Polyoxidometalates (POMs) such as coordination cages and clusters are increasingly applied in a wide range of traditional and interdisciplinary fields.^1-11 These compounds exhibit interesting features in terms of molecular composition, size, solubility, shape, charge density and redox potential. Polyoxidovanadates (POVs) constitute a sub-class of the vast POMs family with remarkable physico-chemical, electronic, magnetic and—particularly—medical properties.^{12, 13} Although the understanding of the POVs binding to proteins is a crucial step for elucidating the mechanism of biological action of these compounds, this research field, due to its high complexity, has been scarcely investigated.^{14, 15} In our continuous efforts to characterize the interaction of proteins with metal complexes,^16-19 we have recently studied the structures of the adducts formed upon reaction of proteins with V^IVOL₂ compounds, where L⁻ is a monoanionic bidentate organic ligand.^20-22 Crystallography works reporting the structures of these adducts are rare^20-24 and the few studies carried out on these systems revealed binding preference of V containing fragments, in particular V^IVOL⁺ and V^IVOL₂, for Asn, Asp, Glu and His side chains.

Here, we report Electron Paramagnetic Resonance (EPR) experiments and high resolution X-ray structures obtained upon exposure of hen egg white lysozyme crystals, grown under two different experimental conditions, to the complex V^IVO(acac)₂, where acac is acetylacetonato. V^IVO(acac)₂ is a water-soluble compound with therapeutic properties,^25-28 that has attracted much interest for its potential use as anticancer and insulin-enhancer agent.^27-29 Lysozyme is a glycoside hydrolase,³⁰ which has been very frequently used as a model protein for metalation studies.^16-19

EPR spectra were recorded as a function of time at 298 K, under the experimental conditions used to grow crystals of lysozyme treated with V^IVO(acac)₂. At pH 7.5, using a solution with the same composition of that from which a crystal of lysozyme in the presence of V^IVO(acac)₂ was isolated (structure A), spectra show that the eight isotropic resonances of V^IVO (I=7/2) disappear after 4 days, suggesting the (almost) complete oxidation to an EPR-silent V^V species in the timescale of crystallization experiments (Figure 1).

In contrast, when the EPR spectra were measured at pH 4.0, namely in the other condition used to grow crystals of lysozyme in the presence of V^IVO(acac)₂ reported in this work (structure B), the reduction of the spectral intensity is evident, but—even after 96 h—the V^IVO signals continue being revealed implying that the oxidation to V^V is only partial (Figure S1 of Supporting Info, SI). Additional EPR experiments were carried out at pH 7.5 and 4.0 allowing O₂ to flow into the solution or completely removing molecular oxygen with Ar. The results indicate that with dioxygen the oxidation is very fast and is almost complete after 24 h at neutral pH (Figures S2 and S3); in contrast, the absence of O₂ slows down significantly the oxidation and the decrease of the EPR signal is negligible at pH 7.5 and not detectable at pH 4.0 (Figures S4 and S5). Overall, these data suggest that the molecular oxygen could be the oxidant in our systems.

Crystals of lysozyme were grown in: a) 2 M sodium formate and 0.1 M Hepes pH 7.5 (structure A) and b) 20 % ethylene glycol, 0.1 M sodium acetate buffer pH 4.0, 0.6 M sodium nitrate (structure B), and then were exposed to stabilizing solutions containing the mother liquors and a saturated solution of V^IVO(acac)₂. X-ray diffraction data were collected on these crystals at 1.18 and 1.79 Å resolution on Beamline XRD2 at Elettra synchrotron, Trieste, Italy (see Supporting Information for further details). Both crystals are isomorphous and belong to the space group P4₃2₁2, with one protein molecule in the asymmetric unit. The structures refine to R-factors and Rfree values of 0.172/0.196 and 0.183/0.232, respectively (Table S1). The overall conformation of the protein is not significantly affected by the presence of the POVs. The Cα root mean square deviations from the metal-free protein structure (PDB code 193L³¹) are within the range 0.19–0.23 Å.

Remarkably and unexpectedly, inspection of the difference Fourier electron-density (2Fo−Fc and Fo−Fc) and anomalous difference electron density maps reveals the presence not of V^IVO(acac)⁺/V^IVO(acac)₂ moieties but of POVs close to the protein surface in both the structures (Figure 2). Notably, structures A and B have been reproduced collecting X-ray diffraction data using replicates of POV/lysozyme crystals.

In the structure A, refined at 1.18 Å resolution to R-factor/Rfree values of 0.172/0.196, non-covalent binding of [V₄O₁₂]⁴⁻ on the protein surface has been found (Figure 2A). Anisotropic refinement of the structure gives R-factor/Rfree values of 0.132/0.167.

The presence of this V species suggests that the metal has changed its oxidation state from +IV to +V under the investigated experimental conditions, confirming the EPR results. An additional V atom close to two oxygens has been also found (Figure S6). [V₄O₁₂]⁴⁻ anion has a cyclic structure with V^V in a tetrahedral geometry and is stable in neutral solutions. [V₄O₁₂]⁴⁻ is held in its position by hydrogen bonds (H-bonds) formed with the N-terminal amine, OH group of Ser86, N atom of Val2, side chain of Gln41, side chain of Asn65 from a symmetry related molecule, and water molecules (Figure 3).

Binding of cyclic tetrameric oxidovanadate [V₄O₁₂]⁴⁻ to proteins was observed upon self-assembling of orthovanadate in the presence of E. coli BtuCD, an ABC transporter,³² and of C3 exoenzyme from Clostridium botulinum.³³ In those structures, [V₄O₁₂]⁴⁻ forms H-bonds with backbone atoms and with the side chains of Asn and Thr residues.

In the structure B, obtained in 20 % ethylene glycol, 0.1 M sodium acetate buffer pH 4.0, 0.6 M sodium nitrate and refined at 1.79 Å resolution to R-factor/Rfree values of 0.183/0.232, a moiety with VO₅ composition close to the side chain of Asp52 (Figure S9) and a [V₂₀O₅₄(NO₃)]ⁿ⁻ anion were found (Figures 2B and 4). As suggested by EPR, this species could be a POV with the simultaneous presence of V^V and V^IV centres. This anion can be described as an unusual octadecavanadate (V₁₈) cage of composition [V₁₈O₄₆(NO₃)]ⁿ⁻ attached to two additional V atoms, each bound to five oxygens in a VO₅ group where one O atom is shared with the cage; this latter group is in its turn coordinated to the side chain of Asp87 and Asp87* (i.e., the residue from a symmetry related molecule) (Figure 4). This POV is reconstituted by its symmetry mate and the occupancy is equal to 0.60. The [V₂₀O₅₄(NO₃)]ⁿ⁻ anion also forms a series of H-bonds with the O atoms of Glu7, Ala10, Ala11 and Ser86, the N atoms of Ala11 and Ile88 and the side chains of Lys1, Glu7, Arg14, His15, Asp87 and Thr89 (Figure 4). It is worth of being highlighted that this is the first time that a protein adduct with a V₂₀ arrangement, and in general with a POV greater than V₁₀, is reported in the literature and the first time that a covalent binding of a protein to a POV is observed.¹⁴ Moreover, a search in the Cambridge Structural Database (CSD) reveals that there are not known structures of POVs with 20 V atoms. Instead, in literature, five types of octadecavanadate (V₁₈) cages with composition [V₁₈O₃₉]ⁿ⁻,³⁴ [V₁₈O₄₁]ⁿ⁻,³⁵ [V₁₈O₄₂]ⁿ⁻,^36-45 [V₁₈O₄₄]ⁿ⁻,^{46, 47} and [V₁₈O₄₆]ⁿ⁻,⁴⁸ have been reported. These present various combinations of V^IV and V^V centres,³⁶ from which the value of the charge n−, the structural features and the magnetic,³⁷ catalytic,^{49, 50} and biological properties depend.^{46, 51} These POVs are able to incorporate in the centre of the cage various types of anions such as Cl⁻, Br⁻, I⁻, HS⁻, HCOO⁻, CO₃²⁻, N₃⁻, NO₂⁻, NO₃⁻ or, also, neutral species like water. To the best of our knowledge, the cage observed in our structure is the second one with composition V₁₈O₄₆, after [V^V₁₆V^IV₂O₄₆(NO₃)]⁵⁻.⁴⁸ Notably, even though the V₁₈ cage observed in the structure B resembles that with V₁₈ composition [V^V₁₆V^IV₂O₄₆(NO₃)]⁵⁻ (reported in the CSD under the accession code UFIQAC⁴⁸), a comparison between the two structures reveals significant differences (Figure 5). In fact, while the anion [V^V₁₆V^IV₂O₄₆(NO₃)]⁵⁻ has all V atoms with square pyramidal geometry based on VO₅ group, the cage found in the structure B has two V atoms with tetrahedral geometry (like the VO₄ group) (Figure 5); notably, the V centers of VO₅ groups, that lead to [V₂₀O₅₄(NO₃)]ⁿ⁻ anion and covalently interact with Asp87 and Asp87* residues, are bound to these tetrahedral vanadium atoms. So, the possibility of a different combination of V^IV and V^V centres in the two structures cannot be excluded nor the influence of protein in the stabilization of V₂₀O₅₄ POV found in this study.

Overall, these data highlight the potential stabilizing role of proteins toward unusual or transient POVs either by the direct coordinative bond or by H-bonds and van der Waals interactions.

In conclusion, here we have reported two structures of lysozyme with POVs which deserve three different types of comments.

Our results provide new structural basis for interpreting experiments carried out with the potential drug V^IVO(acac)₂ and similar V^IVO-based drugs and for understanding their significant biological properties. Indeed, as observed in the literature, to throw light on the mechanism of action of these compounds, their speciation in biological fluids and in the cell milieu and possible subsequent oxidation reactions should be studied in detail.^{52, 53} Here, in particular, we have demonstrated that V^IVO(acac)₂ can degrade, oxidize, and form vanadium(V) oxide polyhedra which—in turn—can interact with the proteins forming covalent or non-covalent adducts stabilized mainly by H-bonds with backbone atoms and side chains of Arg, Asn, Asp, Glu, His, Lys, and Thr residues.