic oxide cluster formation in Fe alloy by ion implantation.