Rémimaurice
email: remi.maurice@subatech.in2p3.fr

Eric Renault

Yu Gong

Philip X Rutkowski

John K Gibson
email: jkgibson@lbl.gov

Synthesis and Structuresof Plutonyl Nitrate Complexes: Is Plutonium Heptavalent in PuO 3 (NO 3 ) 2 -?

Gas-phase plutonium nitrate anion complexes were produced by electrospray ionization (ESI) of a plutonium nitrate solution. The ESI mass spectrum included species with all four of the common oxidation states of plutonium: Pu(III), Pu(IV), Pu(V) and Pu(VI).Plutonium nitrate complexes were isolated in a quadrupole ion trap and subjected to collision induced dissociation (CID). CID of complexes of the general formula PuO x (NO 3 ) y -resulted in the elimination of NO 2 to produce PuO x+1 (NO 3 ) y-1 -, which in most cases corresponds to an increase in the oxidation state of plutonium. Plutonyl species, Pu V O 2 (NO 3 ) 2 -and Pu VI O 2 (NO 3 ) 3 -, were produced from Pu III (NO 3 ) 4 -and Pu IV (NO 3 ) 5 -, respectively, by the elimination of two NO 2 molecules. CID of Pu VI O 2 (NO 3 ) 3 -resulted in NO 2 elimination to yield PuO 3 (NO 3 ) 2 -, in which the oxidation state of plutonium could be VII, a known oxidation state in condensed phase but not yet in the gas phase. Density functional theoryconfirmed the nature of Pu V O 2 (NO 3 ) 2 -and Pu VI O 2 (NO 3 ) 3 -as plutonyl(V/VI) cores coordinated by bidentate equatorial nitrate ligands. The computed structure of PuO 3 (NO 3 ) 2 -is essentially a plutonyl(VI) core, Pu VI O 2 2+ , coordinated in the equatorial plane by two nitrate ligands and one radical oxygenatom. The computations indicate that in the ground spin-orbit free state of PuO 3 (NO 3 ) 2 -, the unpaired electron of the oxygen atom is antiferromagnetically coupled to the spin-triplet state of the plutonyl core. The results indicate that Pu(VII) is not a readily accessible oxidation state in the gas phase, despite that it is stable in solution and solids, but rather that a Pu(VI)-O•bonding configuration is favored, in which an oxygen radical is involved.

Introduction

Accessing high oxidation states of transuranic actinides, particularly plutonium, has gained much attention. [START_REF] Clark | The Chemistry of the Actinide and Transactinide Elements[END_REF] Hexavalent plutonium is common, primarily in the form of PuO 2 2+ in solution. A key issue is whether it is feasible to chemically engage the 5f 2 electrons of Pu(VI) to access higher oxidation states. [START_REF] Dyall | High Oxidation States in Actinide Chemistry: A DHF Investigation of Some Trends Exemplified by the AnO 2 F 4 Species[END_REF] Krot and Gel'man first reported Pu(VII) in alkaline solution in 1967, [START_REF] Krot | Production of Neptunium and Plutonium in Heptavalent State[END_REF] a feat shortly thereafter repeated. [START_REF] Spitsyn | Heptavalent State of Neptunium and Plutonium[END_REF] Keller and Seiffertsynthesized a solid compound containing Pu(VII). 5 More recently, Pu(VIII) has been reported in solution. [START_REF] Tananaev | Plutonium in Higher Oxidation States in Alkaline Media[END_REF] In the gas phase, octavalent PuO 4 has been inferred based on thermogravimetric experiments, [START_REF] Domanov | Refinement of Data on the Volatility of Octavalent Plutonium in the Form of Tetraoxide PuO 4[END_REF] as well as from the apparent vaporization of Pu from alkaline solutions. [START_REF] Nikonov | Plutonium Volatility in Ozonization of Alkaline Solutions of Pu(VI) Hydroxo Complexes[END_REF] However, recent computational studies indicate that PuO 4 as a molecule comprising Pu(VIII) is not stable. [START_REF] Huang | Oxidation States, Geometries, and Electronic Structures of Plutonium Tetroxide PuO 4 Isomers: Is Octavalent Pu Viable?[END_REF][START_REF] Zaitsevskii | On the Existence of Oxide Molecules of Plutonium in Highest Oxidation States[END_REF] Instead, PuO 2 F 4 may be a better candidate as a stable species comprising Pu(VIII). [START_REF] Dyall | High Oxidation States in Actinide Chemistry: A DHF Investigation of Some Trends Exemplified by the AnO 2 F 4 Species[END_REF] In the case of Pu(VII) in the gas phase, it has been predicted that PuO 3 F and Pu 2 O 7 should be stable. [START_REF] Zaitsevskii | On the Existence of Oxide Molecules of Plutonium in Highest Oxidation States[END_REF] The reduction potential in solution for Pu VII [START_REF] Clark | The Chemistry of the Actinide and Transactinide Elements[END_REF] which is only ca. 0.6 V higher than the Ce(IV/III) reduction potential; CeO 2 is a stable molecule [START_REF] Gabelnick | Infrared-Spectra and Structure of Some Matrix-Isolated Lanthanide and Actinide Oxides[END_REF] and it might be feasible to oxidize Pu(VI) to Pu(VII) in the gas phase. An important caveat in this evaluation is that the reported Pu(VII) reduction potential is for highly basic conditions such that direct comparison of these values is not necessarily indicative of comparative intrinsic stabilities of the oxidation states.

O 3 + /Pu VI O 2 2+ is ca. +2.3 V,
In accord with the above assessment, the primary goal of the present work was to investigate the stability of Pu(VII) in gas-phase oxides. The decomposition of gas-phase metal nitrate anions, M(NO 3 ) n -, can result in the elimination of neutral NO 2 , producing MO(NO 3 ) n-1 -. [START_REF] Li | Tandem Mass Spectrometry of Metal Nitrate Negative Ions Produced by Electrospray Ionization[END_REF][START_REF] Schröder | Gas-Phase Model Studies Relevant to the Decomposition of Transition-Metal Nitrates M(NO 3 ) 2 (M =Co, Ni) into Metal-Oxo Species[END_REF][START_REF] Franski | Mass Spectrometric Decomposition of [Mn n+ (NO 3 -) n+1 ] -Anions Originating from Metal Nitrates M(NO 3 ) n[END_REF] In cases where higher oxidation states of the metal are accessible than that in the nitrate precursor, oxidation of the metal center can occur during these processes. An example of oxidation by elimination of NO 2 , and NO,is the conversion of Mn II (NO 3 ) 3 -to

Mn VII O 4 -, in which Mn is in its highest accessible oxidation state. [START_REF] Franski | Mass Spectrometric Decomposition of [Mn n+ (NO 3 -) n+1 ] -Anions Originating from Metal Nitrates M(NO 3 ) n[END_REF] Decomposition of plutonium nitrate anions could be a route to oxidation of Pu, possibly to Pu(VII). It is demonstrated that PuO x (NO 3 ) y -complexes do indeed lose neutral NO 2 molecules upon collision induced dissociation (CID) in a quadrupole ion trap (QIT) resulting in oxidation of Pu, including the formation of plutonyl(V/VI) from Pu(III) and Pu(IV) complexes. Based on analogy with the proposed stable Pu VII O 3 F molecule, it may be expected that PuO 3 (NO 3 ) 2 would similarly comprise Pu(VII). This latter species was produced and its geometric and electronic structures, including the Pu oxidation state, were evaluated by density functional theory. Moreover, the magnetic coupling constant in PuO 3 (NO 3 ) 2 -was also assessed.

Experimental Methods

The experiments reported here were performed using an Agilent 6340 quadrupole ion trap mass spectrometer (QIT/MS) with the electrospray ionization (ESI) source inside a radiological contaminant glove box, as described in detail elsewhere. [START_REF] Rios | Electron Transfer Dissociation of Dipositive Uranyl and Plutonyl Coordination Complexes[END_REF] All the plutonium nitrate complex anions subjected to CID were produced by ESI of methanol solutions of 200 μMPu(NO 3 ) 4 . The plutonium isotope employed was Pu-242, which undergoes alpha-decay with a half-life of 3.8 × 10 5 y. The MS n CID capabilities of the QIT/MS allow isolation of ions with a particular mass-to-charge ratio, m/z, and subsequent insertion of an ion-molecule reaction time without applying ion excitation. The reactions inside the ion trap occur at a temperature around 300 K. [START_REF] Gronert | Estimation of Effective Ion Temperatures in a Quadrupole Ion Trap[END_REF] In the high-resolution mode, the instrument has a detection range of 20-2200 m/z with a mass width (FWHM) of ∼0.3 m/z. Mass spectra were recorded in the negative ion accumulation and detection mode. The instrumental parameters were similar to those employed in previous experiments. [START_REF] Gong | Formation and Characterization of the Uranyl-SO 2 Complex, UO 2 (CH 3 SO 2 )(SO 2 )[END_REF] High-purity nitrogen gas for nebulization and drying in the ion transfer capillary was supplied from the boil-off of a liquid nitrogen Dewar. As has been discussed elsewhere, [START_REF] Rutkowski | Hydration of Gas-Phase Ytterbium Ion Complexes Studied by Experiment and Theory[END_REF][START_REF] Rios | Gas-Phase Uranyl, Neptunyl, and Plutonyl: Hydration and Oxidation Studied by Experiment and Theory[END_REF] the background water and O 2 pressure in the ion trap is estimated to be on the order of 10 -6 Torr. The helium buffer gas pressure in the trap is constant at ~10 -4 Torr.

ComputationalApproach

Quantum mechanical calculations have been performed on the PuO 2 (NO 3 ) 2 -, PuO 2 (NO 3 ) 3 -, and PuO 3 (NO 3 ) 2 -complexes to determine their molecular geometries, and characterize their electronic structures. Various isomers, which may furthermore correspond to different oxidation numbers for the Pu center, are possible, and therefore molecular quantum mechanics is a useful tool to discriminate between the different possibilities. In order to elucidate the role of the coordination of nitrate ligands on the electronic structures of these systems, the corresponding PuO 2 + , PuO 2 2+ , and PuO 3 + cores have also been studied.

Geometry Optimizations

The determination of molecular geometries from quantum mechanical calculations requires the use of a given level of theory. In this work, two levels have been employed,a scalar relativistic (SR) oneand another considering spin-orbit (SO) effects. The SR calculations were performed using the Gaussian09 program package, [START_REF] Frisch | Revision D.01[END_REF] and are based on density functional theory (DFT). Hybrid exchange-correlation functionalslead to accurate geometries and harmonic frequencies in actinide oxides. [START_REF] Kovacs | Computed Vibrational Frequencies of Actinide Oxides AnO (0/+/2+) and AnO 2 (0/+/2+) (An = Th, Pa, U, Np, Pu, Am, Cm)[END_REF] Therefore, in this work, the PBE0 exchange-correlation functional [START_REF] Perdew | Rationale for Mixing Exact Exchange with Density Functional Approximations[END_REF][START_REF] Adamo | Toward Reliable Density Functional Methods Without Adjustable Parameters: The PBE0 Model[END_REF] was used, as in previous work of some of uson actinide carbides. [START_REF] Pereira | Thorium and Uranium Carbide Cluster Cations in the Gas Phase: Similarities and Differences between Thorium and Uranium[END_REF] For the Pu atom, the 60-electron small-core pseudo-potential ECP60MWB was considered, [START_REF] Cao | Valence Basis Sets for Relativistic Energy-Consistent Small-Core Actinide Pseudopotentials[END_REF] and the remaining electrons described using a (14s13p10d8f6g)/10s9p5d4f3g segmented contracted basis set. [START_REF] Cao | Segmented Contraction Scheme for Small-Core Actinide Pseudopotential Basis Sets[END_REF] For the O and N atoms, the (12s6p3d1f)/6s3p3d1f def2-TZVPD basis sets [START_REF] Weigend | Balanced Basis Sets of Split Valence, Triple Zeta Valence and Quadruple Zeta Valence Quality for H to Rn: Design and Assessment of Accuracy[END_REF][START_REF] Rappoport | Property-Optimized Gaussian Basis Sets for Molecular Response Calculations[END_REF] were used. Note that diffuse functions were considered due to the anionic nature of the ligands. Various starting geometries were considered, and relaxed with local optimization algorithms. The nature of the encountered stationary points was checked by computing harmonic frequencies, and for all relevant spin configurations, the lowest energy minima were found. The spin nature of the various considered spin configurations was assessed from the <S 2 > expectation values, computed for the fictitious non-interacting systems of interest. Although no formal relationship exists between Kohn-Sham determinants and true wave functions, these expectation values are used in practice as a measure of the degree of spin state mixings. Note that the numerical grid was defined by using the ultrafine keyword of Gaussian.In order to assess the impact of SO coupling on the molecular geometries, SO-DFT calculations 29 were performed with the NWChem6.3 suite of programs. [START_REF] Valiev | NWChem: A Comprehensive and Scalable Open-Source Solution for Large Scale Molecular Simulations[END_REF] The exchange-correlation functional, the basis sets, and the numerical grid were chosen identical to the ones used in the SR-DFT calculations. The energy adjusted SO pseudopotential of Küchleet al. [START_REF] Küchle | Energy-Adjusted Pseudopotentials for the Actinides -Parameter Sets and Test Calculations for Thorium and Thorium Monoxide[END_REF] was used for these calculations. In NWChem, the collinear approximation is used, which makes the results non-invariant with respect to rotations of the coordinate frame. While the error in total energies can be of the order of 0.1 eV, [START_REF] Van Wüllen | Spin densities in two-component relativistic density functional calculations: Noncollinear versus collinear approach[END_REF] bond distances and atomic charges are not expected to be significantly dependent on the choice of the coordinate frame.

Atomic Charges and Spin Populations

In order to determine the oxidation number of the Pu atom in the studied complexes, the SR Mulliken atomic charges and spin populations of the relevant configurations of the PuO 2 (NO 3 ) 2 -, PuO 2 (NO 3 ) 3 -, and PuO 3 (NO 3 ) 2 -complexes were computed, as well as the SO Mulliken charges. To assess the role of the coordination of the nitrate ligands on the PuO 2 + , PuO 2 2+ , and PuO 3 + cores, these quantities were also determined for these uncoordinated ions.

Note that in the PuO 2 + and PuO 2 2+ cores, the oxidation numbers of Pu are unambiguous, +V and +VI, respectively. Therefore, the comparison of the results obtained in the complexes and with these cores is of crucial importance to determine the oxidation numbers of Pu in the complexes. Mulliken charges are known to be basis set dependent; therefore, the absolute values of these effective charges are not necessarily meaningful. Moreover, effective charges differ drastically from formal charges, and therefore, effective charge values alone cannot be used to assign oxidation numbers. We have chosen this charge model since it was practically available at all the levels of theory considered in this work, i.e. SR-and SO-DFT. The most important criteria concerning the oxidation number of Pu is related to atomic spin density. For high oxidation states of Pu, the Pu spin density is expected to correlate directly with the number of f electrons. Test calculations on the complexes of interest at SR levels showed that our conclusions remain valid if other charge and spin populations models are used, namely CM5 charges [START_REF] Marenich | Charge Model 5: An Extension of Hirshfeld Population Analysis for the Accurate Description of Molecular Interactions in Gaseous and Condensed Phases[END_REF] and Hirshfeld spin densities. [START_REF] Hirshfeld | Bonded-Atom Fragments for Describing Molecular Charge-Densities[END_REF]

Magnetic Coupling in PuO 3 (NO 3 ) 2 -

As will be shown, the PuO 3 (NO 3 ) 2 -complex and the PuO 3 + core possess two local spins at SR levels, localized on the plutonium atomi (S i =1) and on one equatorial oxygen radical j (S j =1/2). Therefore, these local spins can result in two coupled spin states, with either S=1/2 or S=3/2. At a given geometry, the energy difference between these two coupled spin states can be effectively described using the Heisenberg-Dirac-van Vleck Hamiltonian: [START_REF] Heisenberg | On the Theory of Ferromagnetism[END_REF][START_REF] Dirac | Quantum Mechanics of Many-Electron Systems[END_REF][START_REF] Vanvleck | A Survey of the Theory of Ferromagnetism[END_REF] 𝐻 = 𝐽𝑆 𝑖 • 𝑆 𝑗 whereJ is defined as the magnetic coupling constant between the i and j magnetic centers. In this work, the SR calculations are performed within a spin-unrestricted formalism. In this case, solutions for given M S values are obtained, and the spin quantum number S is not imposed. Here, two solutions are computed, corresponding to M S =3/2 and referred to as the high-spin (HS) solution, and the M S =1/2 solution, referred to as thespin-broken-symmetry (BS) solution. In the ideal situation of non-interacting spins (such as at the dissociation limit), the HS solution corresponds to a pure quartet spin state, and the BS solution to an ideal mixture of quartet and doublet components, for which the weights correspond to the square of Clebsch-Gordan coefficients (the BS solution would have 1/3 quartet character, and 2/3 doublet character). If one assumes that such situation is encountered, it can easily be shown that the magnetic coupling constant can be in this case extracted within this weak-coupling scheme (WCS) as follows:

𝐽 WCS = 𝐸 HS -𝐸 BS whereE HS and E BS correspond to the self-consistent field (SCF) energies of the HS and BS solutions, respectively. If one deviates from this ideal case, the expectation values <S 2 > HS and <S 2 > BS deviate from their ideal values (here the ideal <S 2 > HS value would be 15/4, and the <S 2 > BS value 7/4), and one may extract the magnetic coupling constant, within the intermediate coupling scheme (ICS) as proposed by Yamaguchi: [START_REF] Yamaguchi | Electronic-Structures of Biradicals in Unrestricted Hartree-Fock Approximation[END_REF][START_REF] Yamaguchi | Molecular-Orbital (MO) Theory for Magnetically Interacting Organic-Compounds -Abinitio MO Calculations of the Effective Exchange Integrals for Cyclophane-Type Carbene Dimers[END_REF] 𝐽 ICS = 2 𝐸 HS -𝐸 BS 𝑆 [START_REF] Dyall | High Oxidation States in Actinide Chemistry: A DHF Investigation of Some Trends Exemplified by the AnO 2 F 4 Species[END_REF] HS -𝑆 2

BS

Both schemes are considered in this work, and the values obtained for both are compared.

Results and Discussion

ESI of Plutonium Nitrate Solution

ESI of a nominally Pu(IV) solution resulted in a mass spectrum, displayed in Figure 1,in which it is evident that all four of the common oxidation states of plutonium are present: Pu(III), Pu(IV), Pu(V) and Pu(VI). ThePu IV (NO 3 ) 5

-and Pu VI O 2 (NO 3 ) 3 -species were dominant in the ESI mass spectrum; assignments of the minor Pu III (NO 3 ) 4 -and

Pu IV O(NO 3 ) 3 -peaks were confirmed from their CID spectra (see Figures S1 andS2).

Unassigned peaks are attributed to impurities in the plutonium nitrate solution, which is typical under these experimental conditions. [START_REF] Rios | Gas-Phase Coordination Complexes of Dipositive Plutonyl, PuO 2 2+ : Chemical Diversity Across the Actinyl Series[END_REF] Plutonium is known to coexist in its four common oxidation states in solution. [START_REF] Clark | The Chemistry of the Actinide and Transactinide Elements[END_REF] The ESI spectrum illustrates this distinctive phenomenon, but it should be emphasized that ESI mass spectra in general, and this one in particular, do not necessarily directly reflect solution speciation. 41 CID of Pu(NO 3 ) 5 -and PuO 2 (NO 3 ) 3 -are discussed below.

For small mononuclear metal complexes, anion ESI mass spectra are generally dominated by ions with charges of -1 or -2; only ions with the charge state-1 were observed in the present experiments. In addition to the limitation of observable charge states under these experimental conditions, there is the possibility of significant changes in speciation during ESI and ion transport into the ion trap. In the particular case of nitrate ions, facile elimination of NO 2 can result in gas-phase oxides that are not present in solution. As discussed below, fragmentation of Pu(NO 3 ) 5 -results in PuO 2 (NO 3 ) 3 -, which likely accounts for the high abundance of the latter species in the ESI mass spectrum. Despite caveats about quantitative speciation evaluations by ESI, [START_REF] Mcdonald | Failure of ESI Spectra to Represent Metal-Complex Solution Composition: A Study of Lanthanide-Carboxylate Complexes[END_REF] anion complexes present in solution are generally apparent in ESI mass spectra. Thus, the appearance of substantial Pu(NO 3 ) 5 -in the ESI mass spectrum suggests that this Pu(IV) species is present in solution. In contrast, from solution speciation studies it has been suggested that Pu(NO 3 ) 5 -is not significant at any nitrate concentration. [START_REF] Veirs | Characterization of the Nitrate Complexes of Pu(IV) Using Absorption-Spectroscopy, N-15 NMR, and EXAFS[END_REF] Largely based on that work, thermodynamic modeling of plutonium nitrate speciation includes neutral Pu(NO 3 ) 4 and dianionic Pu(NO 3 ) 6 2-, but excludes Pu(NO 3 ) 5 -. [START_REF] Kubic | A Thermodynamic Model of Plutonium (IV) Nitrate Solutions[END_REF] There seems to be no obvious rationale as to why Pu(NO 3 ) 5 -should not be a stable solution species-the present results show that it is a stable gas phase species that results from ESI of plutonium nitrate solutions, suggesting that its existence in solution is probable. It must be emphasized that the importance of Pu(NO 3 ) 5 -at various nitrate concentrations (and other solution conditions) remains uncertain, and furthermore that the solvent in this work was primarily methanol rather than water. However, based on the ESI results in this work,Pu(NO 3 ) 5 -should be considered when modeling speciation in plutonium nitrate solutions.

Gas-Phase Synthesis of PuO x (NO 3 ) y

-

The CID spectra exhibited a variety of processes corresponding to oxidation of Pu by loss of NO 2 , such as reaction 1, which are the focus of this work.Reduction processes such as by NO 3 ligand loss, reaction 2, were also observed but are not pertinent to the quest for higher oxidation states of Pu.

Pu IV O(NO 3 ) 3 - Pu V O 2 (NO 3 ) 2 -+ NO 2 (1) Pu VI O 2 (NO 3 ) 3 - Pu V O 2 (NO 3 ) 2 -+ NO 3
(2) Among the observed redox processes in the CID spectra (Figs. 2,3,S1, S2) were Pu(IIIIV), Pu(IIIV), Pu(IIIVI), Pu(IVV), Pu(IVVI) and Pu(VIV).Theprimary motivations for this work werereactions that convert a Pu(III) or Pu(IV) nitrate anion into plutonyl, and on the formation of a plutonium nitrate complex with an abnormally high number of oxygen atoms bonded to plutonium. The observed reactions that produce these species are presented, followed by a discussion of theirgeometries and electronic structures, including the Pu oxidation states. The -reactions‖ discussed here are induced by CID of an anionic plutonium nitrate complex that has been isolated in the ion trap and is subsequently fragmented after resonant excitation and multiple energetic collisions with helium atoms in the trap prior to acquiring the product mass spectrum.

These -reactions‖ are thus unimolecular decompositions of the specified complex anion, not reactions with any reagent gas in the ion trap. It should be remarked that the instrumental low mass limit for resonant ejection after CID precludes the identification of low m/z anion products such as NO 2 -.

The two reactions that convert plutonium nitrate complexes to plutonyl(V) and plutonyl(VI) complexes are given by reactions 3 and 4, with the corresponding CID spectra shown in Figures S1 and2, respectively.

Pu Rather than 2(NO 2 ), the product in reactions 3 and 4 could alternatively be N 2 O 4 , the formation of which is slightly less endothermic (by ca. 50 kJ/mol) but essentially degenerate in energy when ∆G at 300 K is considered. [START_REF] Lias | NIST Chemistry WebBook[END_REF] There is little doubt as to the general nature and oxidation states of the plutonyl(V/VI) nitrate products in equations 2 and 3, but their geometries and electronic structures were evaluated indetail by the computations discussed below. The ability to produce plutonyl(V) and plutonyl(VI) complexes by CID of plutonium(III) and plutonium(IV) nitrates is enabled by the low enthalpy and favorable entropy for reaction 5, and by the low reduction potentials for Pu(V/III) and Pu(VI/IV), which are both approximately 1 V. 1 NO 3  NO 2 + O (5) ∆H = 211 kJ/mol [START_REF] Lias | Gas-Phase Ion and Neutral Thermochemistry[END_REF] The direct synthesis of plutonylcomplexes from decomposition and oxidation of plutonium nitrates is significant. A second central goal of this work was to explore the possibility of further oxidizing Pu(VI) to Pu(VII) by additional loss of NO 2 . As is seen in Figure 3, CID reaction 6 proceeds efficiently.

III (NO 3 ) 4 - Pu V O 2 (NO 3 ) 2 -+ 2(NO 2 ) (3) Pu IV (NO 3 ) 5 - Pu VI O 2 (NO 3 ) 3 -+ 2(NO 2 ) (4)
PuO Given that the Pu(VII/VI) reduction potential is modest, 0.85 V in basic solution, [START_REF] Clark | The Chemistry of the Actinide and Transactinide Elements[END_REF] it is conceivable that the product of reaction 6 is plutonium trioxide coordinated by two nitrate anions, with an oxidation state of Pu(VII). It should be emphasized that there is not necessarily a direct correlation between reduction potentials in solution, particularly basic solution, and the propensity for redox processes to occur in gas phase complexes. Solution reduction potentials cannot be employed to predict whether a gas-phase oxidation process will proceed, but they can be employed as a qualitative guide for the general feasibility of oxidation. In particular, the oxidation behavior of Pu in basic solution does not necessarily indicate that the Pu(VII) oxidation state should be similarly accessible in the gas phase, but it does suggest this as a reasonable target. An alternative structure is plutonyl(V) nitrate coordinated by an oxygen radical to yield an oxidation state of Pu(VI). This latter scenario is seen in such species asCaO(NO 3 ) 2 -, where the Ca(III) oxidation state is essentially inaccessible. [START_REF] Franski | Mass Spectrometric Decomposition of [Mn n+ (NO 3 -) n+1 ] -Anions Originating from Metal Nitrates M(NO 3 ) n[END_REF] Also apparent in Figure 3 is a small peak that corresponds to PuO 4 (NO 3 ) -, which could conceivably be Pu(VIII) tetraoxide coordinated by a nitrate. However, the octavalent oxidation state is less viable than Pu(VII) and it has furthermore been convincingly demonstrated by theory that the oxidation state in the neutral PuO 4 core is not even Pu(VI), but rather remarkably only Pu(V): (PuO 2 + )(O 2 ) -. [START_REF] Huang | Oxidation States, Geometries, and Electronic Structures of Plutonium Tetroxide PuO 4 Isomers: Is Octavalent Pu Viable?[END_REF] The computed molecular geometries and electronic structures of Pu V O 2 (NO 3 ) 2 -, Pu VI O 2 (NO 3 ) 3 -and PuO 3 (NO 3 ) -are presented below.

As the nomenclature of the first two of these species suggests, they could confidently be assigned as plutonyl(V) and plutonyl(VI) nitrates, which is confirmed and elaborated by the theory results below. The nature of PuO 3 (NO 3 ) 2 -is not similarlyapparent and computations were needed to characterize this exotic species. respectively. Note that the ground spin configuration of the PuO 3 (NO 3 ) 2 -complex is the same the one of the barePuO 3 + ions, i.e.M S = 1/2.It is known from multiconfigurational wave function based calculations that the ground spin state of theplutonyl PuO 2 + core is a quartet, [START_REF] Macchia | A theoretical study of the ground state and lowest excited states of PuO0/+/+2 and PuO20/+/+2[END_REF] and the one of the plutonyl PuO 2 2+ corea triplet. [START_REF] Ismail | Theoretical Studies of the Actinides: Method Calibration for the UO 2 2+ and PuO 2 2+ Ions[END_REF][START_REF] Gendron | Magnetic Resonance Properties of Actinyl Carbonate Complexes and Plutonyl(VI)-trisnitrate[END_REF] Thus,the present DFT calculations are in agreement with these results. Since for the PuO 3 (NO 3 ) 2 -complex and the PuO 3 + core the ground M S = 1/2 configurations do not correspond to pure spin states (<S 2 >=1.74 and <S 2 >=1.72, respectively), but rather to a BS solution (see theElectronic Structures section), the higher-energy HS geometry, which is similar to the ground BS geometrywhile more physically grounded,is also reported, and used for the calculation of the magnetic coupling constants as discussed below.The HS configuration is only 123meV(12 kJ/mol) higher in energy than the BS configuration at the HS geometry (see the Magnetic Coupling section).The optimal geometries of the PuO 2 (NO 3 ) 2 -and PuO 2 (NO 3 ) 3 -complexes, as well as the geometry of the HS solution for PuO 3 (NO 3 ) 2 -are shown in Figure 4, and the most relevant geometrical parameters reported in Table 1 (see Table S1 for the PuO 2 + , PuO 2 2+ , and PuO 3 + cores). In all the considered complexes, a linear O-Pu-O arrangement is observed, while the other fragments (nitrates or oxygen) are located in the equatorial plane, with much longerPu-O distances than for those oxygen atoms in the axial orientation. The structure of the PuO 3 + core is unusual in that it consists of a linear plutonyl moiety with the third oxygen atomin the equatorial plane. Note that a geometry of the PuO 2 (NO 3 ) 3 -complex has been recently computed by Odoh and Schreckenbach [START_REF] Odoh | Theoretical Study of the Structural Properties of Plutonium(IV) and (VI) Complexes[END_REF] and that the geometry obtained in this work is in very good agreement with their reported structure.For all three of the complexes studied here the axial Pu-O distances are close to 1.7 Å, which is typical for plutonyl units.Coordination by equatorial nitrate ligands slightly destabilizes the Pu-O bonds (see the distances reported in Tables 1 andS1). Although all the converged geometries strictly belong to the C 1 symmetry point group, the PuO 2 (NO 3 ) 2 -geometry can be considered as D 2h symmetry, while the PuO 2 (NO 3 ) 3 -and PuO 3 (NO 3 ) 2 -complexes are close to C 3h symmetry and C 2v symmetry, respectively.For all the studied systems, the SO-DFT geometries (see Table S2) are in good agreement with the SR-DFT ones (see Tables 1 andS1), with only a slight shortening of the Pu-O equatorial bond in PuO 3 (NO 3 ) 2 -. However, this change is not expected to modify the oxidation number of the plutonium center (see the following section), such that only the SR results are reported in the main text.

Molecular Geometries

Electronic Structures

In order to further describe the electronic structure of these systems, Mulliken atomic charges and spin populations were computed (see Tables 2 andS7). In the PuO 2 (NO 3 ) 2 complex, as in the PuO 2 + core, the Pu atom possess three unpaired electrons, which indicates a Pu(V) oxidation state. Note that in the PuO 2 + core, these unpaired electrons are located on non-bonding δ u and ϕ u forbitals of the Pu atom. [START_REF] Macchia | A theoretical study of the ground state and lowest excited states of PuO0/+/+2 and PuO20/+/+2[END_REF] In the PuO 2 (NO 3 ) 3 -complex, as well as in the PuO 2 2+ core, two unpaired electrons are associated with the Pu atom, which corresponds to a Pu(VI) oxidation state. In the PuO 2 2+ core, due to the use of a single-determinental picture, SR-DFT leads to a δ u 2 electronic structure instead of the δ u 1 ϕ u 1 one. [START_REF] Ismail | Theoretical Studies of the Actinides: Method Calibration for the UO 2 2+ and PuO 2 2+ Ions[END_REF][START_REF] Gendron | Magnetic Resonance Properties of Actinyl Carbonate Complexes and Plutonyl(VI)-trisnitrate[END_REF][START_REF] Craw | Ab-Initio Quantum-Chemical Calculations on Uranyl Uo22+ Plutonyl Puo22+, and Their Nitrates and Sulfates[END_REF][START_REF] Hay | Theoretical studies of the properties and solution chemistry of AnO(2)(2+) and AnO(2)(+) aquo complexes for An = U, Np, and Pu[END_REF] The δ u and ϕ u levels being non-bonding in this core, this does not affect much computed geometries, atomic charges and atomic spin densities, which are the important quantities reported in this work. However, in order to simplify the discussion and avoid confusion, the Kohn-Sham orbitals bearing the unpaired electrons are not plotted.In the PuO 3 (NO 3 ) 2 -complex (for both the HS and BS geometries), two unpaired electrons are associated with the Pu atom, while the effective Pu charge is very close to thatin the PuO 2 (NO 3 ) 3 -complex. Moreover, the -lone‖ oxygen atom, O 3 ,has one unpaired electron, either coupled ferromagnetically to the plutonyl triplet (HS solution) or antiferromagnetically (BS solution). The same behavior is obtained for the PuO 3 + core. Therefore, it is concluded that the Pu atom is not further oxidized in the PuO 3 (NO 3 ) 2 -complex compared with PuO 2 (NO 3 ) 3 -, i.e. the plutonium oxidation state is Pu(VI), not Pu(VII), in the PuO 3 (NO 3 ) 2 -complex. From these results, one can easily understand that the SO coupling cannot significantlyaffectthe molecular geometries of these plutony systems, since it essentially mixes the ground SO free configuration with configurations that belong tothe same electronic configuration, or with configurations that only differ by the occupation of the essentially non-bonding f orbitals of the Pu center. For instance, in the PuO 2 (NO 3 ) 2 -case,as in the PuO 2 + core, the ϕ u 2 δ u 1 and ϕ u 1 δ u 2 configurations are coupled and mixed in the ground SO state due to SO coupling (with quartet and doublet spin components), while in PuO 2 (NO 3 ) 3 -, as inPuO 2 2+ , the ground SO state, obtained from multiconfigurational wave function calculations, is found to beessentially constituted of the ϕ u 1 δ u 1 configuration [START_REF] Macchia | A theoretical study of the ground state and lowest excited states of PuO0/+/+2 and PuO20/+/+2[END_REF] (with triplet and singlet spin components). In the PuO 3 (NO 3 ) 2 -complex, the lowest energy doublet and quartet components are expected be mixed by SO coupling. These components correspond to a similar electronic structure, with two unpaired electrons on the Pu center, and one unpaired electron on the oxygen radical. For similar reasons, the atomic charges are harlyaffected by SO coupling (see Tables 2, S1, andS8), and therefore, the qualitative conclusions concerning the oxidation states of the Pu centers in these nitrate complexes are independent of the introduction of SO coupling. However, one should note that the SO coupling breaks the spin symmetry, and that its proper inclusion would result in a quartet/doublet mixture in the ground SO state of the PuO 2 (NO 3 )

Magnetic Coupling in PuO 3 (NO 3 ) 2 -

To extend our SR-DFT study, the magnetic coupling constant in the PuO 3 (NO 3 ) 2 complex was computed for the HS geometry. From the results shown in Table 3, we obtain a Jvalue of 123 meVwith both the WCS and the ICS. These values are practically identical, which is not surprising given that the computed values of <S 2 > HS and <S 2 > BS arevery close to the ideal ones of the WCS (3.75 and 1.75, respectively). Therefore, it is concluded that an oxygen radical is present in the PuO 3 (NO 3 ) 2 -complex, and that its unpaired electron is antiferromagnetically coupled to plutonium, with a large coupling constant. Therefore, the ground SO free state is a spin doublet, and the ground SO state is predicted to have a dominant doublet character. Indeed, due to the absence of an inversion center, the doublet and quartet components mix under the effect of SO coupling, which is often referred to as antisymmetric exchange or Dzyaloshinskii-Moriya interaction. [START_REF] Dzyaloshinsky | A Thermodynamic Theory of Weak Ferromagnetism of Antiferromagnetics[END_REF][START_REF] Moriya | Anisotropic Superexchange Interaction and Weak Ferromagnetism[END_REF][START_REF] Maurice | Theoretical determination of spin Hamiltonians with isotropic and anisotropic magnetic interactions in transition metal and lanthanide complexes[END_REF] By comparing Tables 2 andS9, one can see that the magnetic coupling constant is strongly affected by the coordination of equatorial nitrate ligands. This parameter is known to be strongly dependent on the geometry, and it should be noted that the smaller coupling value in the PuO 3 (NO 3 ) 2 complex is associated with a larger Pu-O 3 distance than in the PuO 3 + core.

Conclusions

ESI of a nominally plutonium(IV) nitrate solution resulted in species corresponding to the four readily accessible oxidation states of plutonium: Pu(III), Pu(IV), Pu(V) and Pu(VI). Although changes in oxidation state can occur during ESI, which is an electrochemical process, the coexistence of these four oxidation states is consistent with solution chemistry and the known low reduction potentials for Pu(VI), Pu(V) and Pu(IV). A dominant species in the ESI mass spectrum was Pu IV (NO 3 ) 5 -, which is generally not considered a significant solution species. Recognizing that ESI mass spectra do not directly represent solution speciation, this result nonetheless suggests that Pu IV (NO 3 ) 5

-should be considered when modeling plutonium nitrate solution speciation. CID-induced NO 2 elimination from Pu III (NO 3 ) 4 -and Pu IV (NO 3 ) 5 -resulted in Pu V O 2 (NO 3 ) 2 -and Pu VI O 2 (NO 3 ) 3 -, respectively, demonstrating the direct synthesis of plutonyl from plutonium nitrate. CID of Pu VI O 2 (NO 3 ) 3 resulted in loss of an additional NO 2 molecule to produce PuO 3 (NO 2 ) 2 -; it was postulated that this species could have a PuO 3 + core in which the oxidation state is Pu(VII), and oxidation state known to exist in solution. However, DFT computations revealed that the structure of the PuO 3 + core in PuO 3 (NO 2 ) 2 -is similar to that of the PuO 2 2+ core in PuO 2 (NO 3 ) 3 -, with an oxidation state of Pu(VI) in both. The PuO 3 (NO 2 ) 2 -complex can be represented as PuO 2 2+ coordinated in the equatorial plane by two nitrate ligands and one radical oxygen atom. The results indicate that Pu(VI) is not readily oxidized to Pu(VII) in the gas phase, as might have been expected, but rather that a species comprising a radical oxygen atom bound to Pu(VI) is the more favorable bonding configuration.

Supporting Information

Figure 1 .

 1 Figure 1.ESI mass spectrum of the plutonium nitrate solution. The assignments of the minor Pu(NO 3 ) 4-and PuO(NO 3 ) 3 -peaks were confirmed from their CID spectra (Figs.S1 and S2).

Figure 2 .

 2 Figure 2.CID mass spectrum of Pu(NO 3 ) 5 -. The dominant channel is to produce plutonyl(VI) nitrate. A very small amount of PuO 3 (NO 3 ) 2 -is also produced, presumably by loss of a third neutral NO 2 molecule from the dominant intermediate.

Figure 3 .

 3 Figure 3. CID mass spectrum of PuO 2 (NO 3 ) 3 -. The dominant channel is to produce PuO 3 (NO 3 ) -. Another significant channel is production of PuO 2 (NO 3 ) 2 .

3 -(

 3 shown in Figure 4 for the PuO 2 (NO 3 ) 2 -(left),PuO 2 (NO 3 ) middle) and PuO 3 (NO 3 ) 2 -(right) complexes correspond to M S values of 3/2, 1, and 3/2

Figure 4 .

 4 Figure 4. Ball and stick representations of the PuO 2 (NO 3 ) 2 -(left), PuO 2 (NO 3 ) 3 -(middle), and PuO 3 (NO 3 ) 2 -(right) complexes. For the PuO 3 (NO 3 ) 2 -complex, the high-spin (HS) configuration geometry is shown, the ground-state BS one being practically the same. Color code : Pu(violet), O(red), and N(blue).

  PuO 3 + ions, SO geometrical parameters of the bare ions and of the complexes, SR molecular geometries of the PuO 2 (NO 3 ) 2 -, PuO 2 (NO 3 ) 3 -, and PuO 3 (NO 3 ) 2 plutonium nitrate complexes, SRMullikenatomic charges and atomic spin populations of the bare PuO 2 + , PuO 2 2+ , and PuO 3 + ions, SO Mulliken atomic charges of the bare ions and of the complexes,and energy difference between the HS and BS configurations of the bare PuO 3 + ion (HS geometry) and <S 2 > values for the HS and BS configurations, respectively.

Table 1 .

 1 Scalar-relativistic geometrical parameters of the PuO 2 (NO 3 ) 2 O 1 and O 2 correspond to the oxygen atoms of the plutonyl units. b O 3 corresponds to the oxygen atom that does not belong to a plutonylor nitrate unit.

	-, PuO 2 (NO 3 ) 3 -, and

a

Table 3 .

 3 Energy difference between the high-spin (HS) andspin-broken-symmetry (BS) configurations of the PuO 3 (NO 3 ) 2 -complex (HS geometry) and <S 2 > values for the HS and BS configurations, respectively.

	Parameter	Value
	E HS -E BS (meV)	123
	<S 2 > HS	3.78
	<S 2 > BS	1.77

Pu VI O 2 (NO 3 ) 3 -Pu III (NO 3 ) 4 -Pu V O 2 (NO 3 ) 2 -PuO 3 (NO 3 ) 2 -Pu IV O(NO 3 ) 3 -Ion Intensity

Acknowledgement

The work of YG, PXR and JKG wasfullysupported by the U.S. Department of Energy, Office of Basic Energy Sciences, Heavy ElementChemistry, at LBNL underContract No. DE-AC02-05CH11231.

TOC

The plutonium oxidation states Pu(III), Pu(IV), Pu(V) and Pu(VI) wereidentified in gas-phase nitrate complexes. Fragmentation of the Pu VI O 2 (NO 3 ) 3 -complexresulted in NO 2 elimination to yield PuO 3 (NO 3 ) 2 -, the computed structure of which is a plutonyl(VI) core, Pu VI O 2 2+ , coordinated in the equatorial plane by two nitrate ligands and a radical oxygen atom. It is concluded that Pu(VI) is not oxidized to Pu(VII) by an oxygen radical, revealing the limited stability of oxidation states higher than Pu(VI).


L Fournier

P Delage

E Potin

G Rebuffet

P.-L Michel

Mc Solari

M C Feiters

Czjzek

Catherine Leblanc
email: leblanc@sb-roscoff.fr

H Vilter

J.-B Fournier

L Delage

P Potin

E Rebuffet

G Michel

P L Solari

M Czjzek

Vanadium haloperoxidases: from the discovery 30 years ago to X-Ray crystallographic and V K-edge absorption spectroscopic studies

Keywords: Vanadium-dependent haloperoxidase, vanadium coordination, crystallographic structure, structural evolution, oligomeric state, X-ray absorption spectroscopy on biological vanadium

A c c e p t e d M a n u s c r i p t

Introduction

In Nature, many organic compounds incorporate halogen atoms, which is often an essential step for the biological properties of halogenated antibiotics, drugs or signaling molecules [1]. Halogenated natural products are most commonly chlorinated or brominated, while iodinated and fluorinated compounds are rare, and all structural classes of chemicals are represented, from simple phenolic and aliphatic compounds, to complex alkaloids, polyketides and oligopeptides [2,[START_REF] Gribble | Occurrence of halogenated alkaloids[END_REF]. A large majority of the biogenic halogenated compounds have been isolated in marine organisms, especially marine macroalgae, which seem to be important actors of iodine and bromine transfers in the coastal marine environment [START_REF] Amachi | [END_REF][5][6].

Among halogenating enzymes, haloperoxidases utilize hydrogen peroxide for electrophilic halogenation, via the oxidation of halides according to the following reaction: H 2 O 2 + X -+ H + → H 2 O + HOX, wherein X -represents a halide ion and may be Cl -, Br -or I -. Haloperoxidases have been classified in two groups, according to the nature of their cofactor, vanadium or heme (iron-containing porphyrin) [1,[7][8][9][START_REF] Wever | Vanadium haloperoxidases[END_REF]. Metal-free haloperoxidases have also been described in some bacteria [START_REF] Littlechild | [END_REF]. Since the discovery of the first heme haloperoxidase, in the 1960s, from the terrestrial fungus Caldariomyces fumago [12] a number of other heme haloperoxidases have been characterized [1,9]. In this review, we focus on the second important group of haloperoxidases, the vanadium-dependent haloperoxidases (VHPO). In marine algae, these enzymes could be involved in anti-oxidant protection and in chemical defense and are likely to have a key role in the biochemical part of the iodine and bromine cycling in marine environment [13,14]. Starting with their discovery thirty years ago [15], this review highlights the main progress that has been obtained on the biochemistry and on the global and fine structural characteristics of VHPO, based on molecular cloning, crystallography and X-ray absorption spectroscopy (XAS). They are classified according to the most electronegative halide that they can oxidize, i.e. chloroperoxidases (VCPO) can catalyze the oxidation of chloride as well as of bromide and iodide, and bromoperoxidases (VBPO) react with bromide and iodide, whereas iodoperoxidases (VIPO) are specific for iodide. Until recently, only chloro-and bromoperoxidases from eukaryotic species have been studied, showing a fragmental view of this large enzyme family. Novel types of VHPO discovered in marine bacteria give now some clues to explore new biosynthetic pathways of natural halogenated compounds through in vitro heterologous expression, but also to further

Biochemical and molecular characterization of vanadium haloperoxidases

On the discovery of vanadium-dependent haloperoxidases in the brown alga

Ascophylum nodosum

Interest in peroxidases from brown alga arose in the 1970s, through studies related to the identification of antibiotics from algae reporting that phloroglucinol [16], and then oligomers [17] and polymers of phloroglucinol units [18] were detected in brown algae. These compounds were further characterized as phlorotannins as reviewed in [START_REF] Ragan | Phlorotannins, brown alga polyphenols[END_REF]. It was rapidly hypothesized, that peroxidases may be involved in the formation of these phenolic compounds, unique to brown algae. Despite extraction difficulties, due to the complex materials (anionic mucilages containing alginate and fucans), peroxidase-activity was detected in some crude-extract of brown algae, but the enzyme activity was partially lost during the purification process [START_REF] Vilter | [END_REF]. Intense work finally led to the isolation of the first vanadium-dependent haloperoxidase from knotted wrack, the brown alga Ascophyllum nodosum (Fucaceae, Fucales, Phaeophyta), called AnI in 1984 [15], showing that this enzyme preferentially oxidized iodide and also bromide and thiocyanide in the presence of vanadium (V), but not chloride (reviewed in [START_REF] Vilter | Vanadium-dependent haloperoxidases[END_REF]). The addition of iron and other metal ions had no effect on the activity of the apoenzyme. This algal peroxidase thus was not a hemoprotein, like other iodoperoxidases from plants such as the well-known horseradish peroxidases [START_REF] Gajhede | Horseradish Peroxidase[END_REF].

Anecdotal, in the 1970s, vanadium was not considered to be an essential element in biology or in medicine, and until 1984, no scientific study has reported that vanadium could be a natural component of any enzyme. Consequently the paper on the vanadium-dependence of this new algal haloperoxidase [15] was not accepted in the first instance. However, since vanadium accumulation has been reported in ascidians in 1911 by Henze [START_REF] Henze | [END_REF] experiments to test the effect of vanadium compounds were proposed by Vilter and coworkers. Indeed H. Vilter had observed an effect when solutions were aliquoted using a Hamilton syringe that brought the solutions in contact with vanadium-containing steel. Inspired by these effects, significant increase in haloperoxidase activity was observed in subsequent experiments using vanadate-A c c e p t e d M a n u s c r i p t 6 containing buffers. At that time, the detection of a vanadium-containing nitrogenase was also reported [24,25]. In 1986, the vanadate cofactor was finally confirmed for the vanadiumdependent haloperoxidase from A. nodosum [26] and for the alternative nitrogenase in Azotobacter chroococcum [27]. In parallel, several reports indicated that vanadium was indeed an essential trace element for animals [START_REF] Hopkins | Proceedings of the 2nd International Symposium in Trace Element Metabolism in Animals[END_REF], Chlorella [START_REF] Meisch | [END_REF][30][31][32], Enteromorpha and Fucus [33]. Since its discovery, AnI was used as a laboratory model enzyme for the understanding of VHPO properties. Numerous further studies were carried out to characterize the V coordination inside the active site of AnI (later called An-VBPOI in this review), based on electron paramagnetic resonance [26,34,35], on 51 V NMR [36][37][38], X-ray crystallography [39,40] and on XAS ( [41][42][43] see below).

Biochemical characterization of vanadium haloperoxidases

Vanadium-dependent haloperoxidases (EC 1.11.1.10) catalyze the two-electron oxidation of halides (X -: I -, Br -or Cl -) in the presence of hydrogen peroxide (H 2 O 2 ) by a bi-bi ping-pong mechanism. During the catalytic cycle, the vanadate cofactor, at the resting (V) state, first coordinates one molecule of H 2 O 2 to form a stable peroxovanadate intermediate, which can then react with halide. After halide oxidation, the electron oxidized halogen intermediate "X + ", which is a mixture of different halogen species such as OX -, X 3 -or X 2 , may rapidly halogenate organic substrates in a non-enzymatic reaction (Scheme 1). In the absence of organic substrate, a second equivalent of H 2 O 2 is oxidized to form a single oxygen and X -[for reviews 1,7,8,[START_REF] Wever | Vanadium haloperoxidases[END_REF]44].

Haloperoxidase in vitro assays

For kinetic studies, vanadium and heme haloperoxidases differ from other enzymes by the production of this very reactive and undetectable intermediate "X + ". Standard assays for the determination of VHPO activity are therefore based on the reactivity of this mixture with commercial chemicals. Phenol red and o-dianisidine were first reported as chemical probes for the detection of VHPO-catalyzed halogenation, but are restricted to qualitative studies [START_REF] De Boer | [END_REF][46][47]. The monochlorodimedone (MCD) halogenation provides a simple assay to determine steady-state kinetic parameters of VCPO and VBPO at acidic pH by monitoring the loss of absorbance of MCD at 290 nm [48][49][50]. The triodide (I 3 -) assay (reviewed in [START_REF] Vilter | Vanadium-dependent haloperoxidases[END_REF]) was used to identify the first VIPO, i.e. specific for iodide oxidation, in brown algae of the Laminariaceae A c c e p t e d M a n u s c r i p t 7 family [51][START_REF] Almeida | Phyllariopsis brevipes-a brown algae with vanadium-dependent iodoperoxidases[END_REF][START_REF] Colin | [END_REF][54]. However, this assay presents important limits due to the high chemical instability of I 3 -in solution. For this reason, a colorimetric assay was later developed for the titration of both iodo-and bromoperoxidase activities based on the reactivity of thymolsulfonephthalein, also named thymol blue (TB) toward oxidized bromide and iodide species [55]. Using this assay, novel VIPO were recently characterized in the marine bacteria

Zobellia galactanivorans [56].

Biochemical coordination of V in vanadium haloperoxidases

Vanadate (VO 4 ) is the prosthetic group of VHPO and its incorporation into the enzyme is necessary for the catalytic activity. Indeed the apoenzymes, produced through dialysis, feature a drastic decrease of activity, while the reconstitution of VHPO with aqueous sodium orthovanadate leads to progressive and full recovery of haloperoxidase activity [15,35,[57][58][59][60]. The ratio of vanadate per protein subunit was estimated around one VO 4 per subunit, but was lower in VBPOII from A. nodosum [60]. The affinity of VHPO for vanadate depends on pH [15,61,62], and is much stronger than for oxyanions, such as phosphate or

molybdate (HVO 4 2->> HPO 4 2-> AsO 4 3-> MoO 4 2-
) [34,61]. However, the peroxidase activity decreases in phosphate-containing buffers [START_REF] Vilter | Vanadium-dependent haloperoxidases[END_REF].

Unlike the heme-containing enzymes, there is no change in the oxidation state of the VHPO metal center during the synthesis of the halogenating agent, making them insensitive to oxidative inactivation during turnover. VHPO enzymes are also extremely thermostable, resistant to high concentration of organic solvents such as methanol, ethanol, 1-propanol, acetone [START_REF] De Boer | [END_REF] and dioxane and still active in concentrated solutions of guanidine hydrochloride or SDS [61,63].

Vanadium haloperoxidase -catalyzed halogenation

Because of their biochemical properties, VHPO have received increasing attention as biocatalysts for pharmaceutical and biotechnological applications [44,64] E. coli [65][66][67], in yeast system [58,59], or in other heterologous systems [68][69][70] (see Table 1 for complete references).

VHPO activities have been detected in a very large number of marine macroalgal classes using MCD or I 3 -assays [START_REF] Vilter | Vanadium-dependent haloperoxidases[END_REF], but the corresponding biochemical data are very limited in early studies because of the difficulties to purify such enzymes from algal matrices extremely rich in anionic polysaccharides and polyphenolic compounds. The problem was solved by developing two-phase extraction procedures, and brown algal peroxidases were obtained in gram-amounts [START_REF] Vilter | [END_REF]. Up to now, a number of VHPOs have been characterized at the biochemical and molecular levels from red and brown macroalgae, terrestrial fungi and marine bacteria (see Table 1 for complete references). In the brown alga A. nodosum, the two VBPO isoenzymes present at different locations in the alga [61,71] feature distinct biochemical and structural properties [60], suggesting distinct biological roles in the algal physiology. In vitro halogenation experiments have shown that purified native VBPOI from A. nodosum catalyzes the bromination of pyrroles [72] or phenols [73], whereas the second VBPO is involved in the biosynthesis of methyl 4-bromopyrrole-2-carboxylate, a natural product isolated from the marine sponge [60]. Laminaria digitata also features two isoforms of VHPOs, but with distinct halide specificities. One is a VBPO [START_REF] Colin | [END_REF], whereas the other isoform is specific for iodide oxidation [54]. No specific iodinated substrate has been yet identified in L. digitata, but this particular VHPO may be involved in the very efficient accumulation of iodide in external tissues of L. digitata [14,74,75]. Another VIPO was recently characterized at the structural level in the marine flavobacterium Z. galactanivorans, which belongs to the bacteroidetes and is associated with both red and brown seaweeds [56].

In red algae (Table 1), VBPO were isolated from Corallina officinalis and Corallina pilulifera [76][77][78][79], and more recently VBPO from Delisea pulchra [80] and from Gracilaria changii [81]. The VBPO-catalyzed asymmetric bromination and cyclization of certain terpenes, including nerolidol, lead to the biosynthesis of brominated cyclic sesquiterpenes.

This was established in vitro by VBPO isolated or cloned from Laurencia, Plocamium and

Corallina [82]. The VBPO from Delisea pulchra produces bromofuranones, which are involved in the disruption of quorum sensing of epiphytic bacterial biofilms [80]. Algal halogenated compounds are known to interfere with bacterial signaling systems. Similarly, VBPO from L. digitata can deactivate acylated homoserine lactones, which are the cell-to-cell A c c e p t e d M a n u s c r i p t 9 signaling molecules. Moreover halogenated compounds from this species control the biofilm formation and growth in Gram-negative bacteria [83,84].

VCPO were first characterized in terrestrial fungi, the most studied being that of Curvularia inaequalis ([85], Table 1 for full references). The fungus C. inaequalis is able to chlorinate aromatic rings of lignin in wood during the degradation process and this action is likely to be due to the presence of VCPO enzymes [86]. The VHPO-based biofilm control has been also explored for medical purposes: VCPO from C. inaequalis has an antimicrobial action on Enterococcus faecalis biofilms [87].

VCPOs were also identified in marine actinobacteria [68,88]. Another class of interesting compounds is that of halogenated antibiotics. Interestingly, bromoperoxidase activity was first detected in the chloramphenicol-producing actinomycetes [89]. In Streptomycete bacteria, a VCPO, encoded by the NapH1 gene, catalyzes a highly stereoselective chlorination cyclization reaction in napyradiomycin biosynthesis [68,88].

More recently in vitro biosynthetic experiments established the roles of VCPO from the marine bacterium Streptomyces sp. strain CNH-189 in the final chlorination cyclization step of the merochlorin biosynthesis. The existence of a highly regiospecific and stereospecific reaction in this synthesis of chlorinated antibiotics suggests that in vivo the VHPO bind their natural substrates in a highly specific manner [69,70].

Phylogenetic relationships of vanadium haloperoxidases

VHPO share a relatively low level of overall protein sequence identity. For instance, the fungal VHPO from C. inaequalis [85] and Alternaria didymospora [90] present a protein sequence identity of 68%, and the protein sequence identities between VHPO from brown and red algae are only in the range of 30 to 40%. At the protein level, only one hundred amino acid residues from the carboxyl terminal regions of the fungal enzymes can be aligned with VHPO from macroalgae, with ~25% of similarities. This alignment bears the two following short domains highly conserved in all VHPO proteins and related to vanadate binding (see below).

(1) PxYxSGHA

(2) LxxxxAxxRxxxGxHxxxD A phylogenetic analysis based on the alignment of 26 biochemically-and/or structurally-characterized VHPO (Table 1) was carried out, including four sequences of A few years after this first structural characterization of a fungal VHPO, structures of a VBPO of algal origin were published (see Table 1 for full references), revealing complex multimerization of these latter ones. Indeed, the first characterized VBPO from the brown alga A. nodosum is arranged in dimers, stabilized by two disulfide bridges covalently linking the two monomers (Figure 2C) [39]. These dimers are able to self-arrange in more complex structures, such as hexameric proteins, as shown by gel filtration chromatography results obtained for VBPO from L. digitata [START_REF] Colin | [END_REF] and for the second VBPO from A. nodosum [60]. In contrast, the red algal VHPO dimers are not stabilized by covalent bonds but organized in a hexamer of dimers (Figure 2D) [START_REF] Isupov | [END_REF]94]. This dodecamer is about 150 Å in diameter and forms a cavity in its center. This multimeric complex also presents a high degree of 2-3-point group symmetry that is cubic. Interestingly, so far in all characterized algal VHPOs, the Nterminal helix-bundle is structurally replaced by the C-terminal helix-bundle of a second monomer in the homo-dimeric protein, with the two molecules arranged in a 'head-to-tail' orientation. Multimerization is a biological strategy to stabilize proteins [95]; consequently, in the case of red algal VHPOs, the high stability to organic solvent and high temperature might be a result of the strong interactions present in these highly symmetric multimers. In VBPO dimers, the surface of interaction represents 20 and 46% of the total exposed surface of a monomer for C. officinalis [START_REF] Isupov | [END_REF] and A. nodosum [39], respectively. In contrast, in the red algal VBPO dodecamer, only 12% of a monomer surface is exposed to the solvent [START_REF] Isupov | [END_REF]96,97].

More recently, 3D structures from bacterial VHPOs ( This vanadium binding site structure is perfectly conserved throughout all VHPOs (see below), despite the fact that the algal VHPO lack the helix bundle 1. Indeed the missing helices are replaced by the second monomer in the dimeric forms of VHPOs. Nevertheless, all

VHPOs have roughly the same number of residues, between 450 and 550 amino acids.

Indeed, algal VHPOs display several additional insertions, forming helices and beta-strands that are not present in the fungal or bacterial VHPOs [56]. The comparison of all the known haloperoxidase structures shows that a fungal or bacterial VHPO monomer can be superimposed onto the dimer of an algal VHPO, with the N-terminal helix-bundle 1 of the single peptide chain in the monomeric forms being replaced by the 'back-side' of the Cterminal helix-bundle belonging to the second chain of the homodimeric form (Figure 2C).

This observation, combined with phylogenetic analyses, has allowed postulating a bacterial origin of the ancestral VHPO with a monomeric conformation [56]. In this scenario the loss of helix bundle 1 in algal VHPO, has probably been the key to dimerization: an incomplete gene transfer leading to the deletion of helix bundle 1 exposes a hydrophobic patch at the surface of the protein, necessitating the replacement of this part of the fold by a helix bundle from a second monomer, thus facilitating the dimerization. The dimerization leads to the interesting feature that the homodimeric algal VHPOs have two vanadate-containing active sites, whereas the fungal and bacterial monomeric forms only possess one active site per protein.

Similarities between vanadium haloperoxidases and acid phosphatases

In monomeric VHPOs the two domains of the same protein, the conserved C-terminal helix-bundle and variable N-terminal helix-bundle, can be superimposed on each other at the structural level (Figure 3A), despite the fact that no similarity can be detected between the two helix bundles at the level of primary sequence. Interestingly, the 3D structural fold of acid phosphatases, represented through the monomer structure of a non-specific acid phosphatase from Escherichia blattae [98] is constituted by a single helix-bundle motif. Indeed, when structurally superimposing the five-helix-bundle of an acid phosphatase onto monomeric VHPOs, stronger similarity can be observed with the conserved C-terminal helix-bundle ). This structural domain homology to acid phosphatases has been previously identified by the comparison to Ci-VCPO [98][START_REF] Hemrika | Proc. Nat. Acad. Sci. U.S.A[END_REF][START_REF] Neuwald | [END_REF]. Together with phylogenetic analyses [56], these 3D fold-similarities suggest that acid phosphatases and VHPOs have a common ancestor with an ancestral fold consisting of a single helix-bundle-based core domain, and VHPOs have diverged from this ancestor fold by gene-duplication followed by gene-fusion. In this scenario, the presence of additional ligands provided by the second helix-bundle has led to the fixation of catalytic active vanadate rather than phosphate. However, we can not rule out that ancestral active VHPOs contain only a single helix bundle that have then diverged into two distinct forms, either by dimerization or by gene-duplication followed by gene-fusion. In any case, this evolutionary relationship between phosphatases and VHPOs has some functional consequences: since the phosphate and vanadate binding sites are sufficiently similar, it has been demonstrated that the apo-form of VCPO exhibits phosphatase activity [101], whereas the phosphatases are strongly inhibited by vanadate [102]. In several 3D structures, bound phosphate was located in VHPOs in a similar manner than the vanadate group, either in the native Ci-VCPO [START_REF] Messerschmidt | Proc. Nat. Acad. Sci. U.S.A[END_REF] or in the VBPO of Corallina species [START_REF] Isupov | [END_REF]94,103]. This oxyanion is only weakly linked to the N ε atom of the histidine residue, but a systematic comparison of phosphate-and vanadate-binding proteins in the protein data bank shows high structural similarities and similar binding [104].

VO 4 coordination in the crystal structure of vanadium haloperoxidases

When studying the molecular bases of catalytic properties and halide specificity in For the VCPO from the fungus C. inaequalis the X-ray structures of both native and peroxide forms of the enzyme have been solved at 2.03 Å and 2.24 Å, respectively [105].

These two structures have revealed that the trigonal bipyramidal geometry of the vanadate in the native form is transformed in a distorted tetragonal pyramid after addition of hydrogen peroxide, suggesting the release of the apical oxygen ligand and the coordination of the two peroxide oxygens to the vanadium cofactor in the stable peroxide form of the enzyme ( [105],

see Scheme 1). During the catalytic cycle, at least the histidine and one arginine (His360 and Arg410 in Zg-VIPO1, Figure 4A) are essential residues for the coordination of the vanadate moiety and catalysis, since changing them into alanine or serine totally switched off haloperoxidase activity in Ci-VCPO [58,106] and in Zg-VIPO1 [56]. In the VCPO from Streptomyces sp., the serine residue (Ser427) seems to play a similar role, as its mutation in a histidine residue abolished enzyme activity [68].

Another remarkable difference in the bacterial VCPO and in all algal VBPOs is the presence of an additional histidine, in the proximity of Lys341 (An-VBPOI numbering, Figure 4D) that is replaced by a phenylalanine in the fungal VCPO. Based on the first few available crystal structures [39,[START_REF] Messerschmidt | Proc. Nat. Acad. Sci. U.S.A[END_REF][START_REF] Isupov | [END_REF][94]103], it was postulated that this single residuedifference between vanadium-dependent chloro-and bromoperoxidases, i.e. a Phe397 in VCPO (Figure 4B) replacing a histidine residue in VBPO, was responsible for the difference in halogen specificity [66]. Previously, the mutation of the arginine residue (Arg397) in the red algal VBPO from C. pilulifera (Figure 4F) has conferred significant chloroperoxidase activity to mutant enzymes R397W and R397F [94,[START_REF] Ohshiro | [END_REF]. This residue is replaced by a tryptophan residue in the fungal VCPO. However, the more recent crystal structure of VIPO, having identical residues in the first coordination sphere of the catalytic vanadate as in VCPO [56] including this phenylalanine, showed that the structural factors governing the oxidation potential of halides in this enzyme family is much more complex. In this latter study, sitedirected mutagenesis of residues located in a second sphere around the active site highlighted A c c e p t e d M a n u s c r i p t 15 that some of these could equally well influence the halide-oxidation potential of this enzyme, and even the specificity of the enzyme. Indeed a slight bromoperoxidase activity was obtained in two enzyme mutants, one mutated inside the active site (Ser358, Figure 4A) and the other mutated on a tyrosine residue located outside the active site. The current hypothesis [7,8,55] explains the change of catalytic properties and halide specificity by modifications of H-bond coordination and redox potential of the VO 4 moiety, rather than selective halide binding.

Recent data suggest that this redox potential is finely tuned locally, but also by remote amino acid residues, belonging to the second and third sphere of VO 4 coordination [56].

Biological Vanadium K-edge X-ray absorption spectroscopy

4.1. Introduction to BioXAS and other biological Vanadium XAS results.

X-ray absorption spectroscopy

X-ray absorption spectroscopy (XAS) is applied to study the coordination geometry of transition metals in enzymes, as well as changes of geometry during catalysis [108,[START_REF] Feiters | Practical Approaches to Biological Inorganic Chemistry[END_REF]. The method can also be applied to non-invasively study halogens in whole organisms [14], parts thereof [START_REF] Küpper | [END_REF]111], or isolated enzymes [112]. The X-ray absorption spectra of dilute systems (mM in metal) like biological samples are usually measured in fluorescence mode; for vanadium this is relatively difficult compared to other 1 st -row transition metals, because there is less fluorescence and the K-edge is at low energy, a range where air absorbs significantly. Some examples of vanadium K-edge spectra are shown in Figure 5.

With monochromatic synchrotron radiation, it is possible to scan the X-ray energy around the so-called K edge of an element, for example vanadium (V). At the absorption edge, the X-ray energy corresponds to what is needed to liberate a 1s electron of V into the continuum. The edge energy shifts to a higher value if the V is in a higher oxidation state.

There is usually some fine structure present in this 'X-ray Absorption Near Edge spectrum' (XANES) which can also be represented as the first derivative in order to highlight subtle features (Figure 5A-B). The fine structure is due to the electron being excited to unoccupied higher orbitals with energies just below that of the continuum; the intensities of such transitions depend on their being allowed or forbidden, which in turn depends on the metal ion's coordination geometry. Above the edge, additional features, the so-called 'Extended X- (Å -1 ) as energy axis, and in k 3 -weighting to compensate for the decay in amplitude at higher k.

The EXAFS amplitude depends on the coordination number but this can only be determined with limited accuracy (± 1) because of its correlation with the disorder phenomena, described in the so-called Debye-Waller factor, that are responsible for the aforementioned decay in the amplitude. Fourier transformation (with phase correction) of the EXAFS (FT, Figure 5D) yields a radial distribution function in which the positions of the maxima represent the distances of shells of ligand atoms to the absorber atom, which can be determined fairly accurately (typically ± 0.02 Å). Atom types differ in backscattering amplitudes and in the phase relationship between EXAFS and Fourier transforms; atoms of the same row in the Periodic Table are so similar that they cannot easily be distinguished (Z determined to an accuracy of ± 1). Ligands like the imidazole moiety of the amino acid histidine give a characteristic pattern of shells for which the multiple scattering phenomenon, i.e. the scattering of the electron wave by various ring atoms before it returns to the V absorber, has to be taken into account in the simulation. The interpretation of EXAFS is performed by means of simulation on the basis of parameters of structural models which are iteratively refined to give the optimum agreement between theory and experiment as expressed in a fit index.

Biological vanadium accumulation

Because of the relatively high concentration requirements it is not surprising that the first example of a study of vanadium in a biological context was on organisms that actively accumulate vanadium from seawater, in the form of vanadate (V(5+)O 4 3-), viz. of the aforementioned ascidians [START_REF] Henze | [END_REF], a class belonging to the phylum of tunicates [113]. The XANES of these organisms, when compared to that of suitable model compounds, showed that the vanadium was predominantly in the V(3+) state in an octahedral environment, while the EXAFS revealed the nearest neighbours to be oxygens. In subsequent studies, the V XANES features were used to quantify the amounts of V(3+) ions associated with the biological catecholate-based chelator tunichrome, and water and sulphate, as well as mixtures of the latter in various species [114][115][116][117][118], and a detailed mechanism for the reduction of vanadate to V(3+) was proposed [119]. In this particular context, the role of sulphur in the vanadium metabolism was also investigated with sulphur K edge XANES [120][121][122]. Micro-A c c e p t e d M a n u s c r i p t 17 distribution of V was established in living ascidian blood cells by X-ray scanning microscope in fluorescence mode and local XANES spectra revealed V(3+) and V(4+) in vivo [123]. With X-ray tomography in combination with X-ray fluorescence, three-dimensional maps of the element distribution in objects and biological specimens such as the diatom Cyclotella can be obtained [START_REF] De Jonge | [END_REF]. With scanning X-ray microscopy, it is possible to record short ranges -XANES rather than EXAFS -of X-ray spectra at the edge of an element, and combine them with the image, to map the distribution of that element's oxidation state. This is an interesting approach for vanadium because of the sensitivity of its XANES to oxidation state and ligand geometry. In applications [125][START_REF] Takemoto | Proc. 8th Int. Conf. X-ray Microscopy, IPAP Conf[END_REF] of this technique in relation to the aforementioned biological vanadium accumulation, a scanning X-ray microscopy study of cryo-fixed specimens of one type of blood cell -the so-called signet cell -from Ascidia sydneiensis samea , it was shown that the reduction of V(5+) from seawater proceeds in 2 steps, viz. to V(4+) in the signet ring cells, and to V(3+) in the vacuole. One other occurrence of vanadium in a biological context, besides that of V in haloperoxidases, is that in aforementioned vanadium-dependent nitrogenases [START_REF] Hopkins | Proceedings of the 2nd International Symposium in Trace Element Metabolism in Animals[END_REF][START_REF] Bishop | [END_REF]. The XANES and EXAFS at the V K-edge of the first V-containing nitrogenase from A. chroococcum resembled very much that of a VFe 3 S 4 cubane cluster [128].

Vanadium and other edge EXAFS and XANES of vanadium haloperoxidases

Early vanadium haloperoxidase XANES results

Studies on the vanadium-containing haloperoxidases started with XANES of the brown alga A. nodosum bromoperoxidase [41]. The prominent pre-edge peak at 5469 eV (Figure 5A) is ascribed to a 1s-3d transition which is dipole-forbidden and absent in octahedral VO [129] but very strong in tetrahedral vanadate (Figure 5A). For the native bromoperoxidase of A. nodosum, the feature is somewhat weaker than for vanadate (Figure 5A, [42]) which is consistent with the trigonal bipyramidal coordination symmetry established in the crystallographic study [39]. A similar change was observed in the XANES of vanadate upon binding to the chloroplast H + -ATPase CF1 together with Mg 2+ and ADP; this creates an enzyme-bound transition-state analogue for ATP-hydrolysis in which the V has trigonalbipyramidal coordination geometry [130].

The haloperoxidase XANES is not affected by addition of the substrate Br -(90 molar equivalents), but upon reduction the edge shifts to lower energy and the pre-edge feature is A c c e p t e d M a n u s c r i p t 18 reduced in intensity [42]. Addition of 4 molar equivalents of H 2 O 2 had no effect [42] but a significant shift of the edge to lower energy and a subtle increase in the pre-edge feature were observed with 15 molar equivalents [43]. These changes point respectively to an increase in average V-ligand distance and to a change in symmetry, but not to a change in oxidation state for V. Accordingly, a mechanism was proposed for VHPO-based bromination (Scheme 1)

without valence change for V nor coordination of Br -to V, in which coordination to V(5+) activates H 2 O 2 for attack by bromide, forming the hypobromite which is the halogenating agent.

Vanadium haloperoxidase EXAFS

The first haloperoxidase EXAFS was obtained for the native and reduced forms of A.

nodosum VBPOI in a k range of 3-10 Å -1 [42] at a time when no crystallographic information was yet available. For the reduced enzyme, one short (1.63 Å) and 3 medium (1.91 Å) V-O distances were identified, along with two V-N distances of 2.11 Å, which were inferred to belong to imidazole moieties of histidine residues on the basis of advanced EPR (electron spin echo) studies [43], and simulated as such. The results for the native enzyme were similar, but most distances (1.61, 1.72, and 2.11 Å) were shorter, and the more remote atoms were not explicitly assigned to imidazoles but as N or O. In a later study [131], the distance information was used in a Bond Valence Sum Analysis (BVSA) [132], using known correlations between the valence, ligand distances, and ligand geometry of ions, to correct the coordination number initially found [42] from 6 to 5. If one considers one of the imidazole or N/O ligands from the initial interpretation to be redundant in the 5-coordinate geometry, this outcome is consistent with the crystallographic results, first for the VCPO from the fungus C.

inaequalis [START_REF] Messerschmidt | Proc. Nat. Acad. Sci. U.S.A[END_REF], later for the A. nodosum VBPO [39], that vanadium in haloperoxidases is present as vanadate, bound to the enzyme by coordination to a single imidazole residue.

Although the imidazole ligand was recognized in the EXAFS of reduced enzyme on the basis of the EPR evidence [133], it was almost overlooked in the native enzyme [42].The aforementioned current structural model for the nitrogenase cofactor FeVco also features imidazole coordination, although this is not detectable in the EXAFS. The crystal structure of the [vanadyl(vinylimidazole) [START_REF] Amachi | [END_REF] Cl]Cl complex revealed V-N distances in the range 2.05-2.19 Å, and the imidazoles gave an unambiguous contribution to the EXAFS in the Fourier transform (radial distribution function) in the range 2-4 Å, with an average distance of 2.11 Å [134], in line with the V-N distance found in the A. nodosum VBPOI EXAFS [42]. It is clear from the features in the EXAFS in the range 2-4 Å -1 (Figure 5C) that there are remote shells in A c c e p t e d M a n u s c r i p t 19 addition to the main shell of oxygen (and nitrogen) ligand donors, atoms that also contribute to the EXAFS, but the corresponding features in the Fourier transform at 3-4 Å are relatively small (comparable to the intensity for vanadate in that region) if it is taken over a relatively long data range (Figure 5D). Alternative interpretations have been put forward for the remote haloperoxidase shells, such as a shell of protein heteroatoms that are H-bonded to the vanadate [135], and, by analogy to the archaeal tungstate binding proteins [136], throughabsorber multiple scattering involving the axial ligand donor atoms in the trigonal bipyramid, which would appear in the Fourier transform at the sum of the V-N(imidazole) and V-O(axial) distances [START_REF] Feiters | EUROBIC-8[END_REF].

It was concluded from V EXAFS taken over a relatively short range (up to k = 7 Å -1 ) of the A. nodosum VBPOI in the presence of excess (3 molar equivalents) bromide that there is no direct V-Br interaction (expected distance 2.45 Å) [START_REF] Christmann | [END_REF]139]. This was corroborated by the absence of such a contribution in the complementary Br EXAFS [START_REF] Dau | [END_REF], which instead indicated the presence of Br covalently bound to a low-Z atom, then interpreted as due to a putatively reactive Br-O(Ser) intermediate. It was later found [112] that the enzyme actually contains endogenous bromine in the form of bis-brominated (3,5-dibromo)-tyrosine residues, presumably due to exposure to both Br -and H 2 O 2 before or during isolation. Br EXAFS also revealed the presence of bromine incorporated in other amino acids, possibly tryptophan, in the VHPO of L. digitata [START_REF] Küpper | [END_REF].

As mentioned above, the crystal structures of the native [START_REF] Messerschmidt | Proc. Nat. Acad. Sci. U.S.A[END_REF] and peroxo [105] forms of the C. inaequalis VCPO indicated that two vanadate oxygens are displaced by the peroxide which is bound in a side-on fashion, and that the geometry changes from a trigonal bipyramid with the imidazole-N in one of the apical positions to a square pyramid with an apical oxygen (see Scheme 1 for a schematic representation of the structures, in which the ellipsoids indicate the equatorial planes). While the XANES of A. nodosum VBPOI showed a significant shift to lower energy with a large excess of H 2 O 2 (see above) [43], indicating a longer average Vligand distance, the corresponding spectra for C. inaequalis VCPO with 4 molar equivalents of H 2 O 2 were rather similar. The EXAFS of both enzyme forms was also measured, compared, and analyzed [141] in an approach in which the geometry of the imidazole was constrained [142], i.e. the atom-atom distances within the ring were kept in an idealized geometry, so that the whole imidazole was treated in the refinement of the simulations as a unit. The EXAFS distances agree within 0.1 Å to the crystallographic ones for the native enzyme, but in the peroxo form the crystallography seems to overestimate the V-N and two of the V-O distances by 0.15-0.25 Å. The comparison is complicated by the pH difference, 8. inaequalis VCPO XANES results [141].

Outlook: merging XANES and EXAFS

As mentioned in the introduction, the XANES region of the X-ray absorption spectrum is usually treated as spectroscopy (interpretation in terms of transitions) and the EXAFS as (X-ray induced electron) diffraction. The EXAFS is simulated with 1-dimensional structural models (radial distribution function independent of the arrangement of the atoms in space) or sometimes 2-dimensional (multiple scattering within an imidazole ring) or 3dimensional models (through-absorber multiple scattering involving linear arrangements of absorber and 2 ligand atoms). It has been recognized for some time that it should be possible to simulate the XANES on the basis of the same structural models, but defined in 3 dimensions, that account for the EXAFS; there is, however, as yet no convenient approach for the simulation of both regions of the spectra. The examples given above [42,141] show that it is possible to simulate EXAFS data on the basis of the so-called 'muffin-tin potential' calculations, in which the atoms involved are potential wells separated by an area defined by a common interstitial potential. There is however a growing insight [142,143] that such an approach is too crude to reproduce important features in the XANES, and that the more complex full-potential finite difference method [144] has to be applied to this region. The challenge now is to interpret both ranges of the spectrum with the same underlying theoretical model. One could go back and forth with one or more structural models between simulations of the XANES and EXAFS regions of the spectra, adjusting distance information in the EXAFS and 3-dimensional information in the XANES, until a consistent structural model has been reached. In view of the still increasing power of computational approaches such as the ADF Density Functional Theory package [145], it is also tempting to generate computational structures which can be validated/adjusted in both the EXAFS and XANES part of the X-ray absorption spectrum. and dioxidovanadium (V) stabilized by organic molecules [START_REF] De Boer | [END_REF]152]. However, recent studies [56] have shown that the influence of the protein stabilization of the active center goes beyond the first coordination sphere. At the long term, the biochemical and structural characterizations of known and supplementary VHPOs will be necessary, in particular at very high resolution (beyond 1 Å) and possibly using neutron-diffraction to precisely locate proton 3-bromo-8-epicaparrapi oxide [80], bromofuranone [80], napyradiomycin [68], merochlorin [69,70], bromopyrroles [72] and bromophenols [73]. -VHPO features a large evolutionary and structural diversity from bacteria to algae.

Conclusions and future prospects

-Bacterial VHPO revealed new biosynthesis pathways and enzymatic specificity.

Graphical abstract

The 3D-printed protein structure (*) and vanadium X-ray absorption near edge spectra obtained from the first vanadium haloperoxidase discovered 30 years ago.

(*) http://vilter.de/vanadium-dependent-haloperoxidase-from-ascophyllum-nodosumtouchable-3d-models-from-x-ray-crystallography-to-3d-print/ *Highlights (for review)

A c c e p t e d M a n u s c r i p

A c c e p t e d M a n u s c r i p t 5 study

 5 the structure-function relationships in catalytic and specificity mechanisms, and to shed light on VHPO family evolution.

A c c e p t e d M a n u s c r i p t 10 bacterialFigure 1 ) 3 . Structure/function of vanadium haloperoxidases 3 . 1 .

 101331 nonspecific acid phosphatases as outgroup[7,56]. In this phylogenetic tree (the VHPOs from red and brown algae group together with some VBPOs from Cyanobacteria, suggesting a common origin of all algal VHPOs. Inside the brown algal VHPO sub-group, two branches appear, one including the first VBPO of A. nodosum and the VIPO from L. digitata and the other bearing the second isoform of A. nodosum and the VBPO of L. digitata[56]. The red algal VBPOs form a strong monophyletic group with few cyanobacterial VHPO homologous proteins, one of which has recently been characterized as VBPO in a Synechococcus strain[91]. In the other part of the tree (Figure1) the VIPO from Z. galactanivorans, the fungal VCPO, including that of C. inaequalis, and the first bacterial VCPOs characterized in Streptomyces species merge as different monophyletic groups. A recent larger phylogenetic analysis allowed the identification of numerous VHPO bacterial homologs in public protein databases, suggesting a larger spreading of VHPO in bacterial lineages and a significant bacterial contribution to VHPO-mediated processes[56]. Based on phylogenetic analyses VHPO could derive from a common marine bacterial ancestor, closely related to bacterial acid phosphatases, and their evolution may have been driven by successive lateral transfer events. Moreover the specificity for iodide oxidation results in a convergent evolution of the divergent phyla of bacteroidetes and brown algae[56]. 3D crystallographic structures and quaternary arrangement of vanadium haloperoxidases In 1996, Messerschmidt and Wever published the first crystallographic structure of a vanadium dependant haloperoxidase, which was the chloroperoxidase isolated from the fungus Curvularia inaequalis[START_REF] Messerschmidt | Proc. Nat. Acad. Sci. U.S.A[END_REF]. The overall shape of the monomeric protein has the form of a cylinder of about 80 Å long and 50 Å in diameter (Figure2A). The fold is compact with about 44% of the residues involved in alpha-helices. The helices form two main domains consisting of a helix-bundle each that are related by a pseudo-2-fold axis perpendicular to the axes of the helices. The two helix-bundles are often called bundle 1 and 2, in relation to their occurrence in the N-or C-terminal part of the enzyme, respectively. The site of fixation for A c c e p t e d M a n u s c r i p t 11 vanadate is located at the bottom of a 20 Å deep cavity, situated at the N-terminal ends of helices from bundle 2 (Figure 2A-B).

  A c c e p t e d M a n u s c r i p t 12 one in the second monomer, in an anti-parallel manner, and together they create an interface much smaller than that observed in the complex multimeric algal VHPOs.As explained above, the vanadium containing active site of VHPOs is located at the Nterminal end of helices from bundle 2 in fungal and bacterial VHPOs (Figure2A-B), , and two residues within the second ligand sphere of the VO 4 3-are provided by the helix bundle 1.

A c c e p t e d M a n u s c r i p t 13 domain

 13 than when superimposing onto the variable N-terminal helix-bundle of VHPOs (illustrated by the superimposition onto the C-terminal helix-bundle of Zg-VIPO1 in Figure3B

VHPO, it is

  of interest to compare the 3D active site structures of the different types of VHPO, i.e. the fungal and bacterial VCPO, the algal VBPO, and the recently discovered VIPO from Z. galactanivorans[56]. The active site pockets of the different VHPOs display strong similarities, despite the different oligomeric forms of enzymes, and most of the residues tightly coordinating the catalytic vanadate are strictly conserved within C-terminal helix bundle (Figure4), the active site pocket being also delimited by the neighboring Nterminal helix bundle in fungal and bacterial VHPOs and by the C-terminal helix bundle from the second monomer in algal VHPOs (Figure2).The N  group of a strictly conserved histidine (His416, His496, His494, His486, His553 and His553 in Zg-VIPO1, Ci-VCPO, Ssp-VCPO, An-VBPOI, Co-VBPO and Cp-VBPO, A c c e p t e d M a n u s c r i p t 14 respectively, Figure4A-F) is binding the vanadate, providing the fifth atom in the trigonalbipyramidal coordination sphere of vanadium (V). As mentioned above, the VO4 binding in VHPOs also corresponds to the assumed trigonal bipyramidal transition state of bound phosphate in the phosphatases[104]. All other ligand-atoms of the vanadium-ion are the four oxygens from the prosthetic group. These oxygen atoms are in turn hydrogen bonded by a lysine, a serine, a histidine and two arginine residues that are all conserved, with one exception, viz. the replacement of the histidine (His360 in Zg-VIPO1, Figure4A) by a serine in the bacterial VCPO (Ser427, Figure4C).

A c c e p t e d M a n u s c r i p t 16 ray

 16 Absorption Fine Structure' (EXAFS, Figure5C) are observed, which are due to X-rayinduced electron diffraction phenomena and can be interpreted in terms of distance, number, and type of the surrounding ligands. The EXAFS is usually displayed with the wave vector k

3

 3

A c c e p t e d M a n u s c r i p t 20 vs. 6 .

 206 0 for diffraction and spectroscopy, respectively. While the crystal structures indicate a significant increase (up to 0.25 Å) in the V-ligand distances for the peroxo form, the EXAFS shows a small (up to 0.10 Å) decrease, if a change at all, which is in line with the C.

A c c e p t e d M a n u s c r i p t 21 During

 21 the last thirty years, extensive research on different vanadium-dependent haloperoxidases was undertaken in order to characterize halogenation through VHPOs. Based on information coming from crystal structures, model compounds of the cofactor possessing catalytic center have been analyzed and modeled in order to understand the vanadium coordination occurring in proteins[146], although these models only account for the first coordination sphere observed in proteins. Extensive modeling and chemical analyses have also helped advance on the understanding of the mechanistic reactivity of this metal-organic active center, by predicting the most stable functional model, K[VO(O 2 )(heida)], to be an effective biomimetic catalyst for VHPOs, also providing an explanation for the differences in chloro-and bromo-peroxidases[147]. Much progress has also been obtained by classical biochemical studies, X-ray crystallography and spectroscopic analyses of the global and fine local structures of the native and peroxo-intermediate forms of the enzymes. The resolution of a protein crystal-structure of a VHPO specific for iodide oxidation[56] has recently allowed further exploring the molecular origin of halide selectivity, which still remains an intriguing question. Indeed the halide specificity in VHPO is related to many interacting factors, such as the amino acid composition of the active site and the fine electronic environment of the vanadate pocket. This latter study also showed that the 2 nd coordination sphere of the peroxo intermediate appears to play an equally important role. In order to decipher the complete catalytic mechanisms of VHPOs and their contrasting reactivity towards different halides, it is important to pursue and enhance the combination of approaches, undertaking protein crystallographic studies in parallel to fine local structure analyses by XAS and EXAFS, on the different VHPO available and specific site-directed mutants that show modified biochemical kinetics. For instance, the fine tuning of the VO 4 moiety during the catalytic cycle should be explored through the measurement and interpretation of the EXAFS and XANES of haloperoxidases at various edges in parallel (V, I, Br). These local structural studies not only provide a good starting point for computational studies on the protonation state of vanadate or on the catalytic intermediates[146,[148][149][150][151], but also provide the possibility to test structural hypotheses through analyses of point-mutants. VHPOs are indeed interesting models of research in chemistry to mimic the activity of vanadate centers, using oxidovanadium (IV)

A c c e p t e d M a n u s c r i p t 22 positions

 22 , to infer global evolution of VHPO enzymatic mechanisms among bacterial and eukaryotic lineages. Another black box remains the biological roles of these enzymes in living organisms. The very large panel of halogenated compounds produced by marine organisms, especially by red and brown algae, is likely to have important biological or ecological functions. Moreover the presence of a tremendous number of VHPO-homologs in all bacterial lineages suggest the involvement of VHPO-based halogenation in numerous bacterial biosynthetic pathways. In this biological context, an important bottleneck is the development of functional approaches for deciphering the biochemical functions of VHPO, for finding their specific substrates and for identifying the natural halogenated products, they are producing in vivo. The set-up of heterologous expression and in vitro reconstituted systems are challenging, especially for marine bacteria, but the recent results obtained on Streptomyces sp. [69, 70] have opened interesting perspectives in this research area. In future developments, these advances will certainly give rise to potential biotechnological applications either for generating halogenated biologically active analogues or for improving biological properties of metabolites by enzymatic halogenation.

1 .

 1 Figure legends

Figure 1 .

 1 Figure 1. Phylogeny of VHPO proteins.A protein maximum likelihood (PML) tree was constructed using the multiple alignment of 26 eukaryote and bacterial VHPOs characterized at the molecular and/or biochemical levels and of four Bacterial Acid Phosphatases as an outgroup. 173 informative residues were used for PML analyses and bootstrap values (500 replicates) are provided for main nodes. The NCBI accession numbers of proteins are indicated, except for VBPO from Plocanium cartilagineum (Pcar) and from Delisea pulchra (Dpul) (see[80]). The scale bar represents a difference of 0.1 substitution per site. When characterized at the biochemical level, the VHPO specificity towards halides is mentioned and refers to the legend code, i.e. iodo-(VIPO), bromo-(VBPO) or chloroperoxidase (VCPO) activity. The available 3D structures of VHPO are indicated by a star. Examples of halogenated products are -, -and -snyderol and (+)-

Figure 2 .

 2 Figure 2. Three dimensional structures of VHPO. A. The monomeric VCPO from C. inaequalis (PDB Accession #, 1VNI). B. The monomeric VIPO1 from Z. galactanivorans (PDB 4CIT). C. The homo-dimeric VBPOI from A. nodosum (PDB 1QI9). D. The dodecameric VBPO from Corallina officinalis (PDB 1QHB) and C. pilulifera (PDB 1UP8).In the monomeric VHPOs (A and B), the N-and C-terminal parts of the protein containing the two helix-bundle domains are colored in blue and red, respectively. For the algal VHPOs, the red and blue colors are used to distinguish either the two identical monomers (B) or the different homodimers (D). The VO 4 binding sites are indicated by an arrow.

A c c e p t e d M a n u s c r i p t 33 Figure 3 .

 333 Figure 3. 3D structure comparaison of VIPO1 from Z. galactanivorans (PDB Accession #, 4CIT) and of non-specific acid phosphatase from Escherichia blattae (PBD 1D2T). A. Superimposition of the N-terminal helix bundle (in green) of Zg-VIPO1 onto its C-terminal helix bundle (in blue). B. Superimposition of the monomeric structure of Eb-NSAP (in pink)with a monomer of Zg-VIPO1 (in blue and green). The C-terminal helix-bundle that is conserved with other VHPO is represented in blue and the N-terminal helix bundle is colored in green. The VO 4 or PO 4 binding site is indicated by an arrow.

Figure 4 .

 4 Figure 4. Comparison of vanadate-binding site structures of VHPO. A. VIPO1 from Z. galactanivorans (Zg-VIPO1, PDB Accession #, 4CIT). B. VCPO from Curvularia inaequalis (Ci-VCPO, PDB 1IDQ). C. VCPO from Streptomyces sp. CNQ525 (Ssp-VCPO, PDB 3W36). D. VBPOI from A. nodosum (An-VBPOI, PDB 1QI9). E: VBPO from Corallina officinalis (Co-VBPO, PDB 1QHB). F. VBPO from Corallina pilulifera (Cp-VBPO, PDB 1UP8). The view of the active site is looking inside, towards the axial histidine ligand at the bottom of the 20 Å deep cavity, and amino acid residues are numbered according to the protein sequence. Vanadate and phosphate are represented in the center of the picture. The H-bonds between VO 4 or PO 4 group and the surrounding amino acids are shown as dashed blue lines.

Figure 5 .

 5 Figure 5. Example of vanadium K-edge X-ray absorption spectra. Comparison of (dashed red) Na 3 VO 4 in Tris buffer pH 8.0 and (solid black) native VBPOI from Ascophyllum nodosum. X-ray absorption near edge spectra (XANES, top left, A) and 1 st derivative (bottom left, B); extracted Extended X-ray Absorption Fine Structure (EXAFS, top right, C) and phase-corrected modulus of the Fourier transform of the EXAFS (bottom right, D). Spectra taken with Dr. W. Meyer-Klaucke at the EMBL Outstation at DESY, Hamburg, Germany.

M a n u s c r i p tHighlights-

  3D and XAS data are complementary to study Vanadium coordination and VHPO catalysis.

Figure

  Figure(s)

Table 1

 1

), the VCPO from Streptomyces sp. (Liscombe et al., unpublished) and the VIPO from the marine flavobacterium Z. galactanivorans

[56]

have shed new light on plausible evolutionary relationships between algal multimeric forms and fungal and bacterial monomeric forms of VHPOs. Indeed, the two bacterial VHPOs are similar to the monomeric fungal VCPO, consisting of two helix-bundle domains within a single polypeptide chain in the VIPO from Z. galactanivorans (Figure

2B

). Despite this fold-similarity, the Z. galactanivorans VIPO is a monomer in solution, while VCPO of Streptomyces sp. is dimeric. However, the dimerization mechanism in this latter case is very different to that found in algal VHPOs, and probably carries a different biological function. The Streptomyces sp. VCPO dimer-interface is located above the N-terminal helix-bundle, where two small (3 to 4 residues) beta-strands form an anti-parallel beta sheet. The longest beta-strand of each monomer interacts with the equivalent

Acknowledgements

References Acknowledgements This work was supported by the 'Centre National de la Recherche Scientifique' and 'Université Pierre et Marie Curie, Paris 06', and by the French National Research Agency with regards to the investment expenditure program IDEALG (http://www.idealg.ueb.eu/, grant agreement No. ANR-10-BTBR-04). J.-B. Fournier was financially supported by Region Bretagne and CEA (ARED/TOXNUC-E program). We are very much grateful for assistance and technical help during XAS and EXAFS data collections as well as for very constructive and inspiring discussions during data collections to Stephanie Belin on beamline SAMBA (SOLEIL, Saint-Aubin, France), to Wim Bras and Dipanjan Banerjee on the dutch-belgian beamline DUBBLE from the European Synchrotron Radiation Facilities (ESRF, Grenoble, France), and to Wolfram Meyer-Klaucke from the EMBL Outstation at DESY (Hamburg, Germany). We are indebted to all local contacts for their precious support during crystal data collection at beamlines Proxima 1 (SOLEIL, Saint Aubin, France) and ID23 (ESRF, Grenoble, France).

A c c e p t e d M a n u s c r i p t