and Iron-Pyoverdine Coordination Compounds ... - ACS Publications

Dec 22, 2018 - Carlo Marini,. ⊥. Emanuela Frangipani,. †,#. Daniela Visaggio,. † ... Giovanna Iucci,. † and Chiara Battocchio*,†. †. Depar...
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Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

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Gallium- and Iron-Pyoverdine Coordination Compounds Investigated by X‑ray Photoelectron Spectroscopy and X‑ray Absorption Spectroscopy Chiara Nicolafrancesco,† Francesco Porcaro,‡ Igor Pis,§ Silvia Nappini,∥ Laura Simonelli,⊥ Carlo Marini,⊥ Emanuela Frangipani,†,# Daniela Visaggio,† Paolo Visca,† Settimio Mobilio,† Carlo Meneghini,† Ilaria Fratoddi,∇ Giovanna Iucci,† and Chiara Battocchio*,† Downloaded via IDAHO STATE UNIV on March 28, 2019 at 18:29:32 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Department of Science, Roma Tre University, Via della Vasca Navale 79, 00146 Rome, Italy University of Bordeaux, CNRS, IN2P3, CENBG, UMR 5797, F-33170 Gradignan, France § Elettra-Sincrotrone Trieste S.C.p.A., SS 14, km 163,5 Basovizza, I-34149 Trieste, Italy ∥ IOM-CNR Laboratorio TASC, SS 14, Km 163,5 Basovizza, I-34149 Trieste, Italy ⊥ CELLSALBA Synchrotron Radiation Facility, Carrer de la Llum 2-26, 08290 Cerdanyola del Vallès, Barcelona, Spain # Department of Biomolecular Sciences, University of Urbino Carlo Bo, Urbino, 61029 Province of Pesaro and Urbino, Italy ∇ Sapienza University, P. le A. Moro 5, 00185, Rome, Italy ‡

S Supporting Information *

ABSTRACT: Iron is an essential nutrient for nearly all forms of life, although scarcely available due to its poor solubility in nature and complex formation in higher eukaryotes. Microorganisms have evolved a vast array of strategies to acquire iron, the most common being the production of high-affinity iron chelators, termed siderophores. The opportunistic bacterial pathogen Pseudomonas aeruginosa synthesizes and secretes two siderophores, pyoverdine (PVD) and pyochelin (PCH), characterized by very different structural and functional properties. Due to its chemical similarity with Fe(III), Ga(III) interferes with several iron-dependent biological pathways. Both PVD and PCH bind Fe(III) and Ga(III). However, while the Ga-PCH complex is more effective than Ga(III) in inhibiting P. aeruginosa growth, PVD acts as a Ga(III) scavenger and protects bacteria from Ga(III) toxicity. To gain more insight into the different outcomes of the biological paths observed for the Fe(III) and Ga(III)-siderophore complexes, better knowledge is needed of their coordination geometries that directly influence the metal complexes chemical stability. The valence state and coordination geometry of the Ga-PCH and Fe-PCH complexes has recently been investigated in detail; as for PVD complexes, several NMR structural studies of Ga(III)-PVD are reported in the literature, using Ga(III) as a diamagnetic isosteric substitute for Fe(III). In this work, we applied up-to-date spectroscopic techniques as synchrotron-radiation-induced X-ray photoelectron spectroscopy (SR-XPS) and X-ray absorption fine structure (XAFS) spectroscopy coupled with molecular modeling to describe the electronic structure and coordination chemistry of Fe and Ga coordinative sites in PVD metal complexes. These techniques allowed us to unambiguously determine the oxidation state of the coordinative ions and to gather interesting information about the similarities and differences between the two coordination compounds as induced by the different metal.

1. INTRODUCTION

limits microbial growth, hence the ability of infectious microbes to cause disease. Successful human pathogens have therefore evolved a vast array of strategies to acquire iron and proliferate, the most shared one being the production of highaffinity iron chelators, termed siderophores. Siderophores are low molecular weight iron-binding molecules that are secreted by bacterial, fungal and some plant cells.2 In vivo, microbial siderophores can successfully compete with the host withholding capabilities, displaying extremely high iron-binding constants. Siderophores can chelate Fe(III) in the extracellular

Iron is an essential nutrient for nearly all forms of life, with critical functions in many cellular processes. The crucial biological role of iron depends on its ability to serve as redox catalyst, by cycling between two oxidation states: ferrous [Fe(II)] and ferric [Fe(III)]. However, iron redox potential may also lead to cellular toxicity, as a consequence of the generation of reactive oxygen species. Therefore, the vast majority of iron in vertebrates is either bound to iron-storage proteins, or complexed with the porphyrin ring in hemoproteins.1 In vertebrates, the iron withholding capacity represents the first line of defense against microbial infections, because it © XXXX American Chemical Society

Received: December 22, 2018

A

DOI: 10.1021/acs.inorgchem.8b03574 Inorg. Chem. XXXX, XXX, XXX−XXX

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Ga-PVD complex alleviated the growth inhibitory activity of Ga(III) in P. aeruginosa, suggesting that PVD acts as a Ga(III) scavenger rather than carrier.26,28 The different behavior of GaPVD compared to that of the Ga-PCH complex may reside in the mechanism of siderophore−metal uptake, as well as in the mechanism of metal release from the siderophore carrier in P. aeruginosa cells. Iron is released from the Fe-PVD complex upon reduction to Fe(II); however, since Ga(III) is redox inactive in biological systems, it cannot be removed from GaPVD, which accumulates in the periplasm of P. aeruginosa cells.14 Conversely, although the mechanism of metal release from PCH is not fully elucidated,30 it has been ascertained that PCH delivers metals directly in the cytoplasm of P. aeruginosa where it is more likely to find Ga(III) targets.31 To gain more insight into the different outcomes of the biological paths observed for the Fe-PVD and Ga-PVD complexes, better knowledge is needed of their coordination geometries that directly influence the metal complexes chemical stability. Several NMR studies on the molecular organization of the PVD ligand around Ga(III) suggest an octahedral coordination of the metal with the formation of two diastereomeric structures (differing for the arrangement of three bidentate ligands forming the octahedral complex: Λ configuration, lefthanded; Δ configuration, right-handed).32 Moreover, circular dichroism spectroscopy measurements carried out on Ga-PVD and Fe-PVD complexes confirmed the two diastereomic structures for the gallium compound, and evidenced only one coordination (either Λ or Δ, depending on the specific pyoverdine) for the Fe(III) complexes.33,34 A previous study addressed the crystallographic structure of FpvA-PVD and ferric FpvA-Fe-PVD35 pointing out a PVD conformer selectivity of the FpvA binding site. In the same work, the EXAFS study conducted at the Fe−K and Ga−K edges with and without FpvA pointed out very similar binding mode for Fe(III) and Ga(III). In this work, our objective is to deepen the knowledge about the electronic nature and the local atomic structure of Fe and Ga bound to PVD combining ab initio structural modeling and synchrotron-radiation-induced spectroscopies. Apo-PVD, FePVD, and Ga-PVD were probed by SR-XPS in solid phase (thick films deposited by drop-casting on a substrate suitable for XPS measurements). The Fe and Ga local atomic structures were probed by XAFS in Fe-PVD and Ga-PVD liquid solution, respectively. It is relevant that the chemical selectivity and local order sensitivity of XAFS allow us to specifically probe the local atomic structure around the metal absorber in Ga(III)PVD and Fe(III)-PVD complexes in solution, therefore mimicking their physiological environment. The integrity and stability of the molecular structure was verified applying laboratory spectroscopic techniques, namely UV−visible absorption and FT-IR in total reflection mode (IRRAS). As a start, PVD and Ga(III)-, Fe(III)-PVD molecular integrity and metalation efficacy were assessed by routine techniques as FT-IR and UV−visible absorption. Then, solidphase samples were investigated by SR-XPS to assess the transition metals oxidation state to individuate the functional groups involved in metal coordination. The EXAFS data analysis has been carried out taking as a model of Fe and Ga local coordination geometry the Me-PVD the 3D structure obtained using the Avogadro molecular modeling program. XPS and XAFS analysis allow us unambiguously to assess the oxidation state of the coordinative ions and to gather interesting information about the similarities and differences

milieu, and then, the ferric−siderophore complex is actively transported back into the microbial cell in an energydependent process.3 The opportunistic bacterial pathogen Pseudomonas aeruginosa synthesizes and secretes two siderophores, pyoverdine (PVD) and pyochelin (PCH), both able to bind Fe(III), although endowed with very different structural and functional properties. PVD is a peptidic siderophore containing two hydroxamic groups and a fluorescent dihydroxyquinoline chromophore,4,5 while PCH is a salicylate-based siderophore with a lower affinity for Fe(III) compared to PVD.6,7 Moreover, both PVD and PCH have been shown to bind other metals, with different affinities.8−10 Fe-PVD and Fe-PCH complexes enter P. aeruginosa cells by binding to the outer membrane receptors FpvA and FptA, respectively.11,12 Then, the proton-motive force of the inner membrane drives the transport of the ferric-siderophore complexes across the outer membrane into the periplasmic space, thanks to the inner membrane protein complex TonB, ExbB and ExbD.13 Once in the periplasmic space, PVD releases Fe(III) upon its reduction to Fe(II),14 while the Fe-PCH complex enter the cytoplasm via the inner membrane permease FptX.15 The cellular localization of the Fe-PVD complex, as well as the mechanism of iron release from PVD, were determined using Ga(III), a group IIIA metal with an atomic number of 31 and a molecular weight of 69.72 g/mol.14,16 Fe(III) and Ga(III) ions show extensive similarities in nuclear radius, coordination chemistry, ionization potential, electronegativity, electron affinity, and tendency to ionic bonding, which make many biological systems unable to distinguish between these two metals and leads to Ga(III) incorporation in siderophores as well as in several Fe(III)-containing enzymes.17 Different from Fe(III), Ga(III) cannot be reduced under physiological conditions, and its incorporation in Fe(III)-containing enzymes results in the inhibition of redox processes and/or the alteration of protein conformation, ultimately causing severe impairment of bacterial metabolism. The iron-mimetic properties of Ga(III) have been exploited in clinical medicine for more than three decades. The radioactive 67Ga isotope is a diagnostic tool for localization of malignant cells, while different Ga(III) formulations have been used as antineoplastic drugs.18−20 Interestingly, Ga(III) has also recently attracted attention in the field of anti-infective drug repurposing,21−23 and the effect of Ga(III) formulations have been investigated in several bacterial species (recently reviewed in refs 24 and 25). Ga(III) inhibits both planktonic and biofilm growth of P. aeruginosa cells and protects from P. aeruginosa infection in different animal models.26,27 Some of us have recently investigated ways to improve the antipseudomonas activity of Ga(III) by complexation with suitable carriers, including siderophores. We found that GaPCH was more effective than Ga(NO3)3 in inhibiting P. aeruginosa growth28 and demonstrated that Fe and Ga have the same valence state, the same octahedral nearest neighbor bond geometry, and very similar coordination mode in the next neighbor shells in Fe-PCH and Ga-PCH complexes, using Xray absorption fine structure (XAFS) and synchrotron radiation-induced X-ray photoelectron spectroscopy (SRXPS) investigations.29 The similarities between the Fe-PCH and Ga-PCH metal complexes indicate that Ga(III) can act as a chemical mimic and confirm the role of PCH as a suitable Ga(III) carrier to P. aeruginosa cells, resulting in potentiation of Ga(III) antibacterial activity. Conversely, we found that the B

DOI: 10.1021/acs.inorgchem.8b03574 Inorg. Chem. XXXX, XXX, XXX−XXX

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the continuum, plus a Gaussian peak representing the transitions to localized states close to the Fermi level39 The structural EXAFS signals χ(k), were calculated subtracting the bare atomic absorption background modeled by a polynomial spline.40 Quantitative analysis has been carried via multishell structural refinement procedures41 in order to access information about intermediate range order obtained from next-neighbor shells and multiple scattering contributions that represent a valuable way to individuate the coordination environment of absorber in metallo-proteins.40−42 SR-XPS experiments were carried out at the BACH (Beamline for Advanced DiCHroism)43 beamline at the ELETTRA Synchrotron Radiation facility of Trieste (Italy). The BACH beamline exploits the intense radiation emitted from an undulator front-end. SR-XPS data were collected with a hemispherical electron energy analyzer (Scienta R3000, pass energy = 50 eV, angular mode), 44 with the monochromator entrance and exit slits fixed at 30 μm. Photon energy settings of 1050 and 609 eV, respectively, were used for O 1s, Ga 3d, Fe 2p and N 1s spectral regions, with a total energy resolution of 0.3 eV; C 1s spectra used for calibration were recorded at both photon energies. Calibration of the energy scale was made, referencing all the spectra to the C 1s core level signal of aliphatic C atoms, always found at 285.00 eV.45,46 Curve-fitting analysis of the C 1s, N 1s, O 1s, Fe 2p, and Ga 3d spectra was performed using Gaussian curves as fitting functions. When several different species were individuated in a spectrum, the same fwhm value was used for all individual photoemission bands. 2.3. Building the Coordination Compound Model. In order to obtain a preliminary understanding on how the chelating groups would have interacted with the metal ion, we used the Open Source program Avogadro. In fact, obtaining a three-dimensional model of PVD, both in its “free” and Me+ bound forms, was mandatory for our comprehension on the underlying coordinating mechanisms and thus for proceeding with an effective EXAFS data analysis. Our choice, regarding a 3D molecular rendering program, fell on the C++/Python based Avogadro, given its user-friendly yet effective approach to molecular design.47 This program gives a discrete set of tools for the designing and the preliminary manipulation in silico of a variety of molecules, ranging from small inorganic compounds, to large biomolecules. For the molecule design, it offers both the opportunity of a visual and more user-friendly interface and a command line option, in which the atom coordinates in space can be determined by the user with a higher precision. The model of the PVD molecule was built using the sequence found in refs 32 and 33. We then proceeded minimizing the internal energy of the obtained molecular model, using the steepest descent. The Avogadro default method is relatively fast, as it searches a local minimum for each atom iteratively until the internal energy of the whole system stabilizes.

between the two Fe-PVD and Ga-PVD coordination compounds, as induced by the different metal ion. The data analysis demonstrates that valence state and coordination chemistry is very similar for both Fe and Ga, accounting for the mimetic behavior of the Ga-PVD coordination compound. The model for the EXAFS data analysis was obtained by applying a user-friendly program (Avogadro); the structural analysis results, in excellent agreement with literature findings, confirm the reliability of a such smart approach to the investigation of the coordination geometry of metals in complicated coordination compounds.

2. MATERIALS AND METHODS 2.1. Pyoverdine Extraction and Metalation Procedure. PVD was purified as previously described,36 with minor modification. Briefly, P. aeruginosa PAO1 ΔpchD37 was grown in iron-poor DCAA for 18 h at 37 °C and 200 rpm. The culture supernatant was purified by filtration through a Sep-Pak C18 Vac-Cartridge 3 cm3 (Waters). After the polymer packing was solvated with 10 hold-up volumes of 50% (v/v) methanol, the cartridge was flushed with 10 hold-up volumes of double-distilled water (ddH2O). The filtered culture supernatant containing PVD was loaded, and the unwanted components were eluted with ddH2O. PVD was then eluted with a small quantity of 50% (v/v) methanol, evaporated to dryness in a desiccator, and dissolved in a small volume of ddH2O. The PVD concentration, in its apo-form, was determined by spectrophotometric measurement at OD405 (ε = 1.4 × 104 M−1 cm−136). PVD metal complexes were prepared at the final concentration of 2.25 mM by mixing 1 volume of an aqueous solution of PVD to equimolar concentrations of FeCl3 dissolved in 0.1 M HCl or freshly prepared Ga(NO3)3 dissolved in ddH2O; Tris-HCl was also added to buffer both solutions at pH = 8. 2.2. Spectroscopic Techniques. UV−visible absorption spectroscopy was performed on a Shimadzu UV-2401PC using quartz cells and an approximately 20 μM aqueous solution of the three samples. IRRAS analysis was performed by means of a VECTOR 22 (Bruker) FT-IR interferometer equipped with a DTGS detector and operating in the wavenumber range 400−4000 cm−1. Measurements were carried out by means of a Specac Monolayer/grazing angle accessory GS19650 operating at 70° incidence; a clean Ti surface was used for recording the background. All samples used for FT-IR and XPS analysis (apo-PVD, Fe-PVD, and Ga-PVD) were deposited from aqueous solution onto TiO2/Si(111) substrates by following a dropcasting procedure. XAS measurements were performed on Fe-PVD and Ga-PVD sample solutions 2.25 mM, at the CLAESS Beamline−ALBA Synchroton Radiation facility (Barcelona, Spain)38 in fluorescence geometry at liquid nitrogen temperature. The energy beam was monochromatized using double Si(111) fixed exit monochromator and a couple of X-ray mirrors ensure a harmonic-free X-ray beam; the beam size on the sample was 0.5 × 0.5 mm2. Energy scans have been carried out in quick scan mode (15 min/scan) using a ionization chamber to measure the incident beam intensity I0 and a single channel silicon drift detector by Amptek to collect total fluorescence yield If, in current mode. Reference samples (Fe-foil or Ga2O3 powders) were measured for energy calibration at Fe and Ga K edges. Up to 15 energy scans were collected for Ga and Fe K-edges and averaged up in order to achieve suitable statistics. Samples were slightly shifted in between each scan in order to prevent radiation damage, and edge regions were compared in order to check for radiation damages:38 no changes were observed at Fe or Ga K-edges pointing out absent or negligible radiation damage effects. The total absorption signals were calculated as α(E) = If/I0. After checking for data quality the spectra were averaged up, the pre-edge background subtracted, and the averaged spectra normalized to the edge jump. For quantitative analysis (using X-ray absorption near-edge spectroscopy, XANES), the near-edge regions were modeled as a combination of arctangent raise, taking into account for photoelectron transitions to

3. RESULTS AND DISCUSSION 3.1. Pyoverdine Metalation Procedure Efficiency and Molecular Stability. To ascertain the efficacy of the metalation procedure, and to probe the metal-PVD complexes stability over time, UV−vis absorption measurements were performed twice a week for 1 month on apo-PVD, Fe-PVD, and Ga-PVD solutions (pH = 8). All samples were opportunely diluted in ddH2O (final concentration of PVD 3.73 μM) to ensure that the absorption peak was fit between 0.200 and 1.000 absorbance units (a.u.). The comparison of the three samples spectra showed some interesting shape shifting (Figure 1). In particular, while the spectrum of apo-PVD was in line with the one described previously, showing only one peak at 405 nm,48,49 the Ga- and Fe-PVD spectra displayed a shoulder at 350 nm, and the Fe-PVD spectrum evidenced two more peaks at 450 and 540 nm, respectively, in line with previous studies.48 Interestingly, the spectra of apoand metal-PVD complexes showed the same profile in each C

DOI: 10.1021/acs.inorgchem.8b03574 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 2. IRRAS spectra of apo-PVD (bottom), Ga-PVD (middle), and Fe-PVD (top); the arrows indicate features of interest.

Scheme 1. Schematic Representation of P. aeruginosa PAO1 PVD Moleculea

Figure 1. UV−visible absorption spectra of apo-PVD (top), Fe-PVD (middle), and Ga-PVD (bottom); the arrows indicate features typical of metalated PVD, as reported in the literature for Fe(III) and Ga(III) complexes.46

measurement, thus indicating their stability over time (Figure S1). To shed more light on the PVD molecular structure and stability upon metalation in solid state, IRRAS measurements were performed on the samples of interest (i.e., apo-PVD, FePVD, and Ga-PVD) obtained by drop-casting the solutions on Ti wafers, and left drying out for 4 days. Infrared spectra were collected at different incident angles of 70, 60, and 50°. Since no significant differences were observed between incidence angles (Figure S2), only samples collected at 70° will be discussed herein. The spectra showed that the molecular structure of the PVD was not deteriorated. The spectra of apoPVD, Fe-PVD, and Ga-PVD exhibited similar features (Figure 2), related to functional groups of PVD: νNH and νOH vibrations in the range 3600−3100 cm−1, νCH at 2930 cm−1, νCO (amide I band) at 1650 cm−1, δNH (amide II) at 1530 cm−1, νC−N of the amide group at 1290 cm−1, and νC−O of the alcohol groups at 1080 cm−1. Ga-PVD also showed a broad peak at about 1350 cm−1, due to the presence of NO3− groups derived from the gallium nitrate salt.50 3.2. Pyoverdine Coordination Geometry at Fe and Ga Ions. To gather detailed information on the electronic and molecular structure of the Ga-PVD and Fe-PVD coordination compounds, X-ray emission and absorption spectroscopies induced by synchrotron radiation sources (i.e., SR-XPS and XAS) were applied to the investigation of the solid and liquid samples. As suggested by the representation of apo-PVD reported in Scheme 1, PVD has several functional groups that can act as electron donors in the formation of metal coordination compounds (hydroxyls, carbonyls, carboxyl, and amine functional groups). NMR studies performed on

a

A linear peptide (blue), a cyclic peptide (magenta), and a chromophore (green) are likely to provide the functional groups responsible of metal coordination (red).

pyoverdine extracted from P. aeruginosa PAO132,33 coordinating Ga(III) ions, suggest the formation of an octahedral complex in which four hydroxyl and two carbonyl groups of PVD act as ligands,32 as highlighted in red in Scheme 1. NMR structural studies of metal-PVD usually employ Ga(III) as the coordination compound, since Ga(III) coordinates octahedrally like Fe(III), but contrary to Fe(III) is diamagnetic, thus allowing NMR studies of the complexes.33,34 To perform SR-XPS measurements, thin solid film samples were obtained via drop-casting small aliquots of apo-PVD, GaPVD and Fe-PVD complexes onto TiO2/Si(111) wafers, and then the obtained films were left to dry for 4 days. In order to verify the PVD molecular structure stability before and after metal binding, as well as to get insight into the Fe-PVD and Ga-PVD coordination structures, we compared the results obtained on apo-PVD with those collected on both Fe-PVD and Ga-PVD compounds. On the basis of the PVD chemical structure (see Scheme 1), C 1s, N 1s, O 1s, and Fe 2p or Ga 3d core level signals were acquired. All the XPS spectra were calibrated in energy using the main component of the C 1s, attributed to aliphatic carbons, at 285.00 eV. Moreover, the D

DOI: 10.1021/acs.inorgchem.8b03574 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry core level binding energy (BE) values, full width at halfmaxima (fwhm), atomic ratios, and functional groups assignments of the three samples were measured (Table S1). XPS data collected at the C 1s, O 1s, and N 1s core levels allowed us to assess the chemical structure integrity of the organic molecule. Detailed information about apo-PVD C 1s, O 1s, and N 1s spectral components such as BEs, fwhm values, functional groups assignments, and experimental and theoretical atomic percentages were determined (Table 1).

As also observed in the C 1s spectrum reported in Figure 3a (top), XPS core level signals arising from carbon atoms composing the PVD molecule have BE (indicative for functional groups) and atomic ratio values (experimental atomic percentages) compatible with the expected PVD molecular structure (Scheme 1). Similarly, N 1s and O 1s spectral components (Figure 3a, middle and bottom) fully reflect the pristine PVD composition. These findings confirm the PVD molecular stability already suggested by UV−visible and IRRAS studies and hints that a structural model based on the proposed molecular structure of PVD is adequate for the XAS data analysis. C 1s spectra of coordination complexes Ga-PVD and FePVD are reported in Figure 3b (middle and bottom), as to be easily compared with the C 1s spectrum of PVD, reported in Figure 3b (top). It is noteworthy that the PVD coordination of metal ions determine a splitting and shift of the C 1s spectral components at higher BE, leading to observe five spectral components instead of four. This effect, fully reproducible for Ga-PVD and Fe-PVD in terms of BE shift and atomic ratio trends over different measurements (reproducibility was checked by carrying out measurements on a series of three samples for each coordination compound), indicates that some of the C−OH and HO−N−CO functional groups of PVD participate to the metal coordination, in line with structural models previously proposed.32,33 Qualitatively, the same trend was observed for both metal ions. From a semiquantitative point of view, we compared the observed atomic ratios for the last three C 1s components (modified in intensity and position after the coordination compound formation) as measured in different samples, as to check the reproducibility of the observed trend. Considering the uncertainty of 5% for semiquantitative XPS analysis,51 the

Table 1. C 1s, N 1s, and O 1s XPS Components Atomic Percentages for apo-PVD Compared with the Theoretical Percentages for the Proposed Molecular Structurea BE (eV)

fwhm (eV)

experimental atomic percent (%)a

theoretical atomic percent (%)

assignment

C 1s 285.00 286.10 287.55 288.94

1.4

396.68

1.8

53 30 12 6

38 40 20 1.8

C−C C−OH, C−N N−CO, CO COOH

N 1s

398.37 399,70 401.37

5.6

5.9

29 58 7.7

23.5 59 11.7

C−NH−C, CNH−C HNC−NH2 C−NH−CO C−N(OH)−CO

O 1s 531.65 532.99 534.34

1.8

36.1 45.8 18.1

57 43

CO C−OH physisorbed H2O

a

The statistic error in semiquantitative XPS analysis is of about 5% of the estimated value.51

Figure 3. (a) C 1s (top), N 1s (middle), and O 1s (bottom) SR-XPS spectra of apo-PVD; (b) C 1s SR-XPS spectra of apo-PVD (top), Ga-PVD (middle), and Fe-PVD (bottom). E

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pounds;51,52 satellite peaks are also observed, as expected for this signal.53 The modifications observed in the C 1s XPS spectra profiles suggest that both Fe(III) and Ga(III) ions coordinate PVD, interacting preferentially with −OH and NOH−COH moieties, coherently with the literature findings.32,33 However, a complementary technique is required to unambiguously individuate the coordination geometry. Therefore, X-ray absorption fine structure (XAFS) spectroscopy was used, due to its chemical selectivity and local order sensitivity, which allow to probe the coordination chemistry around a specific absorber.29 The near-edge (XANES) and extended (EXAFS) regions of the XAFS spectra provide complementary information:41 The XANES analysis gives details about the absorber valence state and coordination symmetry; the multishell EXAFS analysis provides information about nearest- and next-neighbors coordination shells in terms of average distances and coordination numbers. Moreover, the multishell EXAFS data refinement approach41 has proved valuable to verify structural models involving intermediate range order, being especially useful to understand coordination geometry in organic molecules. The Fe and Ga K-edge XANES spectra are presented in Figure 5. It is relevant that in the Fe K edge XANES region the area and position prepeak (see inset in Figure 5) is known to provide reliable information regarding the average Fe valence state and coordination number.29,39,54,55 The Fe pre-edge region of Fe-PVD XAFS spectra (inset in Figure 5) was fitted as a combination of arctangent curve, simulating the transitions to the continuum, and a Gaussian which centroid is found at 2.5 eV above the Fe0 edge position with an area of 7.8(5) eV. According to ref 56 Fe ions are in Fe3+ state, according to XPS data analysis results, and in octahedral coordination (coordination number = 6). The Ga-PVD XANES (Figure 5) is consistent with our previous finding on Ga-PCH29 pointing out Ga3+ state in agreement with XPS data analysis. The XANES analysis provides information about the absorber valence state and its coordination geometry, in particular for Fe ions. In order to understand how Fe and Ga ions are incorporated in PVD molecules, it is necessary to extend our structural knowledge to next neighbor coordination shells. To this purpose we exploited a multishell EXAFS data

C 1s peaks intensity differences between the two samples are negligible. All the measured C 1s spectra are reported in Figure S3 (together with N 1s and O 1s spectra, Figures S4 and S5, respectively). Ga 3d and Fe 2p signals, which are of major interest for the study of the electronic structure of the coordination compounds, are reported in Figure 4. In Ga 3d spectrum of

Figure 4. Ga 3d (a) and Fe 2p (b) SR-XPS spectra of respectively GaPVD and Fe-PVD. The Fe 2p signal appears noisier than the Ga 3d one, due to the selected photon energy value (that was the same for the two samples for semiquantitative purposes).

Ga-PVD (Figure 4a), a single pair of Ga 3d spin−orbit components can be observed (not distinguishable, being the expected doublet splitting 3d5/2−3/2 = 0.44 eV for Ga 3d),52 centered at 19.82 eV BE, in excellent agreement with Ga(III) ions binding energy values reported in the literature.53 Corelevel Fe 2p XPS signal acquired on Fe-PVD (Figure 4b) has a single spin−orbit pair with the main Fe 2p3/2 component at 709.24 eV BE, indicative for Fe(III) coordination com-

Figure 5. XANES region of Fe (left) and Ga (right) K edge spectra. Energy scales refer to the Fe0+ (7112 eV) and Ga0+ (10367 eV) edge energy, respectively. The inset (left panel) shows the Fe pre-edge fit (red) and data (black circles); the arctangent background contribution (green line) and Gaussian peak (blue line) are shown for sake of clarity. F

DOI: 10.1021/acs.inorgchem.8b03574 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

further contributions were not found to give statistically significant contributions. Noticeably, the energy relaxed atomic cluster around Ga appears slightly more compressed respect to Fe, which is consistent with the slightly shorter ionic radius of Ga3+ respect to Fe3+. The k2-weighted experimental EXAFS spectra k2χ(k), were fitted39 to the model curve built combining single (SS) and selected multiple scattering (MS) contributions using the standard EXAFS formula within Gaussian approximation, the amplitude, phase, and mean free path functions were calculated using the FEFF8.2 program.56 The statistically relevant SS and MS contributions were selected via a trial and error procedure, the experimental data, best-fit, and partial contributions used in the analysis are presented in Figure 7a, while the relevant structural parameters are resumed in Table 2. Moduli of the Fourier transform (FT) of experimental and best fit curves are also shown in Figure 7b, allowing to better compare the local coordination of Fe-PVD and Ga-PVD: both the FT have very similar shape with a first maximum around 1.5 Å, corresponding to the nearest neighbor coordination shell (phase-shift uncorrected), with an evident shoulder around 2.1 Å and weak oscillations up to about 4 Å. The very similar FT supports the hypothesis of a similar coordination geometry for Fe and Ga. Similar coordination geometry of Fe and Ga was already demonstrated for another P. aeruginosa siderophore, namely, PCH.29 The EXAFS data refinement allows us to quantitatively support this structural hypothesis. In order to refine the spectra, a minimal set of partial contributions (shells) has been selected by individuating structural signals having similar amplitude (neighbor atoms) and phase (scattering geometries). These are grouped together and their multiplicities (N) were constrained to the model cluster geometry, in order to avoid the correlation between N and disorder factors (mean square relative displacement factors σ2). Owing to the close similarity between Fe- and Ga-PVD atomic models, the same contributions were used to fit Ga and Fe EXAFS spectra. After preliminary tests, we found 4 independent contributions required for the analysis: according to the model clusters, 6 O1 atoms were considered in the first shell. In the second shell, we notice that the scattering amplitude of C and N ions are very similar in the EXAFS region and indistinguishable in the data fitting; therefore, a single shell has been considered with 6 C2 atoms. For the sake of verification, we found that the parameters obtained in the refinement of the spectra assuming 6 N2 as second neighbors do not change significantly. The molecular geometry also involves three atoms configuration, providing no negligible multiple scattering contributions enhanced by the focusing effect owing their quasi-collinear geometries: each residue (chromophore and OH-ornitines) contributes with two Fe/Ga−O1−C2(N2) paths which were considered as a single shell including single (SS) and multiple (MS) scattering terms with opportune multiplicities. We also found a contribution around 4.3 Å that we attributed to the SS and MS contributions with similar amplitude and phase from Fe/Ga−O1−C3 and Fe/Ga−C2(N2)−C3 (4 from chromophore and 2 from each OH-ornitine) providing weak but statistically significant signal due to the almost collinear configurations (forward scattering around 160° at the O1 and C2 sites). This signal is weak, therefore affected by large uncertainty. The multiplicity numbers were kept, path lengths (R) and mean square relative displacement (σ2) were refined and reported in Table 2. The coordination structures (Table

refinement procedure that by including next-neighbor coordination shells in the analysis provides a reliable understanding of the absorber coordination mode, starting from possible atomic cluster models for Fe- and Ga-PVD. We built a M(III)-PVD (M = Fe/Ga) 3D molecular model using the NMR information about the PVD extracted from P. aeruginosa PAO132,33 sequence. Notably, the NMR studies individuated the structure of PAO1 PVD bonded to Ga(III) used as a diamagnetic isosteric substitute for Fe(III) in solution. In order to build the atomic cluster representing the Fe/Ga coordination in PVD, we started with the model generated using the 3D open source software Avogadro.47 The PVD in its apo form was generated based on the NMR studies of Wasielewski et al.,32 and the energy was optimized (steepest descent method). In particular, the Ga(III) ion has been placed in the binding site individuated from NMR studies,32,33 being formed by the two chromophore hydroxyl groups, and the carbonyl and hydroxyl groups from the two HO-Orn1,2, the first from the linear peptide and the second from the cyclic peptide, as described in Scheme 1. Finally, the PVD structure has been relaxed (Figure 6a) to a new minimum of the energy.

Figure 6. (a) full PVD structure relaxed around the metal (brown) ion: red = O, blue = N, gray = C, white = H. (b) Local view of the metal site binding mode: HO-ornithine from linear (HO-Orn1), cyclic (HO-Orn2) peptides and chromophore ring (Chr) bound to the metal ion (brown). The neighbor shells are presented in different colors for shake of clarity (see text): red, oxygen nearest neighbors O1; green, C2 and N2; yellow, C3; violet, far away shell above 4.5 Å.

The XPS and XANES analysis suggests Ga(III) and Fe(III) in the same coordination site, according to refs 32, 33, and 35; therefore, in order to model the Fe(III) atomic cluster we used the same procedure placing Fe(III) in the same Ga(III) site and relaxed the energy to a minimum. The relaxed structure is presented in Figure 6a (full PVD), and the detail of Fe/Ga coordination geometry is shown in Figure 6b: Two HOOrnitines (one from the linear and one from the cyclic peptides) and the chromophore, are linked to the metal via oxygen atoms providing octahedral geometry (Figure 6b). The atomic models for Ga(III)- and Fe(III)-PVD are very similar; both the ions have 6 O1 nearest neighbors (red in Figure 6 b) located around 1.90 Å from Fe and slightly closer to Ga at 1.83 Å and 4 C2 and 2 N2 as second neighbors shell (green in Figure 6 b) around 2.59 Å (Fe) or 2.53 Å (Ga). The third shell is composed by 4 C3 around 3.95 Å (Fe) or 3.89 Å (Ga); G

DOI: 10.1021/acs.inorgchem.8b03574 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

Figure 7. (a) k2-weighted experimental (circles) EXAFS spectra k2χ(k), fitted to the model curve (red line) built combining single and selected multiple scattering (MS) contributions calculated using the standard EXAFS formula within Gaussian approximation. Partial contributions are shown (blue lines) vertically shifted for sake of clarity. (b) Moduli of the Fourier transforms (FT) of experimental (circles) and best fit curves (red lines).

Table 2. Results from EXAFS Data Analysis of the Ga and Fe K Edgesa Fe K-edge N O1 C2 O1−C2(MS) O1−3(MS)

6 6 6 8

Ga K-edge σ2 (× 103 Å2)

R (Å) 1.97(1) 2.81(2) 3.07(2) 4.35(4)

[1.83−1.93] [2.5−2.7] [2.8−3.0] [4.0−4.2]

7.0(5) 5.8(4) 5.5(5) 10.7(9)

R (Å) 1.95(1) 2.73(2) 3.07(2) 4.16(4)

[1.79−1.88] [2.5−2.6] [2.7−2.9] [3.9−4.2]

σ2 (× 103 Å2) 4.5(5) 4.8(4) 3.2(5) 8.4(7)

a Maximum 8 parameters were refined together, the correlations among the free parameters were less than 0.7, the uncertainties on the last digit of refined parameters are reported in parentheses. Multiplicity numbers (N) were kept fixed to the atomic cluster model to avoid the strong correlation between N and σ2. The range of distances from the 3D atomic cluster models are shown in italics in squared parentheses for sake of comparison. The third and fourth signals around 3 and 4.3 Å mainly derives from multiple scattering contributions (see text).

and Ga-PVD, both in solid phase and liquid solution. We coupled state of the art probes (XAFS, XPS) with advanced molecular modeling program (Avogadro) to achieve a reliable description of the electronic nature, oxidation state, and atomic coordination structure around the Fe/Ga hosting site in PVD. In particular, Ga(III) and Fe(III) are demonstrated to share a closely similar local structure in Me(III)-PVD and the model coordination made by the dihydroxyquinoline chromophore and two HO-Orn (form linear (Ser2) and cyclic (Thr1)) fit well with the experimental EXAFS data further supporting the use of Ga(III) as diamagnetic isosteric substitute for Fe(III) for the NMR studies. Noticeably, the coordination geometry involving Ga is slightly tighter and less distorted respect to Fe, likely related to the redox equilibrium Fe3+/Fe2+, responsible for its biological relevance. Overall, our physicochemical findings corroborate the iron-mimetic properties of Ga(III) in biological systems. Furthermore, we here demonstrated that the application of complementary X-ray probes associated to structural modeling provides a reliable and smart approach to the in depth investigation of the structural, coordination and geometrical parameters of metal complexes.

2) derived from EXAFS analysis are in good agreement with previous studies,35 giving confidence in the analysis and demonstrating that Ga and Fe in addition to the isosteric behavior32 also share a closely similar coordinative environment in PVD. The coordination distances appear all similar to those of the 3D clusters obtained from molecular models, giving confidence on the modeling procedure. Consistent with previous studies35 and the structural model, the coordination geometry involving Ga is slightly tighter and less distorted (reduced disorder factors in Table 2) with respect to that of Fe, presumably due to the smaller Ga3+ ionic radius being more electron-dense than Fe. The less dense and more disordered structure of Fe-PVD complex likely favors the redox equilibrium Fe3+/Fe2+, responsible for the metal biological relevance. We note that Fe-PVD may present two diastereomeric structures; neither XAS or XPS may distinguish diastereomeric structures.

4. CONCLUSIONS In the present study, the structure of the P. aeruginosa siderophore PVD complexed with Fe(III) and Ga(III) was investigated, by using complementary spectroscopic techniques. In particular, we thoroughly determined the electronic structure and coordination geometry of the Fe-PVD complex and compared it with the analogous Ga-PVD coordination compound, applying up-to-date synchrotron-radiation-induced spectroscopies as SR-XPS and XAFS to apo-PVD, Fe-PVD,

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ASSOCIATED CONTENT

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b03574. H

DOI: 10.1021/acs.inorgchem.8b03574 Inorg. Chem. XXXX, XXX, XXX−XXX

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UV−visible absorption spectra; IRRAS spectra; XPS C 1s, N 1s, and O 1s spectra; XPS data (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]. ORCID

Francesco Porcaro: 0000-0001-6506-1398 Igor Pis: 0000-0002-5222-9291 Silvia Nappini: 0000-0002-4944-5487 Ilaria Fratoddi: 0000-0002-5172-0636 Giovanna Iucci: 0000-0002-6478-3759 Chiara Battocchio: 0000-0003-4590-0865 Funding

MIUR; Italian Cystic Fibrosis Research Foundation; Department of Sciences Roma Tre University. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Grant of Excellence Departments, MIUR (ARTICOLO 1, COMMI 314−337 LEGGE 232/2016), is gratefully acknowledged. This work was supported by grants from the Italian Cystic Fibrosis Research Foundation (grants FFC#21/2015 and FFC#18/2017). F.P. is grateful for the hospitality at the CLAESS beamline (ALBA).

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ABBREVIATIONS PVD, Pyoverdine; SR-XPS, Synchrotron Radiation induced Xray Photoelectron Spectroscopy; XAFS, X-ray Absorption Fine Structures spectroscopy; XANES, X-ray Absorption Near Edge Spectroscopy; NMR, Nuclear Magnetic Resonance

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REFERENCES

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