Báo cáo khoa học: Neptunium uptake by serum transferrin potx

Neptunium uptake by serum transferrin Isabelle Llorens 1 , Christophe Den Auwer 1 , Philippe Moisy 1 , Eric Ansoborlo 1 , Claude Vidaud 1 and Harld Funke 2 1 CEA Marcoule, Bagnols sur Ce ` ze Cedex, France 2 Rossendorf beamline, Grenoble, France Metallobiomolecules are considered as elaborated inor- ganic complexes with well designed metal active sites. Although the various interaction processes between essential metallic cations and proteins have been widely studied, focus on the actinide family is more seldom [1,1a,2]. In particular, the interaction of these cations in the biologically active sites is only partially understood. Sequestration and transport of iron in vertebrates are carried out by transferrin (Tf), a monomeric glyco- protein of  80 kDa. Crystal structures of transferrins reveal that these proteins consist of a polypeptide chain folded in two similar but not identical lobes. Each of them contains one metal binding centre [3,4]. Serum Tf is reported to bind a wide variety of d-block transition metals, as well as actinides and lanthanides [3,5–7]. Furthermore, Taylor et al. have suggested that the protein is able to stabilize the tetravalent state and forms stable (M 4+ ) 2 –Tf complexes [8]. This is why Tf contamination by actinide cations is a critical issue of nuclear human toxicology. The oxidation state IV of neptunium (Np) has been of particular concern for its relative stability in physiological conditions and reacti- vity similarities with both Pu(IV), Th(IV) and Fe(III). Pu(IV) as well as most of the transition metal cations reported to be complexed by Tf are assumed to be located in the iron sites [9]. In the case of Fe the donor atoms are provided by two tyrosyl phenolates, one hist- idyl imidazole and one aspartate carboxyl. The require- ment of a synergistic bidentate carbonate anion has been confirmed [10]. Other synergistic anions have also been reported as the nitrilotriacetic anion (NTA) in the crystallographically characterized structure of Fe(III)(NTA)(Tf) [11]. Aspartate has a dual role: it provides an oxygen ligand to the metal and allows the formation of a hydrogen bond, helping stabilization of the lobes in a closed configuration. Other residues not directly coordinated to iron also play important roles through hydrogen bonding as described by the crystal structure of MacGillivray et al. [12] in which arginine stabilizes the synergistic carbonate. Combined extended X-ray absorption fine structure spectrometry (EXAFS) and absorption spectrometry are ideal complementary probes to characterize the Np coordination site in the metalloprotein. On the one hand Keywords neptunium; serum transferrin; XAS Correspondence C. Den Auwer, CEA Marcoule, DEN ⁄ DRCP ⁄ SCPS, 30207 Bagnols sur Ce ` ze Cedex, France Fax: +33 4 66 79 63 25 Tel: +33 4 66 79 62 89 E-mail: christophe.denauwer@cea.fr (Received 26 November 2004, revised 19 January 2005, accepted 8 February 2005) doi:10.1111/j.1742-4658.2005.04603.x Although of major impact in terms of biological and environmental haz- ards, interactions of actinide cations with biological molecules are only par- tially understood. Human serum transferrin (Tf) is one of the major iron carriers in charge of iron regulation in the cell cycle and consequently con- tamination by actinide cations is a critical issue of nuclear toxicology. Combined X-ray absorption spectroscopy (XAS) and near infrared absorp- tion spectrometry were used to characterize a new complex between Tf and Np (IV) with the synergistic nitrilotriacetic acid (NTA) anion. Description of the neptunium polyhedron within the iron coordination site is given. Abbreviations EXAFS, extended X-ray absorption fine structure spectrometry; NIR, near infrared; NTA, nitrilotriacetic acid; Tf, transferrin; XAS, X-ray absorption spectroscopy. FEBS Journal 272 (2005) 1739–1744 ª 2005 FEBS 1739 spectrometric data provide a fingerprint of the specific complexation mechanism and on the other hand, in the EXAFS regime of X-ray absorption spectrometry (XAS), a quantitative description of the cation coordi- nation sphere can be achieved. Results and Discussion To avoid hydrolysis at physiological pH, Np(IV) in initial stock solution was complexed by NTA. Among the Tf synergistic anions as carbonate, oxalate or citrate, nitrilotriacetic acid is a well known chelat- ing agent that can be used for actinide(IV) [13,14]. Fig. 1 shows the spectrometric near infrared (NIR) spectra exhibiting the characteristic absorption band of Np(IV) aq , Np(IV)(NTA) and Np(IV)(NTA) 2 com- plexes at 960, 973 and 980 nm, respectively [14]. The band at 960 nm for Np(IV) aq is spectroscopically described as an internal 5f)5f transition and is very sensitive to coordination modification [15]. In the visible range, the spectra also exhibit characteristic bands at 724, 732 and 740 nm, respectively. The presence of a band at 724 nm and the absence of any band at 610 nm confirm that the spectral evolu- tion cannot be attributed to the oxidation of Np(IV) to Np(V) but is characteristic of the Np(IV)–NTA complex. To identify the formation of a complex between apoTf and Np(IV) with the NTA synergistic anion (a) the titration of Np(IV) with 2.8 equivalents of NTA by apoTf (Fig. 2A) and (b) the titration of apoTf by Np(IV) with 2.8 equivalents of NTA (Fig. 2B) has been followed by spectrometry in the NIR and visible regions. Fig. 2A shows the evolution of the absorption band at 980 nm of Np(IV) in the Np(IV)(NTA) 2 com- plex upon apoTf titration. The band at 980 nm decrea- ses and a new band appears at 995 nm from [Tf] ⁄ [Np] ¼ 0 to [Tf] ⁄ [Np] ¼ 1.27. In the visible region (data not shown) the band at 740 nm decreases and new bands at 747, 732 and 727 nm appear. Again, the absence of any band at 610 nm precludes the presence of Np(V). The L III edge XANES spectrum of Np in Tf(Np(IV)NTA) 2 (data not shown) also confirms the oxidation state (IV) of Np in the complex [absence of any shoulder at around 15 eV above the edge charac- teristic of the transdioxo form in which Np is at oxida- tion state (V) or (VI)]. This result and particularly the isobestic point at 987 nm is a characteristic fingerprint of the formation of a new Np(IV) complex with Tf and NTA as synergis- tic anion. Moreover, the total disappearance of the absorption band that is characteristic of Np(IV)(NTA) 2 for two equivalents of apoTf suggests the stoechiometry 1 : 2 for the new complex, as observed for Fe(III). Accordingly, the reaction between apoTf, Np(IV) and NTA can be written as shown in Eqn (1). 2NpðIVÞþ2NTA þ apoTf () Tf ðNpðIV ÞNTAÞ 2 ð1Þ Fig. 2B presents the spectrometric evolution of the titration of apoTf by a mixture of Np(IV) with 2.8 equivalents of NTA from [Tf] ⁄ [Np] ¼0.68–0.26. At the beginning of the titration process, the two complexes Np(IV)(NTA) 2 and Tf(Np(IV)NTA) 2 are in equilib- rium according to Eqn (1), as characterized by the two absorption bands at 980 and 995 nm. Upon titration and decrease of [Tf] ⁄ [Np], disappearance of the charac- teristic absorption band of the Tf(Np(IV)NTA) 2 com- plex at 995 nm, the isobestic point at 987 nm and the concomitant increase of the band at 980 nm indicates the formation of Np(IV)(NTA) 2 because of the large excess of NTA in the solution. This confirms the equi- librium described by Eqn (1). The Np L III edge EXAFS spectrum and corresponding Fourier transform of Tf(Np(IV)NTA) 2 are presented in Fig. 3A and B. From the pseudo radial distribution function (R + F ), backscattering contributions from the first Np neigh- bours (I) and from second shell contributors (II) are clearly observed. In the adjustment of the EXAFS spectrum, the typical coordination number of eight was set for Np(IV). Two oxygen shells with 5.2 atoms at 2.34 A ˚ (r 2 ¼ 0.007 A ˚ 2 ) and 2.8 atoms at 2.56 A ˚ (r 2 ¼ 0.025 A ˚ 2 ) and one carbon shell of 7.4 atoms at 3.37 A ˚ (r 2 ¼ 0.009 A ˚ 2 ) were necessary to obtain a Fig. 1. NIR spectrometry of Np(IV) aq , Np(IV)(NTA) and Np(IV)(NTA) 2 . Experimental conditions are as described in Experimental proce- dures. Neptunium uptake by serum transferrin I. Llorens et al. 1740 FEBS Journal 272 (2005) 1739–1744 ª 2005 FEBS satisfactory adjustment (R factor ¼ 0.06). The fit qual- ity is very poor above 3 A ˚ because of the high signal- to-noise ratio and the short data range (only EXAFS data up to 8.5 A ˚ )1 were considered because of the presence of a glitch at 9 A ˚ )1 ). These results will be compared to the structure of the coordinating lobe in TfFe(III)(NTA) [11] for which iron is coordinated to two tyrosines at a mean distance of 1.83 A ˚ and a tetradentate NTA (three O at 1.99 A ˚ and one N at 2.76 A ˚ ) in a highly distorted octahedral symmetry. From a steric point of view, the global increase of the size of the Np coordination sphere vs. that of iron(III) can be explained by the increase in ionic radii although the coordination symmetry is radically different between the two cations. A putative model based on the crystal structure of TfFe(III)(NTA) [11] with Np in the Tf iron binding site with two tyrosines, one tetradentate NTA and two additional water molecules was tested. Note that it is Fig. 2. NIR spectrometry of Np(IV). (A) Titration of Np(IV) in the presence of 2.8 equivalents of NTA per Np by apoTf from [Tf] ⁄ [Np] ¼ 0–1.27. (B) Titration of apoTf by Np(IV) in the presence of 2.8 equivalents of NTA per Np from [Tf] ⁄ [Np] ¼ 0.68–0.26. Experimental conditions are as described Experimental procedures. Fig. 3. k 3 -Weighted EXAFS spectrum (A) and corresponding Fourier transform (B) of Np(IV) in the Tf(Np(IV)NTA) 2 complex (straight line, experimental data; dots, fit). I. Llorens et al. Neptunium uptake by serum transferrin FEBS Journal 272 (2005) 1739–1744 ª 2005 FEBS 1741 impossible to achieve distinction between each coordi- nation site of each lobe with EXAFS because of their structural similarity. Thus, the two Np cations in the Tf complex were considered equivalent. The localiza- tion of Np in the iron binding site is in agreement with previous studies, as referenced above [3]. Because the data resolution is equal to 0.2 A ˚ and the technique averages the signal over all the contributors of similar backscattering factor (i.e. O, N, C), the data fitting is only indicative of the validity of the putative model coordination site. According to this model, a satisfac- tory agreement with an experimental spectrum (R fac- tor ¼ 0.07) was achieved with single scattering contributions from the coordination of a tetradentate NTA molecule as in the Nd(III)(NTA) 2 (H 2 O) complex [16], two tyrosines and two water molecules (no signifi- cant multiple scattering contributions were needed). The three carboxylate oxygen atoms of NTA were refined at 2.35 A ˚ (r 2 ¼ 0.009 A ˚ 2 ) and the correspond- ing nitrogen atom at 2.63 A ˚ (r 2 ¼ 0.001 A ˚ 2 ), the two distances being linked together according to the struc- ture of Nd(NTA) 2 H 2 O. The two tyrosines were refined at 2.34 A ˚ (r 2 ¼ 0.005 A ˚ 2 ) and the two additional water molecules at 2.47 A ˚ (r 2 ¼ 0.014 A ˚ 2 ). In the sec- ond sphere, the eight carbon atoms were linked to the corresponding first coordination sphere atoms (three plus three adjacent to O and N of NTA, plus two adjacent to O of the tyrosine) and only one Debye Waller factor was used for all the carbon scattering paths (r 2 ¼ 0.001 A ˚ 2 ). The average of these distances compares satisfactorily with the two-shell fit described previously: five oxygen atoms at 2.35 A ˚ (2.34 A ˚ in the two-shell fit) and three oxygen ⁄ nitrogen atoms at 2.52 A ˚ (2.56 A ˚ in the two-shell fit). From the bond distance point of view, the Np(IV)–Tf interaction may be compared on the one hand to that of Nd(III)–NTA in the crystal structure of Nd(NTA) 2 (H 2 O) (2.42 for Nd–O and 2.67 A ˚ for Nd–N) [16] and on the other hand to that of Ce(IV)–Tf in the crystal structure of TfCe(IV) 2 [7]. The shortening of the Np(IV)–Tf distances in the Tf lobe compared to neodymium is in agreement with (a) the shortening of the ionic radii at CN ¼ 8 from Nd 3+ (R(Nd 3+ ) ¼ 1.107 A ˚ [17]) to Np 4+ (R(Np 4+ ) ¼ 0.980 A ˚ [17]), and (b) the increase of the ionic charge from three to four if mainly electrostatic interactions are considered. According to the crystal structure of TfFe(III)(NTA), the two tyrosine residues are the only side chain functions available because NTA forces the Tf lobe to be locked in the open form. Thus, the aspartate and histidine residues are unavailable [11]. The short Np-O(Tyr) distance (2.34 A ˚ ) is in agreement with the strong basicity of the phenolate group and agrees with the average value of 2.3 A ˚ in the crystal structure of TfCe(IV) 2 [7]. The overall average Np(IV)–Tf bond distance is equal to 2.42 A ˚ and com- pares well with the Ce(IV)–Tf average distance of 2.46 A ˚ although the comparison must be taken with care given the difference in coordination numbers [six for Ce(IV)] and the differences in coordination pattern (there is no NTA in the case of Ce). More generally speaking, it compares satisfactorily with other actin- ide(IV) coordination complexes as neptunium oxalate (CN ¼ 8, 2.45 A ˚ [18] or plutonium-siderophore com- plex (CN ¼ 9, 2.38 A ˚ [19]. See also reference [1]). We have shown here that the interaction of apoTf with Np at formal oxidation state (IV) leads to the uptake of the cation by the protein. A putative model that places the Np cation in the iron binding site with concomitant binding of NTA synergistic anion has been tested by EXAFS and further spectroscopic and theoretical investigations are needed to support this model. The average Np–ligand distances are in agree- ment with comparable crystallographic structures in the literature. The protein conformation may also be affected by the size of the synergistic anion as in the open form of the coordinating lobe in TfFe(III)(NTA). In that case, binding to the Tf receptor is impossible and cation transfer inside the cell is disabled. Conse- quently, comparison between the lobe conformation in the holo and Np form is also essential. Experimental procedures Np(IV) stock solution preparation The stock solution of Np(IV) (24 mm) was prepared by hydroxylamine (310 mm) reduction (60 °C) of Np(V) obtained by dissolution of Np(V)O 2 OHxH 2 O in hydro- chloric acidic solution. nitrilotriacetic acid complexation was achieved with 2.8 equivalents of ligand at pH ¼ 4. For protein samples, the buffering solution was Hepes. Note that Np ( 237 Np, CEA stock) is a radioactive nucleus and must be manipulated with specific radiological shielding. Human serum Tf was provided by Sigma-Aldrich (Paris, France), 97% purity (Aldrich ref T2252). NIR absorption spectrometry Data acquisition was carried out at room temperature with a Shimadzu 3101 spectrophotometer with a 10-mm path length. In Fig. 1, the spectra of the Np–nitrilotriacetic acid complexes have been obtained by variation of the solution acidity in the presence of 20 mm NTA from an initial solution of aqueous Np(IV) [Np(IV)] ¼ 1.5 mm; HCl, 1 m; Neptunium uptake by serum transferrin I. Llorens et al. 1742 FEBS Journal 272 (2005) 1739–1744 ª 2005 FEBS Np(IV)(NTA) 1 [HCl, 1 m; NTA, 20 mm]; Np(IV)(NTA) 2 [HCl, 0.8 m; NTA, 20 mm]. Fig 2A presents the titration of Np(IV) in the presence of 2.8 equivalents of NTA per Np by apoTf [Hepes, 0.4 m (pH ¼ 7.5); Np(IV), 1.79 mm; NTA, 5.0 mm]. The succes- sive addition of apoTf (powder form) was carried out from [Tf] ⁄ [Np] ¼ 0 to 1.27. Fig. 2B presents the titration of apoTf by Np(IV) in the presence of 2.8 equivalents of NTA per Np [Hepes, 0.4 m, (pH ¼ 7.5); apoTf, 2.35 mm]. The volumetric successive addition of Np(IV)(NTA) 2 was carried out from [Tf] ⁄ [Np] ¼ 0.68 to 0.26. The composition of the titrating solution was: Np(IV), 24.4 mm; NTA, 68.3 mm (pH ¼ 5.5). EXAFS Data acquisition Np L III -edge EXAFS spectra were recorded at the ROBL beamline (BM20) of the European Synchrotron Radiation Facility (Grenoble, France). The ring was operated at 6 GeV with a nominal current of 200 mA. The beamline is equipped with a water-cooled double crystal Si(111) monochromator. Higher harmonics were rejected by two Pt coated mirrors. A Ge solid state detector was used for data collection in the fluorescence mode. Dead time corrections were not necessary because of the low sample concentration. Monochromator energy calibration was carried out with yttrium K-edge at 17052 eV. All measurements were recorded at room tempera- ture, 298 k. The composition of the solution is [Np(IV)] ¼ 0.28 mm; NTA, 0.77 mm; apoTf, 0.14 mm (pH ¼ 6.5). Data analysis and fitting Data treatment was carried out using EXAFS98 code [20]. Background removal was performed using a pre-edge linear function. Atomic absorption was simulated with a cubic spline function. The extracted EXAFS signal was fitted in k 3 CHI(k) without any additional filtering using ARTEMIS code [21]. Due to the low signal to noise ratio above 10 A ˚ )1 and a glitch at 9 A ˚ )1 , Fourier transform (Kaiser window) was done between 2.0 and 8.5 A ˚ )1 . Fitting was carried out in R space between 1.0 and 3.5 A ˚ . Theoretical phases, amplitudes and electron mean free path were calcu- lated with FEFF82 code [22] based on the crystallographic structures of TfFe(III)(NTA) (PDB code 1NFT) and Nd(NTA) 2 (H 2 O). 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