Assist Prof Maria Rosa Beccia

Prof Gaëlle Creff

Prof Den Christophe
email: christophe.denauwer@univ-cotedazur.fr

Auwer

Prof Di Christophe

Giorgio

Dr Aurélie Jeanson

Prof Michel Hervé

Gaston Bachelard

Environmental Chemistry of Radionuclides : Open Questions and Perspectives

Since the discovery of nuclear fission, atomic energy has become for mankind a source of energy, but it has also become a source of consternation. This Perspective presents and discusses the methodological evolution of the work performed in the radiochemistry laboratory that is part of the Institut de Chimie de Nice (France). Most studies in radioecology and environmental radiochemistry have intended to assess the impact and inventory of very low levels of radionuclides in specific environmental compartments. But chemical mechanisms at the molecular level remain a mystery because it is technically impossible (due to large dilution factors) to assess speciation in those systems. Ultra-trace levels of contamination and heterogeneity often preclude the use of spectroscopic techniques and the determination of direct speciation data, thus forming the bottleneck of speciation studies. The work performed in the Nice radiochemistry laboratory underlines this effort to input speciation data (using spectroscopic techniques like X ray Absorption Spectroscopy) in environmental and radioecological metrics.

Introduction

Since the discovery of nuclear fission by O. Hahn, F. Strassmann and L. Meitner in 1938, mankind has come to rely on atomic energy as a source of power. [START_REF] Gosling | The Manhattan Project: Making the Atomic Bomb (DOE/MA-0001)[END_REF] Since then the nuclear industry, both military and civil, expanded dramatically after World War II and it globally continues to do so, although at different pace (depending on the country) until today. This, of course, has always raised social, political and scientific concerns over an eventual accident or misuse of nuclear power. Anecdotally, early misuse of radioactivity indeed occurred as early as in the 30s as exemplified by the Tho-Radia adventure, among others. [START_REF] Zaga | [END_REF] In 1947 a group of nuclear physicists created the Doomsday Clock to warn politicians about the use of nuclear weapons. This clearly demonstrated that the threat of nuclear disaster was felt throughout the world. In addition to concerns about defense applications, questions have also been raised regarding nuclear toxicology, legacy, long-term management of waste and the impact on the environment. Although exposure to radionuclides and radiation does not constitute a day-to-day issue of public health, concerns become more acute in the case of a nuclear event or of a deficiency in waste management. Most importantly, the possibility of exposure raises constant questioning from society and underlines the need for information and scientific input. Therefore, the risks related to radionuclides environmental contamination and its impact on ecosystems and humans must be better understood and assessed in order to prevent catastrophe. Of all the elements naturally occurring on Earth, uranium and thorium are the last on the Periodic Table (although ultra trace levels plutonium are also produced from uranium spontaneous fission and further neutron capture) and are used for the nuclear civil industry (thorium to a much lesser extent). Plutonium is one of the most emblematic elements of this industry and was synthesized in 1942 by G. T. Seaborg in Berkeley using a cyclotron accelerator. The three major civilian accidents of Three Mile Island, Chernobyl and Fukushima in March 1979, April 1986 and March 2011 respectively contributed significantly for the need to assess the impact of radionuclide release in the environment. At the same time, remediation technologies have always been a challenging operation since they are highly dependent on the physical-chemical history of the site under consideration. Finally legacy of historical nuclear sites like Mayak, Sellafield and Hanford (not exhaustive) also contributes to radionuclides dissemination. These factors, in addition to the general, social and political concerns on environmental impact, continue to drive fundamental scientific questions regarding any further accidental release of radionuclides on the Earth's surface. Today, on the other hand, in the context of climate change and the goals established in the 2015 Paris Agreement, nuclear power has been identified as a viable alternative (although not unique) to the use of fossil fuel-based energies. [3] Most studies in radioecology and environmental radiochemistry have intended to assess the impact and inventory of very low levels (see definition further down) of radionuclides in specific compartments (e.g., minerals, sediments, water, plants, organisms). This very helpful approach allows monitoring the transfer and accumulation momenta at very large scales but often fails to describe the chemical form of the element in question because such information is unavailable at this concentration level. Therefore, chemical mechanisms at the molecular level remain hardly decipherable because it is technically impossible (due to large dilution factors) to assess direct speciation. From the IUPAC gold book, speciation is defined as the "distribution of an element amongst defined chemical species in a system". [START_REF]IUPAC gold book[END_REF] In consequence a consequence, not only the quantity (the dose) but also the chemical form (speciation) are essential input data. [START_REF] Wolfe | Radioecology: Proceedings of the First National Symposium on Radioecology Held at Colorado State University[END_REF] Despite those limitations, attempts to better understand the migration and accumulation transfers to the environment and ecosystem have raised further questions. Data on the subject are indeed largely abundant in the literature and this article does not intend to review it. The following references published over the last decade may be considered as more exhaustive for the reader: Hu et al., [START_REF] Hu | [END_REF] Sachs et al., [7] Walter et al., [8] Kersting, [9] Maher et al., [10] Romanchuk et al., [11] Metian et al., [12] G. J-P. Deblonde et al.. [13] This opinion article presents the methodological evolution of the work performed in the Nice radiochemistry laboratory (Nice, Alpes Maritimes, France), now part of Institut de Chimie de Nice (ICN), in environmental chemistry of radionuclides and lies on a selection (not exhaustive) of literature data. Since the beginning of environmental chemistry research in Nice in 1980, the approach has shifted over time from pure radioecology to a radioecology combined with environmental radiochemistry and even biochemistry. This could only be done with the use of chemical, biological and spectroscopic tools in conjunction with analytical tools. The major difficulty in achieving this goal is to combine two orthogonal scales: the ultra-trace concentration scale on the one hand (in atoms per volume or surface units) and the large spatial heterogeneities of the field on the other hand (in length units).

Foreword, chemistry at the ultra-trace levels.

The chemistry of elements at the ultra-trace levels (< 1 ppb, vide infra) is strongly linked to the discoveries in radiochemistry in the search for trace radionuclides (Marie Curie's work) or for very short life radionuclides. At this level of concentration (in atoms per cm 3

), reactivity may be shifted away from the mass action law and classical thermodynamics toward statistical thermodynamics. Kinetics might also be affected, although the border between traditional and ultra-trace chemistry is not well defined. It is not the purpose of this note to discuss the reactivity of ultra-trace levels and the reader can refer to the article of Guillaumont et al.. [14] Nevertheless the limits of the classical description of chemical reactivity described for ultra-trace radiochemical studies also find justifications in environmental studies because of enormous dilution factors that often occur in the environment.

Arriving at a definition of environmental studies with radionuclides

In environmental chemistry studies, the term Heavy Metals has been the subject of long controversy in the scientific literature because it is ambiguous and does not reflect the long list of elements that are of concern for general environmental studies. In 2021, Pourret et al. stated that Heavy Metals is "a very imprecise term, never rigorously defined by any authority, and loosely used to refer to an element and its compounds". [15] Consequently, Metallic Trace Elements (MTE) or Metalloid Trace Elements are also frequently used. Both terms are not strict synonyms and this might be a source of ambiguity. In the case of environmental radiochemistry or radioecology, the elements of interest for environmental studies cover almost all the families of the periodic table, from alkaline to noble gas (i.e., fission products, activation products, actinides). The generic term Trace Radionuclides (TR) might therefore be perfectly appropriate together with the sub ensemble Metallic Trace Radionuclides (MTR) which could be synonymous with radioactive MTE. When dealing with TR or MTR, two types of properties are to be considered: their chemical behavior (as chemical elements) and their radiation impact (as radionuclides). The first one refers to electronic properties, the second one to nuclear properties and both properties are almost disconnected. One should also keep in mind that most elements possess stable and unstable isotopes while only elements after Bi in the Periodic Table possess only unstable isotopes with half-lives ranging from a few picoseconds to billions of years (from 83 Bi to 118 Og to date). The first property obviously affects the chemical equilibria and reactivity; the second property mainly affects the biological mechanisms but also chemical states in some cases because of the impact of radiation (type and dose). Depending on the element and its isotope, both aspects (chemistry and radiation) might be worth considering, or one aspect might be neglected over the second one. On one hand, the environmental chemistry of radionuclides refers to the chemical study of TR or MTR in environmental systems. On the other, radioecology is a subdivision of ecology that "emerged as a scientific discipline at the end of World War II, in response to environmental problems from radioactive fallout associated with nuclear weapons testing". [16] This historical view of radioecology seems to exclude, although by omission, possible effects of natural radioactivity (radiations) on ecosystems. But the origin of MTR does not directly affect the dose effect, nor its thermodynamics or kinetics. In short, the environmental chemistry of radionuclides and radioecology are distinct but complementary approaches that may overlap in some cases in their goal to study the impact of MTR and their radiation on ecosystems (which may or may not include humans). The main factors under consideration are the radionuclide (its chemical and radiation properties), the object of interest (environmental compartments, biotopes, living species, humans included or not, dose-effect relationships at individual organism level), and its concentration scale. Although radioecology emerged in the 50s, one of the first radioecology symposia was held at Colorado State University in 1961. [START_REF]Radioecology: Proceedings of the First National Symposium on Radioecology Held at Colorado State University[END_REF] Introductory remarks from J. N. Wolfe implicitly paved the way for complementarity between ecology and environmental chemistry within the scope of nuclear sciences: "The last assessment of experimental result in biology must be ecological, and the understanding of the environment and its working complex is likely to be essential for survival". [START_REF] Jorgensen | Mammals of the Nevada Test Site[END_REF] This approach contributed to the development of accumulation data. In 1966 in Stockholm, the symposium on "Radioecological concentration process" further defined the use of concentration factors (CF) to characterize the accumulation processes in living species. [START_REF] Aberg | Radioecological concentration processes In[END_REF] Since then, IAEA is constantly updating CF data in order to assess the impact of MTR dissemination. [START_REF]IAEA Library Cataloguing in Publication Data, Sediment distribution coefficients and concentration factors for biota in the marine environment[END_REF] In a recent article, Carvalho reviews the use of CF and their limitations as a useful concept to quantify the uptake and accumulation of radionuclides in aquatic biota. [21] In the Nice radiochemistry laboratory, environmental studies started in 1980 and ramped up shortly after the Chernobyl accident (1986) in order to track impact on land and water biota. One of the first articles published on the subject was issued in 1987. [22] This was shortly before what would become a long-lasting collaboration with IAEA Monaco on the determination of metrics in marine biota like "Radionuclides in macro algae at Monaco following the Chernobyl accident". [23], [24] In 1995, another article was published on TR transfer in trees with similar methodology. [25] Concentration factors (or direct concentration data) appeared therefore as key descriptive data for radioecological assessment. In those papers and in most radioecological studies, TR and MTR are present at the ultra-trace or trace scales (ultra-trace scale <1 ppb, trace scale <1 ppm) because very large dilution factors occur in the environment. Therefore, chemical toxicity might be considered negligible versus long-term radiation effects (radiotoxicity) although this might not be clearly defined. This is a point where the environmental chemistry of radionuclides and radioecology may become complementary: the input of speciation data is essential to understanding the biochemical pathways at the origin of possible chemical and radiological toxicities. However, this is not straightforward because chemical reactivity and speciation largely depend (although not exclusively) on the concentration scale whereas the nucleus decay process does not. Speciation is therefore a key parameter to be defined if chemical mechanisms underlying transfer and accumulation are to be described. Therefore, two difficulties immediately arise: concentration and heterogeneity.

Speciation in field studies [dealing with large dilution factors]

As mentioned above, most often large dilution factors of the contaminants appear when drifts or water flux are involved. Large heterogeneities and presence of hot spots may occur. Speciation at ultra-trace scale is also limited to analytical techniques that will give in best cases indirect speciation data. In consequence, radioecology and environmental chemistry of radionuclides have largely been based on Mass Spectrometry. Sequential extraction techniques may also bring valuable information, although indirectly. [26] At very low levels (below fg/g) recent advanced mass spectrometry techniques like Accelerated Mass Spectrometry (AMS) may be very useful. [27], [28] The use of radiometric techniques often requires preliminary radiochemical separation when direct and in particular Pu). [29] In tight collaboration with the International Atomic Energy Agency (IAEA) in Monaco, [30] such radiochemical separation techniques were implemented in the Nice radiochemistry laboratory for environmental samples by G. Ardisson in the 80s using co-precipitation steps. [31] In the 90s, Ardisson and coworkers implemented in Nice a separation methodology using a unique ion [32] The technique has been used, for instance, in the case of Belarus soils in the Chernobyl region. [33] For gamma emitters like 137 Cs or 110m [34], [35] Although speciation could not be directly addressed with those techniques and at such a level of concentration, inventories were performed and migration processes could be investigated.

In order to answer the question of direct speciation i.e., information on metal oxidation state and coordination sphere, spectroscopic techniques are needed. Most spectroscopic techniques (X-ray Absorption Spectroscopy, Vibrational Spectroscopy, Spectrophotometry to name a few) hardly detect ppm values. XAS spectroscopy, for instance, allows direct speciation analysis at a given electronic edge of a given element (central atom) in a complex matrix. However, the signal is averaged by all the central atoms lightened by the photon beam. Therefore, an optimum between beam size and sample heterogeneities must be found (see next section). Also, the technique remains restricted by detection limits in the range of ppm values. Laser Fluorescence Spectroscopy (time resolved) may be used at sub-ppm levels but only with elements that are fluorescent (UO 2 2+ , Eu 3+ and Cm 3+ notably). One possibility for overcoming the intrinsic limitation of concentration is to dope (or spike) the analyzed medium with a salt of the element of interest. It is important to underline some of the criteria and limitations of this methodology : i) the concentration after doping must be a compromise between the detection limit and the avoidance of any significant modification of the chemical equilibria; ii) the solubility limit of the element must not be reached in the given medium; iii) the chemical and physical form of the element must mimic, if possible, the source term in the environment (soluble form, particles etc.), iv) equilibria that are favored at ultra low concentrations in the environment might become invisible after doping at macro concentrations. Since 2011 at ICN, we have been working in the marine environment in collaboration with CEA DAM Ile de France. Seawater represents the largest proportion of the hydrosphere (ca 96.5%) and covers about 71% of the Earth's surface. Oceans and seas are also the geographical endpoints of rivers. Therefore, these large bodies of water act as a long-term repository for organic and inorganic pollutants. For this reason, the marine environment has often been monitored as a pollution bellwether. The origin of radionuclides in seawater is either anthropogenic (atmospheric nuclear tests from the Cold War, crash of satellites, accidental or industrial releases, etc.) or natural from the Earth crust composition (mainly uranium, but also thorium). In the case of uranium, over the last two decades, all of the speciation models have reported on the formation of carbonate species as predominant. [START_REF] Choppin | Actinide speciation in aquatic systems[END_REF] However, the speciation of uranium in seawater has been lacking experimental spectroscopic data. Using the doping strategy described above combined with XAS measurements, Maloubier et al. have determined uranium (VI) and neptunium(V) speciation in natural seawater samples that were previously doped with uranium and neptunium salts (the doping value of [U, Np] = 5x10 -5 M was used in this study). [37] Figure 1 shows the concentration scale of most major ions in seawater compared to the selected doping concentration in this study.

Figure 1: Schematic scale representing the major components of natural seawater compared to the working range in the described methodology. Scale is not linear, values are taken from the MBARI web site. [38] Direct structural data were then obtained with EXAFS at the uranium L III edge and neptunium L II edge. The occurrence of an uranyl (UO 2 2+ ) carbonate species has been confirmed for uranium in seawater, very similar to the uranium local environment in the crystal structure of Liebigite and to uranyl carbonate species in pure aqueous solution. For neptunyl (NpO 2 + ), EXAFS data indicate the presence of at least one carbonate ligand, but a mixing with the aquo form cannot be precluded. 37 The case study of uranium speciation in seawater shows that the main limitation of XAS (as for other spectroscopic techniques) may not only be linked to low concentrations of the investigated element, but also to interference from other elements in the matrix (in aqueous medium heterogeneities are excluded). In fact, at the uranium L III edge, difficulties in obtaining data with a very good signal-to-noise ratio in those conditions come also from the bromine emission line (Kβ1) and the strontium emission present in seawater (Figure 1).

Speciation in field studies, [dealing with spatial heterogeneities]

Environmental compartments and samples are intrinsically defined by strong spatial heterogeneities bioaccumulation occur, km when seawater drifts or catchment basins are studied, to name a few). The radionuclide speciation is therefore subject to spatial definition, because different kinetic and thermodynamic conditions may tune the speciation at very low spatial scales (a few nm). Several reviews have summarized the different migration and accumulation pathways associated with heterogeneous (at the sub-micron scale) uranium and plutonium field samples using complementary imaging and spectroscopic micro-and nano-probes. [9], [39], [40] Brown et al. showed at Hanford Site (Washington State, USA) that contaminants are pluming in the zone beneath various Hanford Tank Farms. In this example, uranium is pluming at hundreds of meter scale from the Tank. Speciation le using X-ray Spectroscopy showed that uranium occurs in fractures dominantly as the uranophane group mineral. [41] In this specific example, speciation could be performed directly on (heterogeneous) environmental samples because the source contamination term is large enough (leading to hot spots and/or diffuse contamination). With similar spectroscopic tools, Bargar et al. described how biogeochemical processes can tune the uranium speciation within domains that are of the order of a few nm.

[42], [43] At a much larger spatial scale (few cm), the implementation in situ of DGT (Diffusive Gradient in Thin film) and DET (Diffusive Equilibrium in Thin film) in wetlands impacted by former mining activities in the French Massif Central region provides a vertical mapping of Nat U gradient. The depth profiles with fairly good spatial resolution (a few cm) show the correlation with the soil nature, the organic content matter, all factors combined with the history of the site. [44] In the Nice radiochemistry laboratory, a large program was launched in 2000 by G. Barci-Funel and H. Michel to investigate the fallout from the Chernobyl accident in the Boréon sector, located in the Alpes-Maritimes mountains, less than 100 km North of Nice (Alpes Maritimes, France). The study zone was an alpine wetland and surroundings, which consisted of a small lake, a clearing and a steep catchment in which MTR from nuclear test fallout and the Chernobyl accident are still detectable: from nuclear weapon tests ( 238,239+240 Pu and 241 Am) and from the Chernobyl nuclear power plant accident ( 137 Cs). The main question was to understand the large scale distribution of selected MTR in various environmental compartments (soil, sediments, water) and their mobility from the aqueous phase to the soil and sediment surface and subsurface. In the lake sediments, direct deposits of the radioactive fallout were detected in depth due to the effect of sedimentation causing MTR to migrate from the ground surface to the sub-surface. The transfer between those two natural compartments has shown the importance of water as a transport vehicle and the prevalence of leaching phenomena. Leaching carries MTR from the drainage basin slopes, to the clearing and then to the sediment through the aqueous phase. [45] In sediment samples, MTR have a low relative mobility, 241 Am being the most mobile element and 137 Cs the less mobile, 238,239+240 Pu having an intermediate behavior. [46] The vertical distribution of Cs was explained by its low mobility due to important adsorption capacities of solid matrices leading to its immobilization. In short, the order of mobility Am > Pu > Cs reflects their various chemical and biochemical reactivities. This shows the intricacy of the migration mechanisms with various processes that can compete: mechanical (wind, rainfall, snow), thermodynamic (complexation and sorption equilibria, hydrolysis, leaching), all phenomena being linked. In all cases, large dilution factors or significant concentration factors (rarer) modify the localization and speciation. To further address this question in complex and heterogeneous environmental systems, a combination of radiometric, spectroscopic and imaging micro-probes would certainly be necessary as already pointed out. This is the methodology recently implemented in the Nice radiochemistry laboratory in collaboration with the MARS beam line of SOLEIL synchrotron and CEA in order to follow the spatial speciation of anthropogenic uranium in model columns of simulated soils.

[47]

Speciation of radionuclides, transfer to living species

During the first decades after World War II, radioecology studies were driven by the study of the impact of nuclear radiation and transfer of radionuclides to ecosystems. As stated in a recent viewpoint on radioecology from Rhodes and coworkers, "There is a growing desire to incorporate attributes of ecosystem science into radiological risk assessment and radioecological research […]" [16] putting radioecology (mostly at very low doses) into the larger perspective of ecological risk assessment (see also publications from the International Commission on Radiological Protection, ICPR [48] ) in which humans are considered as part of their ecosystems. Considering our recent interest in the marine system, our approach certainly deviates from this position mainly because of our methodology to dope the natural systems with the MTR of interest as the only methodological pathway for studying speciation in living systems. But the way we are considering the possible uptake of MTR from marine species may complement current radioecological approaches. In one of our recent case studies, we considered the sea urchin (here Paracentrotus lividus) often used as a sentinel species of local pollution because of its sedentary habits and wellknown sensitivity to pollutants (accumulation of MTE). [START_REF] Chiarelli | Marine Invertebrates as Bioindicators of Heavy Metal Pollution[END_REF] We investigated the chemical mechanisms of MTR ( Nat U, 60 Co, 137 Cs) uptake by P. lividus using a well-controlled system of aquaria doped with the MTR of interest. For Nat U, for instance, and using XAS spectroscopy, we provided evidence of the metabolization of uranium (under its most stable uranyl UO 2 2+ chemical form) in living P. lividus. [50] Furthermore, contaminated gonads of P. lividus were analyzed by Scanning Transmission X-ray Microscopy (STXM), a technique that couples XAS and microscopy in order to localize the element of interest in a 2-D space and define its speciation at the same time. Such a technique is suitable for element-selective imaging studies with a spatial resolution down to a few tens of nm. In this work, measurements performed at the uranium N V edge (4d-nf transition) showed the absence of precipitated inorganic phases of uranium. Instead, it appeared to be homogeneously distributed on the gonad cell membranes, leading to the conclusion that uranyl was bound to specific biomolecules (Figure 2). EXAFS data analysis at the U L II edge of contaminated gonads further suggested that the metal is coordinated to carboxylate residues of a biomolecule. The toposome protein, the main protein in P. lividus was further identified as a possible target for U complexation because of its ability to store Ca 2+ ions with carboxylate chemical functions. To complement this downscale molecular approach "from the animal to the metalloprotein", we also explored the bioinorganic actinide chemistry of some possible target metalloproteins involved in the iron homeostasis (because Fe 3+ and An 4+ bear some chemical similarities like their propensity for hydrolysis): the transferrin and the ferritin. Using tools from bioinorganic chemists (bio-XAS for instance), we investigated the modification of the complexation site when an actinide cation is replacing the endogenic cation (like iron in ferritin). [51] Considering humans as part of the ecosystem, direct or indirect human contamination by radionuclides may occur as a result of an incident (nuclear power plant and/or ships), an act of war or terrorism, or after an injury (plant, mining industry). In such cases, the available treatments are very limited and mostly consist of i.v. administration of sodium bicarbonate in the case of U, DTPA (triaminopentaacetic acid, as calcium or zinc form) or more innovative chelating molecules in the case of plutonium.

[52], [53],

.

To target the main biological retention sites defined by biokinetics data (liver, kidneys, lungs and bones) and therefore provide a more complementary therapy, a macro-or supramolecular approach makes sense and can constitute a fully complementary alternative to the current treatments, to passively target these reservoirs. For the past ten years, the Nice radiochemistry laboratory has been developing such approaches for human decorporation, i.e. decontamination of the incriminated actinide(s) resulting from excretion, by natural means, linked to the utilization of the countermeasure. In that sense, we have developed two polymeric analogues of DTPA: Polyethyleneimine functionalized by acetate (PEI-CH 2 COO -) or phosphonate groups (PEI-CH 2 PO 3 2-) to vectorize the chelates. Furthermore, dose-response experiments showed promising actinide complexation efficiencies comparable to the reference DTPA.

[54], [55]

Conclusive remarks

Studies on radionuclides in environmental chemistry took place in the Nice radiochemistry laboratory in order to analyze nuclear fallout after the Chernobyl accident. The methodology developed in this context, at first strongly tied to IAEA, enabled the determination of inventory data and migration mechanisms. Such data are essential for better understanding and assessing operational CFs, although initial conditions (exposure, delivery, etc.) must be carefully taken into consideration. But ultra-trace levels of contamination often preclude the use of spectroscopic techniques and the determination of direct speciation data. Heterogeneity at various scales also requires adapting the size of the probe to the size of the sample. This always questions the representativeness of the measurement. Those two limiting conditions (ultra-trace environmental levels on the one hand, and heterogeneity on the other) have up until now formed the bottleneck of speciation studies. In our effort to input speciation data in environmental (and radioecological) metrics, we have developed a doping methodology that fills this methodological gap. Our recent work on marine species in collaboration with CEA uses this approach which overlaps radioecology and environmental radiochemistry. The major limitation is certainly the validation of representativeness for contaminated ecosystems. For that purpose, much care should be taken to complement such approaches with field data, as much as possible.

In conclusion, obtaining speciation data calls for a methodological compromise. Nevertheless, the input data so gathered is essential and can impact calculation codes developed on a larger scale.

Figure 2 :

 2 Figure 2: STXM image of sea urchin gonad cell obtained at 725eV, above U NIV edge (left), and contrast obtained between the images recorded at 725 eV, above U NIV edge, and 738 eV, below U NIV edge (right). Reprinted (adapted) with permission from B. Reeves, M. R. Beccia, P. L. Solari, D. E. Smiles, D. K. Shuh, L. Mangialajo, S. Pagnotta, M. Monfort, C. Moulin, C. Den Auwer, Environ Sci. Technol. 2019, 16, 7974-7983. Copyright 2022 American Chemical Society.

in La formation de l'esprit scientifique, contribution à une psychanalyse de la connaissance, G. Bachelard, 1938. Transl. "It would be better to be completely ignorant than to have knowledge deprived of its fundamental principle"

Acknowledgements

The authors would like to greatly acknowledge the MARS beam line of SOLEIL synchrotron where most of this work has been performed. They also would like to thank C. Moulin, M. Monfort and J. Aupiais from CEA DAM DIF for extremely fruitful collaboration and discussions. The authors acknowledge CEA DAM DPN, CNRS MITI and DGA for financial support.


Dr Cyril Zurita

Satoru Tsushima
email: s.tsushima@hzdr.de

Dr Pier Lorenzo Solari

Dr Denis Menut
email: denis.menut@synchrotron-soleil.fr

Dr Sandrine Dourdain
email: sandrine.dourdain@cea.fr[

Dr Aurélie Jeanson

Gaëlle Creff
email: gaelle.creff@univ-cotedazur.fr

Christophe Den Auwer
email: christophe.denauwer@univ-cotedazur.fr

Prof Asst Gaëlle Creff

Prof Den Christophe

Auwer

Prof Satoru Tsushima

Interaction between the transferrin protein and plutonium (and thorium), what's new ?

Keywords: Metalloprotein, Plutonium, Transferrin, XAS, SAX

Transferrin (Tf) is a glycoprotein that transports iron from the serum to the various organs. Several studies have highlighted that Tf can interact with metals other than Fe(III), including actinides that are chemical and radiological toxics. We propose here to report on the behavior of Th(IV) and Pu(IV) in comparison with Fe(III) upon Tf complexation. We considered UV-Vis and IR data of the M2Tf complex (M = Fe, Th, Pu) and combined experimental EXAFS data with MD models. EXAFS data of the first M-O coordination sphere are consistent with the MD model considering 1 synergistic carbonate. Further EXAFS data analysis strongly suggests that contamination by Th/Pu colloids seems to occur upon Tf complexation, but it seems limited. SAXS data have also been recorded for all complexes and also after the addition of Deferoxamine-B (DFOB) in the medium. The Rg values are very close for apoTf, ThTf and PuTf, but slightly larger than for holoTf. Data suggest that the structure of the protein is more ellipsoidal than spherical, with a flattened oblate form. From this data, the following order of conformation size might be considered : holoTf < M2Tf (M = Th, Pu) < apoTf < M2Tf-DFOB (M = Fe, Th, Pu).

Introduction

Since the discovery of nuclear fission in 1938, the nuclear industry in both the civil and military sectors has dramatically expanded, most notably in the Northern Hemisphere. These developments have given rise to new geostrategic, geopolitical and environmental challenges that also lead to concerns regarding security, the environment, and indeed the entire biosphere. Nuclear energy has also become a key strategy for many nations, who must therefore develop policies that mitigate these potential dangers. It is well known that fission products, activation products and actinide elements associated with nuclear fuel can be chemically and radiologically toxic if accidentally released into the environment. [1] Of these, actinides are the heaviest elements that may be involved and have no essential biological function. They are thus transported in the organism towards target organs, and are retained or excreted, while expressing their dual radioactive and heavy metal toxicity. [2] The main routes of contamination for actinides during internal exposure are inhalation of aerosols (pulmonary route), ingestion (gastrointestinal), and penetration through an open wound (subcutaneous) and skin. In vivo, Pu is mainly present in the +IV oxidation state. [3] It is a hard acid cation on the Pearson scale, it has a large coordination number (8-12) and a large propensity for hydrolysis. [START_REF] Morse | The chemistry of the actinide and transactinide elements[END_REF] The intake of Pu therefore poses considerable health risks if contamination occurs. Early studies on Pu accumulation in rats have provided evidence of the importance of the metal speciation and have described its biokinetic characteristics. [START_REF] Durbin | [END_REF]6] Pu(IV) injection has shown that its distribution is similar in all animal species studied (mammals), with very little excreted in the urine. [7] After ingestion, post-mortem radiometry in humans has shown that 90% of the Pu activity in extra pulmonary sites is accumulated in the liver and skeleton. [8,9] Furthermore, the percentage deposited in the skeleton increases with time. In various mammals species, 85% of the injected Pu(IV) is deposited in bone and liver, with a boneliver distribution varying according to speciation. [2] In the absence of steric constraints, Pu 4+ tends to form complexes as stable as those of the first-row trivalent transition metals, especially Fe 3+ due to their comparable charge-radius ratios (Fe(III) = 0,645 Å, CN = 6 ; Pu(IV) = 0,96 Å, CN = 8 to 10) and have a high affinity for carboxylate functions. [10] In mammals, the large majority of Fe is associated with the hemoglobin protein, allowing the transport of oxygen in our organism. [11] . The small amount of Fe that remains can also interact with the transferrin protein (Tf) to be transported from the serum to the cells. [12] Previous reports have suggested that Tf is also a possible target of Pu that would be transported into the cells. [13,14 15]] Tf, once bound to Pu, could cross the cell barrier (endocytosis) and thus could enter the cell. Jensen et al. have proposed a mechanism for Pu-Tf recognition by cells in which the proposed model involves a FePuTf mixture, with Pu bound to the C lobe and Fe to the N lobe. [16] This is why the interaction of Pu(IV) with Tf would give key indications on its possible internalization mechanism in the cells. Tf is a glycoprotein with a molecular weight of around 80 kDa. The role of this protein is to transport iron from the serum to the various organs. [17,18] Tf consists of a single polypeptide chain of 670-700 amino acids and has two lobes, named N and C connected by a short bridge. [19] These two lobes have a similar but not identical affinity for metal cations. [20] Each of the two lobes of the protein contain a metal binding site that can bind one ferric ion. [21,22] The bio-distribution of the different Tf complexes is 27% of Fe2Tf (also called holoTf), 23% of FeNTf, 10% of FeCTf and 40% apoTf (Tf with no metal). [23] Each Fe(III) atom binds similarly one lobe of the protein. The coordination of Fe(III) in each lobe involves four amino acids consisting of two tyrosine, one histidine and one aspartate residue. [START_REF] Baker | Advances in Inorganic Chemistry Ed[END_REF][START_REF] Sargent | [END_REF] It also binds with a synergistic anion, usually a bicarbonate or carbonate anion to complete the Fe coordination sphere. The 4 amino acids and carbonate anion fulfill the Fe coordination sphere (CN = 6) leading to the lobe closure. [26] The Fe2Tf complex (pKD = 20.9 ± 0.6) [22] is thus in the appropriate closed conformation to be recognized by the transferrin receptor protein (RTf) on the cell surface and to cross the cell membrane. [27] Several studies have highlighted that Tf can interact with metals other than Fe(III) such as titanium, vanadium, chromium, ruthenium and bismuth. [15] Other metals including certain actinides can also interact with Tf. [28] For instance, Tf binds strongly with Pu(IV) with a reported conditional constant pKD = 21.75 ± 0.75 comparable to that of Fe(III). [29] In vivo, the exact conformation of complexed MnTf (M = metal, n = 1,2) and the occurrence of this conformation is often not known. A previous report by Jensen et al. has shown that the interaction of the Pu2Tf complex with the transmembrane receptor RTf can only occur in presence of Fe within the protein to form a mono-plutonium mono-iron Tf complex in the PC12 cells. [15] In the active form of the Pu-loaded Tf, i.e. a conformation that is able to bind to RTf and cross the cell membrane barrier, Pu(IV) is complexed to the binding site of the C-lobe of the protein, and Fe(III) to the N-lobe of the protein (PuCFeNTf). According to this report, all other forms of Pu2Tf and PuFeTf complexes that may exist in vivo do not have a suitable conformation to subsequently bind with the RTf receptor. The slight binding differences between the two lobes of Tf therefore turns off its complexation with RTf and by extension, the cellular uptake of Pu. [16,[START_REF] Sargent | [END_REF] In the present report we propose to revisit the interaction of Pu(IV) as well as Th(IV) with Tf with a methodology that addresses the conformational change of the protein after complexation by both metals. 232 Th has been used here as a surrogate of 239 Pu for the test experiments (it is less radioactive) but also to allow a comparison between the two actinides. Th(IV) and Pu(IV) local environments in M2Tf (M = Th, Pu) complexes were first explored with XAS (X-ray Absorption Spectroscopy) complemented with IR (Infra-Red) and spectrophotometry. A macromolecular model obtained with MD (Molecular Dynamic) calculations allowed us to identify which amino acids are involved in this interaction and to understand the change in the overall conformation of the protein after complexation. MD calculations were also used to interpret structural information obtained from SAXS (Small Angle X-ray Scattering) data on M2Tf and holoTf complexes compared to apoTf. Based on these results, we propose a model of complexation and of conformation between Pu/Th and Tf with respect to Fe.

Results and Discussion

To avoid hydrolysis at physiological pH, Th(IV) and Pu(IV) were protected with carbonate anions which are found in the serum at 22 mM to 28 mM levels. The limit complexes M IV (CO3)5 6-(M = Th, Pu) were prepared and used as stock An solutions (see experimental section). In the text that follows, the carbonate anions will be omitted from the notation M2Tf (M = Th, Pu) for clarity but are nevertheless present. The notations apoTf and holoTf are related to the commercial Tf without and with iron respectively.

Thorium, Plutonium complexation, spectrophotometry

The complexation of Tf with Pu(IV) in presence of carbonates (Pu2Tf) has been monitored by spectrophotometry (Figure 1) and compared to the spectra of apoTf and holoTf. The same comparison has been performed for the complexation of Tf with Th(IV) in presence of carbonates (Th2Tf). All spectra have been normalized to the absorption band at 280 nm. This band is characteristic of π → π* transitions in the protein, and mainly due to the tyrosine and tryptophan chromophore groups. [30,31] The spectrum of holoTf shows an absorbance band in the range 463-472 nm attributable to a tyrosyl phenolate (pπ)  Fe(III) (dπ*) charge transfer transition. [32] The spectra of Th2Tf and Pu2Tf are different from each other but also from the spectrum of holoTf and apoTf. Furthermore, in the 300-375 nm range, the presence of an absorption band not observed in the apoTf spectrum may be a signature of the formation of a Tf complex with the different metals. [33,34]

Thorium, Plutonium complexation, Infrared spectroscopy

In order to obtain a first description of the chemical environment of the metal within the Tf complex, IR spectra were recorded with Fe(III), Th(IV) and Pu(IV). Figure S1 of SI shows a comparison between the spectra of apoTf (black line), holoTf (blue line), Th2Tf complex (red line) and Pu2Tf complex (orange line). Note that all the Tf complexes were micro-filtrated at 3 kDa and then washed to remove the excess of the synergic anion (see experimental section). The IR transmission spectra were normalized to the amide II band at 1550 cm -1 as this band is less strongly influenced by complexation with metals than the amide I band. [35] Figure S1 of SI shows that the spectrum of apoTf is very similar to that of holoTf. The spectra of M2Tf (M = Th, Pu) have a similar (although not identical) signal in the region 1450 -1200 cm -1 that is however little different from both spectra of holo and apoTf. Note that the spectrum of Pu2Tf was recorded using a specific ATR module, which makes the acquisition conditions different from the other three spectra (see experimental section). This first examination of the spectra shows that in our working conditions there is no evidence of protein degradation. Indeed, the structure of the amide I band is affected by the presence of the metals evidencing modifications in the protein conformation upon complexation. The large band centered at 1366 cm -1 on the two M2Tf spectra could be attributed to the antisymmetric v3 vibration of carbonate groups present in the sample.

Thorium, Plutonium local environments, X-ray Absorption Spectroscopy

To cast light on the complexation site of Pu/Th in M2Tf complexes (M = Th, Pu), XAS data at the cation LIII edge have been recorded in the EXAFS (Extended X-ray Absorption Fine Structure) regime. A first comparative observation of the XANES part of the spectra with MO2 oxide references (spectra not shown) confirms the +IV oxidation state, not surprisingly. A qualitative comparison between the EXAFS spectra of Th2Tf and Pu2Tf in Figure 2 indicates that the local environment in both protein complexes is very similar. Additional comparison with the M IV (CO3)5 6-(M = Th, Pu) complexes at pH 10 shows unambiguously that the EXAFS signature of the M2Tf complexes in our working conditions is different from the pure carbonate complexes signature. A shift in low k values and a modification of the wave shape are visible in Figure 2. In order to adjust the EXAFS data with ad hoc structural models and attempt to go beyond a description with averaged scattering shells, models of Pu2Tf were created by MD calculation. To realize these models, a Ce(IV)-transferrin structure (PDB reference 1FCK) was used as a starting point. [36] Ce(IV) is an interesting element since it can be considered as a non-radioactive analogue of Pu(IV) (Ce is the only lanthanide stable at oxidation state +IV in aqueous solution). In these calculations, Ce(IV) has been substituted by Pu(IV) while varying the number of metal-interacting carbonate groups in each lobe. These calculations were performed for 100 ns mimicking physiological pH (pH 7.4) by adjusting the protonation states. Each model has a different carbonate stoichiometry with respect to Pu, ranging from 0 to 3 (a stoichiometry of 4 or above lead to instabilities and unrealistic results), reflecting that several carbonate anions can bind to the metal within the Tf binding site. Carbonate anions are also known to bind through Hbonding to the amino acids of the protein to close the complexation site. [37] On the other hand, in the absence of any carbonate (simulation with 0 carbonate) the Pu2Tf complex was found to be unstable, to stay in a folded state and the lobe opened up in the middle of the simulation. Therefore, the result with 0 carbonate is excluded from further discussion. The three models (1-3 carbonates) are compared to the crystallographic structure of Fe2Tf (PDB 1N04) and Ce2Tf (PDB 1FCK) in Figure S2 and Table S1 of SI. In the N lobe, Pu is bound notably to Asp60 as in the case of the 1FCK (Ce) and 1N04 (Fe) structures. In the C lobe, Pu is bound to Asp395 residue as in Ce2Tf and Fe2Tf models (see Table S1 of SI). [36] More precisely, in the Pu2Tf model with a carbonate in each lobe, denoted Pu2Tf(1carb), although the amino acids interacting with the metal are different, the interaction mode is close to that of 1FCK and 1N04 crystallographic structures. In the Pu2Tf models with two or three carbonates, named Pu2Tf(2carb) and Pu2Tf(3carb), respectively, a decrease in the number of amino acids bound to the metal is observed compared to the Pu2Tf(1carb), 1FCK and 1N04 models. This decrease, in particular for 3carb can be explained by the increase of the number of carbonates interacting with Pu which induces the repulsion of the amino acids of the protein making this interaction with Pu(IV) more difficult. Overall, the molecular models reveal that local environments in each lobe around Pu that are not very similar to that of natural Fe. However, we note in the different models the presence of residues of glutamate, serine, alanine, proline or threonine which do not intervene in the holoTf. On the other hand, tyrosine and histidine residues, present in the case of Fe(III), do not appear in the close environment of the Pu(IV) atoms. However, the presence of the same aspartate residue in each of these lobes indicates that Pu(IV) is indeed suggested in the same site as Fe(III). Because the sidechain of tyrosine has a large pKa value (> 10), in the MD simulations, they were all assumed to be protonated. They may get deprotonated upon coordination to metal as pointed out previously. [38] Deprotonation occurs only after the coordination of Tyrosine, and if we assume it to be deprotonated from the beginning, it would certainly add bias to the calculations. Therefore we restricted ourself to the use of protonated tyrosine. Overall, direct coordination of tyrosine to Pu was rarely observed during the simulation (exception for one Tyr coordination in C-Lobe for two carbonate system), though the tyrosine involved in Fe(III)-binding always remained in the vicinity of Pu(IV). Therefore, despite that Pu(IV)-Tf does not share the same metal coordination environment as Ce(IV)-Tf, there is hardly any conformational change upon Ce-to-Pu substitution. When comparing the calculated EXAFS spectra for each lobe N and C for a given model (see Figure S3 of SI), the presence of several carbonates greatly modifies the environment around Pu(IV). In order for it to be compared more precisely with the experimental spectrum of Pu2Tf, the average of the calculated spectra for the two lobes for each model was calculated and is presented in Figure 3. It compares the experimental EXAFS spectra of Pu2Tf, Pu(CO3)5 6-and the calculated spectra for each model (both lobes averaged). Table 1. Best-fit structural parameters obtained from the fit of EXAFS Pu2Tf (exp), Th2Tf (exp) data. Comparison with [Th(CO3)5] 6-and [Pu(CO3)5] 6-from ref 39 . σ 2 is the Debye Waller factor of the considered diffusion path. S0 2 is the global amplitude factor, ∆E0 is the energy threshold, Factor-R is the fit factor in % and Factor-Q is the quality factor (reduced χ 2 ) of the fit. The uncertainties in brackets.

The calculated EXAFS spectrum of Pu2Tf(1carb) is qualitatively close to the experimental spectrum of Pu2Tf in Figure 3. On the contrary, the model with three carbonates shows significant differences and does not resemble any of the experimental spectra, neither Pu(CO3)5 6-nor Pu2Tf(exp). The spectrum of Pu2Tf(2carb) shows an intermediate behavior. The difference between the calculated spectra and that of the experiment may be estimated point by point according to the formula: |Δk| = χ(k) Pu2Tf (ncarb) -χ(k) Pu2Tf (exp). This average difference between the model signal and the experimental signal is only indicative over the entire spectra but it corroborates the qualitative comparison : 18 % for Pu2Tf (1carb); 22 % for Pu2Tf (2carb) and 44 % for the Pu2Tf(3carb). Clearly, the distinction between both models 1carb and 2carb is difficult to make at this stage, but 3carb can be excluded (similarly 0carb has been excluded). In order to use a model as simple as possible and also to match with the holoTf, the 1carb model was chosen and is shown in Figure 4. For comparison, models Pu2Tf (1carb) and Pu2Tf (2carb) are presented in Table S1 of SI). Accordingly, the N-lobe site of PuTf(1carb) was chosen to generate the scattering paths for EXAFS data fitting (lobe C of this model was not selected as shown in Figure S4 of SI). Comparison to the best fit data of the Pu(CO3)5 6-reference shows a significant shrinking of the O first coordination sphere (the same remark will be valid for Th2Tf compared to Th(CO3)5 6-). [39] In the Pu2Tf(1carb) model, oxygens are present at distances spanning from 2.20 to 2.68 Å, very disperse. ) is likely to be EXAFS sensitive. The EXAFS data could indeed be fitted with one layer of C atoms and attempts to add a second layer at a larger distance as in the model did not affect the fit. The experimental fitted average distance is about 0.2 Å larger than in the model, suggesting a significant difference in the amino acid conformation. Attempts to reduce this distance led to a significantly degraded fit. Similarly, very similar Th-O distances were obtained for Th2Tf, although slightly larger because of the larger ionic size of Th 4+ compared to Pu 4+ . On the other hand, the average Th…C distance is significantly smaller than for Pu and in better agreement with the Pu2Tf(1carb) model. At this point we have no explanation for these discrepancies in the M…C coordination sphere except that significant second sphere rearrangements from the amino acid carbon atoms compared to the static model are not surprising. As shown in Figure S6 of SI, an additional contribution at R+Ф = 3.6 Å is not reproduced well by the fit. This contribution indicates that another interaction is visible and could signal a Th/Pu...Th/Pu interaction as in colloidal Pu(IV) (localized around 3.8 Å in Rspace). [40] This contribution was filtered and could be fitted with a path of Pu...Pu scattering in the case of Pu2Tf. The Pu...Pu distance at 3.85(2) Å (see Table 1) is consistent with the distance of a colloidal plutonium compound. [40] The same observation was made for Th2Tf. Since this contribution is not present in the fitting of Th/Pu(CO3)5 6-it suggests the formation of a small quantity of colloids in our conditions for Tf complexation. The small amplitude value associated with this scattering path in Table 1 strongly suggests negligeable colloidal contamination as discussed in the Discussion section.

Transferrin conformation upon Pu/Th complexation

Additional SAXS (Small Angle X-ray Scattering) data were acquired to cast light on the tertiary structures of the M2Tf (M = Fe, Th, Pu) complexes. Furthermore, we also investigated the influence of a competing ligand in order to observe the effect of complexation and decomplexation on the Tf conformation. Deferoxamine-B (DFOB) siderophore was chosen because it is known to interact strongly with Fe(III) (β1,1 = 31.0 ± 0.3 in KCl 0,1 M) and Pu(IV) (β1,1 = 35.8 ± 0.9 in NaNO3 0.1 M). [41,42] Thus, the addition of DFOB to Pu2Tf may induce a total or partial decomplexation of Pu or the formation of a Pu2Tf-DFOB ternary complex.

Sample c a Fitting error χ²

The UV-Vis spectrum of DFOB measured in the presence of Pu2Tf complex (blue line), is plotted in Figure S7 and compared to the spectra of apoTf (black line), free DFOB molecule (orange line), Pu2Tf (red line) and of a Pu-DFOB complex (purple line). All spectra are normalized to the absorption band at 280 nm. Figure S7 indicates that the addition of 10 DFOB to Pu2Tf (1:5 Pu:DFOB ratio) does not lead to the total decomplexation of the Pu(IV) bound to the protein. The spectrum is still dominated by the signal of Tf while the band of Pu2Tf around 340 nm is clearly disappearing. Although it is difficult to draw any further conclusions, one may hypothesize that DFOB is either partially decomplexing Pu from Tf or forming a ternary complex Pu2Tf-DFOB.

Table 2.

Best fits results of SAXS spectra with an oblate structure. a: is the longer dimension called equatorial radius (in Å) c: is the smaller dimension called polar radius (in Å).

The SAXS spectra of Pu2Tf, Th2Tf, holoTf, apoTf, Th2Tf-DFOB, Pu2Tf-DFOB and holoTf-DFOB are shown in Figure 5 and standard deviations are provided in Table S2 of SI. The spectra of the different complexes are compared with the spectrum of the pure apoTf (see SI paragraph 3 for details). The shape of these spectra shows a rise of scattering signal towards the small Q vectors. This increase of signal is attributed to the scattering of the protein. Two decreases of intensity can moreover be observed, indicating two distinct dimensions in the protein structure. The first, located between 0.03 nm -1 to 0.09 nm -1 , and the second, between 0.1 nm -1 and 0.22 nm -1 , are characteristic of a large and a small dimension of the protein, respectively. They are denoted as 1 st and 2 nd regime in Figure 5. It can be noticed that the first drop of intensity appears similar for all the complexes, while the second shows distinct behaviors depending on the complex. For instance, the decrease in intensity of the spectrum of holoTf is clearer than that of the spectrum of apoTf, while spectra obtained for Th2Tf and Pu2Tf also show these two drops of intensity but are more attenuated than for holoTf. These first apoTf 16 indications confirm that the complexation of the protein influences its conformation. The addition of DFOB in the medium induces a significant modification of the SAXS spectra. Structural changes upon complexation are therefore detectable with SAXS.

In order to interpret quantitatively these changes, gyration radii were first fitted with the Guinier approximation applied on the 1 st regime of the curves (see Table S3 of SI). The radii of gyration determined in this work are compared to the literature as well as to the radii of gyration of the different models of Pu2Tf, with 1,2 or 3 carbonates, also in this work. First, we observe that the holoTf complex adopts a smaller conformation than the apoTf, with a conformational difference of 1.1 ± 0.2 Å. For instance, the radii of gyration obtained by Yajima et al. [43] , although with a different buffer media, was greater than ours, but still had a difference of 1.0 Å between apoTf and holoTf. For Th2Tf and Pu2Tf complexes, the radii of gyration of Table S3 of SI are very close and comparable to that of apoTf. In their study, Jensen et al. [16] observed (although absolute values are different) a larger radius of gyration for Pu2Tf than for holoTf, indicating again a different structure and thus an open conformation for Pu2Tf. This is consistent with our results. Moreover, the radius of gyration obtained from MD simulations are close regardless of the carbonates number. Finally, the addition of DFOB to the M2Tf (M = Th, Pu) complex increases the gyration radius. These values suggest that DFOB partially binds to the metal in interaction with transferrin, thus increasing the overall gyration radius of what would be best described as a ternary complex. In the case of holoTf (Figure 5), DFOB appears to partially interact with Fe, resulting in an increase in the gyration radius measured between holoTf and holoTf-DFOB. This increase could be explained by an opening of the protein lobes when the holoTf-DFOB ternary complex is formed. Indeed, Bellotti and Remelli also observed that DFOB does not fully decomplex the protein from Fe. [44] In our study, Pu and Th also appear to interact with DFOB, and may also form a ternary complex with the protein.

To provide a more quantitative analysis of the SAXS data, a form factor considering a non-spherical shape of the scattering objects was used. Martel et al. studied the structure of holoTf and showed that a flattened ellipsoid of revolution (oblate) form factor is a simple structure similar to that of this protein. [45] Two parameters were used: a polar radius c (the shortest distance) and an equatorial radius a (the longest distance). The fit of the data of holoTf, Pu2Tf, Pu2Tf-DFOB and apoTf complexes are shown in Figure 6. The fitted parameters of both dimensions of the protein are given in Table 2. The slight structural changes between the open and closed conformations of the protein are further highlighted by these fits using the oblate structure. The polar radius c is the most affected by the conformational change of the analyzed complexes. Indeed, a decrease of this radius, from apoTf to holoTf is observed in agreement with the conformational change of the protein. Similarly, the polar radius of Pu2Tf is slightly smaller than that of apoTf, but larger than that of holoTf, which is consistent with a slight closure of the protein in the presence of Pu. In contrast, the polar radius of the Pu2Tf-DFOB complex is larger than that of apoTf. In comparison, the equatorial radius of the different fitted complexes is only slightly impacted by the conformational changes. Martel et al. have also analyzed the shape of human holoTf by SANS (Small Angle Neutron Diffusion) using a flattened ellipsoidal structure and obtained values of 46.6 ± 1.4 Å and 15.8 ± 3.8 Å for the equatorial and radial radii respectively. [45] In addition, they also observed that the oblate ellipsoidal structure is the best simple approximation to the shape of the Tf molecule. Our data are in good agreement with those published by Martel et al. and confirm the choice of this form factor.

Discussion

Models of Pu2Tf obtained from MD calculations show that increasing the number of carbonates (from 1 to 3) interacting with Pu prevents the amino acids of the protein from entering the coordination sphere. Indeed, with 3 carbonates or higher, they seem to repel the amino acids, which end up at a distance larger than 5 Å from the metal. Furthermore, the MD model suggests that the A careful comparison of these simulation data with experimental EXAFS spectra supports the presence of Th and Pu in the Fe coordination site. It also suggests that only 1 carbonate (an assumption with 2 cannot be excluded but seems less likely) is present in the local environment of Pu and Th, as is the case of Fe. Comparison of our best fit EXAFS data of Pu2Tf can be made with data published by Jeanson et al. in 2010, however using NTA as the synergistic anion. [46] The parameters of the first oxygen coordination sphere are very similar ( ). But further comparison with the present system is difficult because of the presence of NTA in Jeanson's study whose size is significantly larger than that of the carbonate and probably resulted in a rearrangement of the amino acids conformation. Finally the presence of Pu or Th atoms at a distance of 3.8 -3.9 Å is required here for the fits. Our assumption is that a "contamination" by colloids or another type of condensed metallic cluster occurs upon Tf complexation since the metal carbonate complexes used at the start do not show any "contamination". Therefore, an estimate of this "contamination" can be made according to the structure of the colloids reported by Micheau et al.. [47] In this case 0.8 Pu (0.7 Th) would represent about 10% of colloidal species in our sample. During the preparation of Th/Pu2Tf, a Th/Pu : Tf ratio of about 2.2 : 1 was used in order to saturate both lobes of the protein. The minor excess of free Th/Pu is then likely to occur in the solution. And after adjustment to physiological pH, the carbonate being no longer predominant at this pH range would not sufficiently protect the Pu against hydrolysis. Micheau et al. also showed that plutonium particles, obtained in an acidic medium, have a radius of gyration Rg of about 10.4 ± 0.5 Å, [47] characterized by a bend at about Q ~ 2 nm -1 in the SAXS data. This was not observed in all our spectra. A careful look at Jeanson's data from 2010 shows a similar contribution at R+Ф = 3.6 Å that was difficult to fit because it may have had the same origin. Differences in the metal coordination sphere also have an impact on the potential closure of Tf after complexation. The two decreases in intensity observed on our SAXS spectra are characteristic of the Tf shape as they are also observed in the absence of Pu and in the literature. [16] Fitting the scattering spectra with a simple oblate ellipsoid model led to a good reproduction of the experimental data until Q= 0.2nm -1 . For higher Q range, additional oscillations were identified, which cannot be fitted with a simple form factor. The fitted data of Table 2 suggest that the Pu2Tf complex is larger than holoTf but slightly smaller than apoTf. On the other hand, Rg values given in Table S3 of SI are very close for apoTf, ThTf and PuTf, but slightly larger than for holoTf. Furthermore, it is interesting to observe that the gyration radii of both calculated Pu2Tf models (1,2carbs) are similar to each other, confirming a close or partially close conformation in both cases with Pu (absolute values of Rg must be considered with caution). Finally, it is interesting to note that the addition of DFOB with the already existing M2Tf complex (M = Fe, Th, Pu), favors the formation of the ternary complex M2Tf-DFOB whatever the metal with a clear open conformation. Indeed, the radius of gyration measured for these ternary complexes is higher than that of apoTf, which seems to indicate that DFOB partially binds with the metal in the protein. In addition, these ternary complexes were also observed by UV-Vis analysis.

In conclusion, the above data complement previous literature on actinide(IV)-Tf interaction and suggest the following order of conformation size : holoTf < M2Th (M = Th, Pu) < apoTf < M2Tf-DFOB (M = Fe, Th, Pu). This order is in full agreement with the expected behavior of Tf as explained above.

Conclusion

The interaction between transferrin (Tf) and exogeneous metals is still an open question despite the vast literature on the subject. In this work, we proposed to further characterize the behavior of Th(IV) and Pu(IV) in comparison with Fe(III) upon Tf complexation. We first considered UV-Vis and IR data of the M2Tf complex (M = Fe, Th, Pu) and combined experimental EXAFS data with MD models in order to better describe the metallic coordination site. In all cases, the iron coordination site is involved although MD models suggest that the local environments of Fe and Pu are not identical due to the presence of residues in the Pu environment that are not found in the Fe environment. EXAFS data of the first M-O coordination sphere are consistent with the MD model considering 1 synergistic carbonate. Further EXAFS data analysis strongly suggests that contamination by Th/Pu colloids seems to occur upon Tf complexation, but this contamination seems limited to c.a. 10%. SAXS data have been recorded for all complexes and also after the addition of DFOB in the medium. The Rg values are very close for apoTf, ThTf and PuTf, but slightly larger than for holoTf. To provide a deeper quantitative analysis of the SAXS data, a form factor considering a non-spherical oblate shape of the scattering objects was considered as for holoTf. Data suggest that the structure of the protein is more ellipsoidal than spherical, with a flattened oblate form rather than an elongated ellipsoid in agreement with Martel's data on holoTf. [45] From this data, the following order of conformation size might be considered : holoTf < M2Th (M = Th, Pu) < apoTf < M2Tf-DFOB (M = Fe, Th, Pu).

The interaction with DFOB is therefore an interesting subject for investigation as we seek means of reducing the affinity of a Pu2Tf-DFOB ternary complex with the RTf1 transmembrane receptor, which is the entry pathway of Tf into the cell.

Experimental section

All chemicals used were of analytical grade except for the protein. Human apo-Transferrin and holo-Transferrin were purchased from Sigma-Aldrich. The commercial batch was dialyzed prior to use: apo-Transferrin or holo-Transferrin was placed in a 12 kDa membrane and dialyzed for 24-hours 3 times in buffer medium 5x10 -2 M MES, 1.5x10 -1 M NaCl, pH 7.4 ± 0.1.

Actinide (Th, Pu) stock solutions

239 Pu (96% 239, 4% 240) was obtained from CEA stock. Pu(CO3)5 6-stock solution was prepared by mixing 120 µL of Pu(IV) at 6 x10 -3 M and 120 µL of carbonate at 3 M. Pu(IV) concentration in the carbonate medium was determined from the absorbance of the 486 nm peak for which the molar extinction coefficient is 66 L.mol-1.cm -1 . [39] A 0.15 M solution of Th(IV) nitrate (Th(NO3)4.5H2O, Sigma Aldrich) was dissolved in a 3 M carbonate solution to obtain a Th(CO3)5 6- solution.

Transferrin complexes spectrophotometry

Absorption spectra were recorded over the wavelength region 260-1000 nm with a single beam Agilent Cary 60 UV/Vis spectrophotometer and using a small volume 10 mm pathlength quartz cuvette. The plutonium transferrin complex was prepared by mixing 50 µL of Pu(CO3)5 6-stock, [Pu] = 3 x10 -3 M, with 250 µL of Tf in the buffered solution: 5x10 -2 M MES + 1.5x10 -1 M NaCl; pH 7.4 ± 0.1. An addition of 30 µL HNO3 2 M followed by a 50 µL addition of the buffer solution maintained the pH at 7.4 ± 0.1. After tree centrifugal steps (12 000g; 6 minutes; 4°C) followed by rinsing with the buffer medium was carried out. The final volume is adjusted at 250 µL with a buffer solution. The different complexes were prepared using the same protocol. For samples containing DFOB, 25µL was added with an initial concentration of 3 x 10 -3 M and another centrifugal step was performed (12 000g; 6 minutes; 4°C). The finals concentrations of samples analyzed by UV/Vis are about 3.10 -6 M for and about 6.10 -6 M for Pu.

Transferrin-Pu MD simulation

MD simulations were performed using AMBER 15 program package [48] with ff99SB force field applied on the protein. For Pu 4+ , 12-6-4 LJ-type parameters developed by Merz et al. have been employed. [49] For carbonate ions, additional parameters were employed. [50] The starting structure was taken from Ce2Tf (PDB:1FCK). Missing atoms including hydrogens were added and the protonation state of the protein was adjusted to mimic physiological pH (pH 7.4). Additional CO3 2-as well as Na + ions were added to mimic the presence of bulk carbonate and to make the system electrostatically neutral. TIP3P waters were then added with a minimum water layer thickness of 10 Å. 500 steps of steepest descent and 500 steps of conjugate gradient with 500 kcal mol -1 Å -1 harmonic restraint on the protein was initially conducted after which 1000 steps of steepest descent and 1500 steps of conjugate gradient were performed without constraints. 40 ps of heating of the system from 0 to 298 K with 10 kcal mol -1 Å -1 harmonic restraint on the protein, after which another 1 ns preconditioning run was performed at 298 K without restraint on the solutes. Finally, 100 ns MD run was performed in a periodic boundary condition in the NPT ensemble at 298 K. Simulations were terminated and restarted every 5 ns to avoid artificial convergence to a particular geometry. The SHAKE algorithm, a 2 fs time integration step, 12 Å cutoff for non-bonded interactions, and the particle mesh Ewald (PME) method were used. MD trajectory was recorded at each 50 ps. Simulations were performed with different numbers of carbonate ions (0 to 3) bound to Pu. RMSD of all simulations are given in Figure S8 in SI of the Supporting Information.

Transferrin complexes IR spectroscopy

ATR-FTIR spectra were recorded on a Bruker Tensor 27 instrument by using a nitrogen cooled mercury cadmium telluride (MCT) detector. A six-reflection diamond crystal was used as the refection unit. 50 µL of each sample were put in a micro-volume cell placed directly on the crystal. Spectra were acquired with 4 cm -1 resolution and 1024 scans were averaged for each sample. The slit opening for the incident beam was optimized at 3 mm.

Fe, Th and Pu XAS measurements

XAS experiments were carried out on the MARS beamline at the SOLEIL synchrotron facility which is dedicated to the study of radioactive materials. The optics of the beamline consisted of a water-cooled double-crystal monochromator for incident energy selection and horizontal focalization, and two large water-cooled reflecting mirrors for high-energy rejection (harmonic part) and vertical collimation and focalization. All the measurements were recorded in double-layered solution cells (200µL) specifically designed for radioactive samples at room temperature. The calibration of the incident beam energy was performed at the K threshold jump of Yttrium corresponding to 17.038 keV. A specific cell was used for the radioactive samples and consisted of Teflon sample holders containing three 250 µL slots closed by two layers of Kapton® (polyimide) film on each side. EXAFS data were acquired at the Th LIII edge (16 300 eV), at the Pu LIII edge (18 057 eV). Data were processed by using the ATHENA code of Demeter 0.9.25 package. [51] EXAFS data were adjusted using phases and amplitudes calculated with Feff7 code embedded in ARTEMIS code of Demeter 0.9.25. The site of Pu3(B) obtained in the above MD B calculations was used as a model. EXAFS signal was fitted in R space between 1 and 6 Å without any additional filtering after Fourier transformation using a Hanning window in k 2 (2.7 -10.8 Å -1 ). In all the fits, only one global amplitude factor S0 2 and one energy threshold e0 factor were considered for all the contributions. The agreement factor R (in %) and quality factor reduced χ 2 (Q) are both provided as an indication of the fit quality.

Simulation of the Pu sites of different models calculated by MD calculation were performed with Feff9 code.

Fe, Th and Pu SAXS measurements

SAXS experiments were subsequently performed with an X-ray energy of 16.0 keV corresponding to an average wavelength of 0.7749 Å-1, and a sample-to-detector distance of 763.67 mm. This configuration allows a Q range of approximately 0.02 Å-1 to 2 Å-1. A tank filled with He gas containing the centered photodiode beam stop was placed between the sample holder and the Pilatus 3 2M CdTe detector to reduce air diffusion. [52] Sample transmissions were recorded for 5 s using photodiodes and scattering patterns were recorded for 45 s per sample. The isotropic 2D scattering data were azimuthally averaged to obtain the scattering intensity I(Q) using the PyFAI software. [53] The scattering intensity of the samples was then normalized by acquisition time, transmission and thickness, and multiplied by a normalization constant determined using the scattering intensity of polyethylene (PE) at Q= 0.36 nm-1 which corresponds to 4.9 cm-1. The absolute intensity scattered by the samples was finally obtained by subtracting the contributions of the empty cells, the solvent and the background. The data was simulated using SasView 4.2.0 software. The solvent signal was subtracted, prior to fitting these spectra with an ellipsoid form factor.

3-SAXS measurements

Firstly, a statistical study was carried out, measuring each complex ten times, in order to calculate a mean spectrum and standard deviation and to check that the samples have not been damaged by the beam. The buffer medium used to make the complexes and the empty cells were analyzed under the same conditions. For all the samples analyzed, the average relative standard deviation between spectra is less than 3% except for the Pu 2 Tf n°2 (4.57%) and Th 2 Tf n°1 (34.50%) measurements (see Table 4 of SI). Beyond a probable variation of the beam intensity over the measurement, this problem seems to be due to the data flow transfer, linked to the repetition of short acquisitions and the real time of responses between the different devices of the beamline. For Th 2 Tf n°1, the coefficient of mean variation is reduced from 34.50% to 2.17% by omitting the divergent spectrum measurement. If degradation was the cause of this large variation, large mean coefficients of variation would have been observed for all consecutive measurements. Overall, the acquisition of the spectra for the same sample is repeatable and shows that the protein was not damaged under the beam as the calculated deviations are similar to those observed in our buffer medium alone and in the empty cells.

Table S2: Average relative standard deviation, in percent, of the ten recorded spectra for each sample.

Sample Relative standard deviation (%)

Empty tube n°1 The average spectrum, at each point, of the different samples is calculated according to the formula:

=

Then the variance, which represents the dispersion of the values of a sample, was calculated, at each point, of the different samples according to the formula:

= ( )
The standard deviation is deduced from the variance, at each point, according to the formula:

=

Finally, the relative standard deviation, noted RSD, was calculated, at each point, according to the formula: =

Figure 1 :

 1 Figure 1: UV-Visible spectroscopy comparison of Th2Tf (red line), Pu2Tf (orange line), holoTf (blue line) and apoTf (black line).

Figure 2 :

 2 Figure 2 : Comparison of experimental EXAFS spectra in k 2 χ(k) of Pu2Tf with [Pu(CO3)5] 6-complex and Th2Tf with [Th(CO3)5] 6-complex.

Figure 3 :

 3 Figure 3: Comparison of theoretical EXAFS spectra in k 2 χ(k) of the different models: Pu2Tf (1 carb) model (red line), Pu2Tf (2 carb) model (green line) and Pu2Tf (3 carb) model (purple line) with the experimental spectrum of Pu2Tf (top black line) and of [Pu(CO3)5] 6-complex (bottom blue line).

Figure 4 :

 4 Figure 4 : Superposition of the PDB structure of Ce2Tf (1FCK, grey) with the structure of Pu2Tf(1carb) (blue) calculated by molecular dynamics. In red are the 2 metallic complexation sites of the N-and C-lobes.

  Figure S5 of SI shows the radial distribution of the 20 oxygen atoms from lobes N and C in Pu2Tf(1carb) model. The EXAFS data represented by an arrow on the graph of Figure S5 are very consistent with the model, although on the shorter side of the dispersion. The second shell of carbon atoms in the model contains two distinct layers of 8 carbon atoms from lobes N and C, but only the first set (from 2.7 to 3.1 Å

Figure 5 :

 5 Figure 5: Scattering spectra of apoTf, holoTf M2Tf, holoTf-DFOB and M2Tf-DFOB complexes (solid lines) at physiological pH (M = Th, Pu) and their Guinier fits (dotted lines).

Figure 6 :

 6 Figure 6: SAXS spectra of holoTf, Pu2Tf, Pu2Tf-DFOB and apoTf complexes (solid line) and their fitting by an oblate ellipsoidal structure (dotted line).

  and Asp 395 in the N and C lobes respectively, are present in the local environment of Fe and Pu. The local environments of Fe and Pu are not seen as identical due to the presence of residues in the Pu environment that are not found in the Fe environment. This would also support the differences observed in the UV-Vis data that are very sensitive to the metal coordination sphere.

Figure S7 :

 S7 Figure S7: UV-Visible spectroscopy comparison of Pu 2 Tf (red line), DFOB (orange line), Pu 2 Tf-DFOB (blue line), Pu-DFOB (purple line) and apoTf (black line).

Figure S3 :

 S3 Figure S3: Theoretical EXAFS spectra of N and C lobe of each model along with experimental spectrum.

Figure S4 :Figure S5 :

 S4S5 Figure S4: Comparison of theoretical EXAFS spectra in k 2 χ(k) of the two lobes N and C (orange and blue line respectively) of the Pu 2 Tf (1carb) complex model with the experimental spectrum of the Pu 2 Tf complex (black line) and the average Pu 2 Tf (1carb) model (red line)

Figure S8 :

 S8 Figure S8: RMSD (Root-mean square deviation) of atomic positions of four independent MD simulations with different number of carbonate ions bound to Pu. Large fluctuations as seen in the simulation without carbonate (black line) suggest that the structure gets distorted when carbonate ion is absent. The structure of Pu 2 Tf without carbonate at t = 50 ns (right top) shows that the binding pocket fully opens during the simulation and Pu get easily "excreted". Proteins in simulations with 1 to 3 carbonates remain folded and show overall stability.

Table 1

 1 The best fit data for Pu2Tf reveals a first shell composed of 2.8(3) and 6.2(8) O with distances of 2.21(1) Å and 2.36(1) Å.

  2.21 / 2.36 this work, 2.21 / 2.37 Jeanson et al.) but are markedly different in the second carbon sphere (3.23 this work, 3.33 / 3.47 Jeanson et al.

Th 2 Tf n°1 34

  1.99 ± 0.01 .50 ± 0.25 / 2.12 ± 0.01 (after removal of the different spectrum)

	Empty tube n°2	1.66 ± 0.01
	Buffer n°1	1.99 ± 0.01
	Buffer n°2	1.29 ± 0.01
	Pu 2 Tf n°1	2.22 ± 0.01
	Pu 2 Tf n°2	4.57 ± 0.01
	Pu 2 Tf n°3	2.12 ± 0.01
	Pu 2 Tf-DFOB	1.85 ± 0.01
	Th 2 Tf n°2	2.25 ± 0.01
	Th 2 Tf n°3	2.86 ± 0.01
	Th 2 Tf-DFOB	1.82 ± 0.01
	holoTf n°1	1.25 ± 0.01
	holoTf n°2	1.55 ± 0.01
	holoTf-DFOB	1.45 ± 0.01
	apoTf n°1	1.44 ± 0.01
	apoTf n°2	1.81 ± 0.01
	apoTf n°3	1.50 ± 0.01

Table S3 :

 S3 Guinier fitting approximation of the SAXS data of apoTf, holoTf, M 2 Tf, holoTf-DFOB and M 2 Tf-DFOB complexes (M = Th(IV), Pu(IV); pH ~ 7.4; I = 0.05 M), this work, using SASView© software. χ 2 represents the error of fit. Comparison with the radius of gyration (in Å) calculated from MD model of Pu 2 Tf with 1,2 or 3 carbonates. Comparison also with the radius of gyration (in Å) of Yajima et al.43 , Jensen et al.16

	Sample	Rg (Å)	Fitting error χ²	MD models	Yajima et al.	Jensen et al.
	apoTf	28.7 ± 0.1	3.3.10 -5		30.3
	Th 2 Tf	28.9 ± 0.3	4.7.10 -6
	Pu 2 Tf	28.8 ± 0.2	30.4 ± 0.1 (2carb) 1.7.10 -5 30.1 ± 0.2 (1carb)		34.2± 0.6
	holoTf	27.6 ± 0.1	3.9.10 -5		29.3	33.2
	Th 2 Tf-DFOB	30.5 ± 0.3	7.3.10 -7
	Pu 2 Tf-DFOB	30.4 ± 0.2	3.6.10 -6
	holoTf-DFOB	30.3 ± 0.2	9.9.10 -7

Acknowledgements

All MD calculations were performed at Zentrum für Informationdienste und Hochleistungrechnen of Technische Universität Dresden, Germany. The generous allocation of computing time is gratefully appreciated. XAS and SAXS data were collected at the MARS beam line of SOLEIL synchrotron, Saint-Aubin, France. This work benefited from the use of the SasView application, originally developed under NSF award DMR-0520547. SasView contains code developed with funding from the European Union's Horizon 2020 research and innovation programme under the SINE2020 project, grant agreement No 654000.PhD fellowship of C. Zurita was provided by the Ministry of Higher Education and Research, France

Supporting information file

3 sections provided :

1-Spectroscopy 2-MD simulation and EXAFS data analysis 3-SAXS measurements

1-Spectroscopy