Journal of Inorganic Biochemistry 105 (2011) 1293–1298 Contents lists available at ScienceDirect Journal of Inorganic Biochemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i n o r g b i o Terbium, a fluorescent probe for investigation of siderophore pyochelin interactions with its outer membrane transporter FptA Yang Binsheng a,⁎, Françoise Hoegy b, Gaëtan L.A. Mislin b, Philippe J. Mesini c, Isabelle J. Schalk b,⁎⁎ a b c Institute of Molecular Science, Shanxi University, Taiyuan, PR China UMR7242, Université de Strasbourg-CNRS, ESBS, Blvd Sébastien Brant, F-67413 Illkirch, Strasbourg, France Institut Charles Sadron, Université de Strasbourg-CNRS, 23 rue du Loess, 67034 Strasbourg Cedex 2, France a r t i c l e i n f o Article history: Received 22 December 2010 Received in revised form 2 March 2011 Accepted 24 March 2011 Available online xxxx Keywords: Terbium Pyochelin Siderophore Pseudomonas aeruginosa Metal traffic Fluorescence Resonance Energy Transfer a b s t r a c t Pyochelin (Pch) is a siderophore and FptA is its outer membrane transporter produced by Pseudomonas aeruginosa to import iron. The fluorescence of the element terbium is affected by coordinated ligands and it can therefore be used as a probe to investigate the pyochelin–iron uptake pathway in P. aeruginosa. At pH 8.0, terbium fluorescence is greatly enhanced in the presence of pyochelin indicating chelation of the metal by the siderophore. Titration curves showed a 2:1 (Pch:Tb3+) stoichiometry and an affinity of K =(2 ± – 1)× 1011 M− 2 was determined. Pch–Tb interaction with the transporter FptA could be followed in vitro and in vivo in P. aeruginosa cells, by Fluorescence Resonance Energy Transfer (FRET) between three partners: the tryptophans of FptA (donor), Pch (acceptor for the Trps and donor for Tb3+) and Tb3+ (acceptor). Pch–Tb binds to the Pch–Fe outer membrane transporter FptA with a dissociation constant (Kd) of 4.6 μM. This three-partner FRET is a potentially valuable tool for investigation of the interactions between FptA and its siderophore Pch. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Bacteria produce low-molecular-weight ligands, called siderophores, to obtain iron [1,2]. Iron is a cofactor for many redox-dependent enzymes and is essential for almost all living organisms. Despite its abundance on earth, iron is not freely available to microorganisms under aerobic conditions, because it forms poorly soluble ferric hydroxides in the environment or is tightly bound to transport and storage proteins in mammalian hosts. Consequently, many bacteria produce siderophores which chelate and thereby solubilize iron from minerals or organic substances. Consistent with this function, siderophores have very high affinity for ferric iron (for example Ka = 1043 and 1032 M− 1 for enterobactin and pyoverdine respectively [3,4]). Ferrisiderophores are formed in the extracellular medium and are transported back into the bacterial cytoplasm by specific transporters. Siderophores also form complexes with metals other than Fe3+, although with lower affinity [5–8]. There is recent evidence that siderophores are used by bacteria not only to acquire iron but also to protect themselves against the ⁎ Correspondence to: Institute of Molecular Science, Shanxi University, Taiyuan 030006, PR China. Tel.: +86 0351 7016358. ⁎⁎ Correspondence to: UMR7242, ESBS, Blvd Sébastien Brant, BP 10412, F-67413 Illkirch, Strasbourg, France. Tel.: +33 3 68 85 47 19; fax: +33 3 68 85 48 29. E-mail addresses: yangbs@sxu.edu.cn (B. Yang), isabelle.schalk@unistra.fr (I.J. Schalk). 0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2011.03.016 toxicity of heavy metals: siderophores sequester these metals outside the bacteria and thereby avoid their entry into cells by diffusion through the outer membrane porins [9]. The siderophore pyochelin (Pch) was first isolated from an irondeficient culture of Pseudomonas aeruginosa by Liu and Shokrani in 1970 [10]. It is produced not only by numerous strains of P. aeruginosa but also by many strains of Burkholderia cepacia [11,12]. The structure of Pch has been established by Cox et al. [13] as 2-(2-o-hydroxyphenyl)-2thiazolin-4-yl)-3-methylthiazolidine-4-carboxylic acid. The absolute configuration of the three chiral centers in the native Pch is 4’R, 2”R,4”R (Fig. 1), but the 2”-center, adjacent to the free carboxyl group in the third ring, isomerizes readily to the S form, resulting in an equilibrium mixture of the two epimers [14]. Pch is biosynthesized in P. aeruginosa from salicylate and two molecules of cysteine by a thiotemplate mechanism [15,16] involving proteins encoded by the biosynthetic operons pchDCBA and pchEFGHI [16,17]. This biosynthesis is autoregulated by a positive-feedback loop [17] requiring the transcriptional regulator PchR and Pch as an effector molecule [18,19]. Pch chelates Fe3+ in the extracellular medium with a 2:1 (Pch:Fe3+) stoichiometry [13,20,21], with one molecule of Pch tetradentately coordinated to Fe3+ and the second molecule bound bidentately to complete the octahedral geometry [22,23]. Pch has poor water solubility, and its Fe3+ affinity was determined in ethanol to be 2× 105 M [13]. This is lower than values for other siderophores, and probably in aqueous solution at physiological pH the affinity for iron is higher. Once loaded with Fe3+, Pch is recognized at the cell surface of P. aeruginosa and 1294 B. Yang et al. / Journal of Inorganic Biochemistry 105 (2011) 1293–1298 2.3. Fluorescence spectroscopy Fig. 1. Structure of pyochelin (Pch). transported into the periplasm by a specific outer membrane transporter, FptA. The structure of FptA is typical of this class of transporters; a protein consisting of a transmembrane 22-β-stranded barrel occluded by an Nterminal domain containing a mixed four-stranded β-sheet [24]. The Pch binding pocket is principally composed of hydrophobic and aromatic residues, consistent with the hydrophobicity of the siderophore, and is highly stereospecific. The X-ray structure of FptA and binding studies have shown that one molecule of Pch is sufficient for a ferriPch complex to be recognized by the FptA transporter, the second chelator providing the remaining bidentate coordination does not interact with FptA and can therefore be a molecule other than Pch, for example another siderophore [24,25]. Docking experiments using the FptA structure and in vivo binding assays showed that the stereospecificity of ferriPch recognition by FptA was mostly due to the configuration of the Pch chiral centers C4 and C2 and was only weakly dependent on the configuration of the siderophore at the C4 carbon atom [26,27]. Transport of ferriPch across the outer membrane by FptA requires the protonmotive force of the inner membrane and the energy-transducing TonB protein that transverses the periplasm and interacts with FptA [28]. The inner membrane transporter of ferriPch may be a permease [29]: mutation of fptX was found to cause a Pch utilization-defective phenotype. Nothing is known about the mechanism of iron release from Pch. Lanthanides have a diversity of biological effects, and they are potentially valuable as tools in medicine and biology [30–32]. Lanthanide ions emit fluorescence of particular wavelength when they are coordinated with particular ligands. Lanthanide coordination compounds have various properties that compare well with those of traditional organic fluorescent materials: long lifetime, large Stokes shift and the fluorescent peak profiles are sharp. Here, we report an analysis of the Pch iron uptake pathway in P. aeruginosa using the lanthanide terbium (Tb3+) as a fluorescence probe. 2. Experimental procedures 2.1. Chemicals Pch was synthesized and purified as described previously [27,33]. TbCl3·6H2O was purchased from Strem, Sodium N-lauroylsarcosine from Sigma and oPOE (n-octylpolyoxyethylene) from Bachem. 2.2. Bacterial strains and growth media The strains used in this study were wild-type P. aeruginosa strain PAO1 [34], PAD07, a strain deficient in Pch and pyoverdine (the major siderophores produced by P. aeruginosa) synthesis [35], PAD14, a Pvd deficient strain mutated for TonB1 [28] and PAO6541, a strain deficient in Pch and pyoverdine synthesis and deleted for fptA [36]. The strains were grown overnight in a succinate medium (composition: 6 g/l K2HPO4, 3 g/l KH2PO4, 1 g/l (NH4)2SO4, 0.2 g/l MgSO4.7H2O, and 4 g/l sodium succinate, with the pH adjusted to 7.0 by adding NaOH). The antibiotics streptomycin (100 μg/mL) and tetracycline (50 μg/mL) were added for cultures of PAD07 and PAD14, and kanamycin (50 μg/ mL) for PAO6541. Fluorescence experiments were performed with a Fluorolog spectrofluorometer. For all experiments, the samples were stirred at 29 °C in a 1 mL cuvette. The excitation wavelength (λex) was set at 295 nm (for the FRET experiments with Trps) or 355 nm (for direct excitation of Pch or FRET between Pch and Tb3+), and fluorescence emission (λem) was measured at 450 nm (for Pch) and 546 nm (for Tb3+). For experiments using bacteria, the bacteria were cultured overnight in succinate medium, and the cells were harvested, washed with 2 volumes of 50 mM Tris–HCl (pH 8.0) and resuspended in the same buffer to an OD at 600 nm of 1. To determine the affinity of Tb3+ to Pch, Pch was incubated in the presence of increasing concentrations of Tb3+ in 50 mM Pipes pH 7.0 buffer and the fluorescence at 546 nm (excitation at 355 nm) monitored. The fluorescence F was fitted with the following function: 2 FP;K;F∞ ðT Þ = F∞ Kp T 1 + Kp2 with T and P the total Tb3+ and Pch concentrations respectively, K the association constant and p the concentration of free Pch, that is given by: p=   1 pffiffiffiffi13 B B pffiffiffiffi 3 1 − + Δ + − − Δ − ð2T−P Þfor positive values of Δ 2 2 3 and rffiffiffiffiffiffiffiffiffi "   1 !# A 1 B A −3 p = − cos acos − for positive values of Δ − 3 3 2 27 with A= 1 1 2 − ð2T−P Þ K 3 B= 2ð2T−P Þ3 P 1 − − ð2T−P Þ 27 K 3K Δ= B A + : 4 27 2 3 The curves were fitted with IgorPro from Wavematrics, with a home made macro. The fit was adjusted with 2 parameters F∞ and K, but the first parameter is always found close to the initial slope. The fit shown Fig. 2B was done with a total concentration of 30 μM Pch so that P2 ≫ K. In such conditions, the equivalence for T = P/2 is clearly visible and the 2:1 complexation is unambigously demonstrated. The measured value of K is 11 −2 K = 2  1•10 mol •L 2 A titration curve obtained for P = 1 μM (P2 b K) could also be fitted with the same K value even if the plateau is reached after the 2:1 equivalence (data not shown). The dissociation constant (Kd) of Pch–Tb to FptA was estimated by incubating FptA in the presence of increasing concentration of Pch–Tb and using the following equations: FptA + TbPch⇄FptA−Pch–Tb B. Yang et al. / Journal of Inorganic Biochemistry 105 (2011) 1293–1298 1295 750 μL of ice-cold water. Next, 1 μL of benzonase was added and the samples incubated for 1 h at 30 °C with shaking. The cytoplasmic fraction was separated from the insoluble material by centrifugation at 40,000 ×g for 40 min at 4 °C. The inner membranes were extracted from the pellets by incubation at room temperature in the presence of 1% sodium N-lauroyl sarcosine in 20 mM Tris, pH 8.0, and isolated by a second centrifugation at 40,000 g. The pellets containing the outer membranes were resuspended in 20 mM Tris, pH 8.0. 3. Results 3.1. Pch chelates terbium Fig. 2. A. Fluorescence emission spectra of Pch–Tb. The excitation wavelength was set at 355 nm. Pch (black) was dissolved at 30 μM in 50 mM Pipes pH 7.0. Aliquots of 0.1 molar equivalent of Tb3+ were added successively and the fluorescence monitored every 4 min (blue to yellow). The fluorescent emission of 300 μM Tb3+ in 50 mM Pipes pH 7.0 in the absence of Pch is presented in green. B. Titration curve. Aliquots of 0.1 molar equivalents of Tb3+ were added to 30 μM Pch in solution in 50 mM pipes, pH 7.0 at 25 °C every 4 min. Fluorescence intensity at 546 nm (excitation wavelength: 355 nm) is plotted against the amount of Tb3+ added. or Pch is a fluorescent siderophore with a maximum fluorescence emission at 430 nm (excitation wavelength: 355 nm) in 50 mM Tris– HCl pH 8.0 [37]. The ability of Pch to chelate Tb3+ was investigated by following the fluorescence of both the siderophore and the metal ion. Tb3+ was added to the siderophore in solution in 50 mM Tris–HCl pH 8.0; this resulted in quenching of the Pch fluorescence at 430 nm and the apparition of four new peaks of fluorescence at 490 nm, 546 nm, 590 nm and 623 nm, corresponding to electron transition from 5D4 to 7 F6, 7F5, 7F4 and 7F3, respectively (Fig. 2A). The fluorescent peak produced by the 5D4 to 7F5 transition (at 546 nm) was very sensitive to the coordination environment of Tb3+, and appears as a doublet. This doublet peak can be considered as characteristic of the complex between Pch and Tb3+. These various spectral changes indicate that Pch can chelate Tb3+ and the energy transfer from Pch to bound Tb3+ enhances the fluorescence of Tb3+. The emission of fluorescence at 546 nm by the Pch–Tb3+ complex was used to monitor the formation of this complex. Titration experiments showed an increase of fluorescence at 546 nm with increasing added Tb3+ concentration (Fig. 2). There was showed a sharp breakpoint in the curve near [Tb3+]/[Pch] = 0.5, revealing a 1:2 ligand stoichiometry for the Pch–Tb3+ complex, consistent with the stoichiometry of the Pch–Fe complex [22]. The binding constant, K was estimated to be K = 2 ± 1 · 1011 M− 2 (see in Experimental procedures). P + L⇄P−L   Kd = ½PŠt ½LŠf = ½P−LŠ   ½P−LŠ = ½PŠt ½LŠf = Kd + ½LŠf [P] t is the total concentration of FptA, and [L] f the free concentration of Pch–Tb. At 546 nm, the fluorescence of Pch–Tb can be neglected compared to the fluorescence of FptA–Pch–Tb. Therefore the concentration of [P–L] or [FptA–Pch–Tb] is directly proportionnal to the fluorescence intensity at 546 nm.   F546nm = Bmax ½LŠf = Kd + ½LŠf 2.4. Preparation of periplasmic, cytoplasmic, inner membrane, and outer membrane fractions PAD07 and PAO6541 cells were grown in succinate medium overnight. Cells were pelleted and resuspended at an OD at 600 nm of 2.6 in 20 mL of 50 mM Tris–HCl pH 8.0 buffer. Four aliquots of 1600 μL of 400 μM Pch–Tb were added at intervals of 10 min, and the samples incubated for 30 min at 37 °C. The cells were pelleted and the pellets were gently rinsed with 10 mL of Tris–HCl, pH 8.0, and resuspended in 500 μL of Tris-sucrose buffer (0.2 M Tris, pH 8.0, 20% sucrose). Lysozyme (5 μL of 100 mg/mL) was added and the mixtures incubated on ice for 1 h. Periplasmic fractions were separated from the spheroplasts by centrifugation at 6700 g for 10 min. The pellets, which were firm and showed no signs of lysis, were gently rinsed twice with 250 μL of Tris–sucrose buffer and then resuspended in 3.2. Binding of Pch–Tb to FptA FptA, the Pch–Fe outer membrane transporter in P. aeruginosa, copurifies with Pch bound to its binding site [37]. When such purified FptA is excited at 355 nm, it emits fluorescence at 450 nm due to the presence of Pch [37] (Fig. 3A). Pch–Tb was added to copurified FptAPch in 50 mM Tris–HCl, pH 8.0, and 0.1% OPOE: all the fluorescent peaks characteristic of Pch–Tb appeared and the fluorescence of Pch at 450 nm was enhanced (Fig. 3A). FptA-Pch was excited at 295 nm resulting in the emission of fluorescence at 338 nm, corresponding to the fluorescence of the Trp present in the protein, with a shoulder peak at 450 nm due to the presence of Pch (Fig. 3B). After addition of Pch–Tb, the emission of fluorescence at 338 nm was quenched and the characteristic doublet peak of Pch–Tb at 546 nm appeared (inset in Fig. 3B). The emission of fluorescence at 546 nm was undetectable when the experiment was repeated with the same concentration of Pch–Tb but in the absence of FptA. In the presence of only Tb3+ (in large excess) and no Pch, the emission of fluorescence at 546 nm appeared as a single peak (data not shown). These various observations indicate that Pch–Tb binds FptA in 50 mM Tris–HCl, pH 8.0, 0.1% OPOE. The quenching of fluorescence at 338 nm and the enhancing of fluorescence at 546 nm is due to energy transfer in the FptA–Pch–Tb3+ complex from Trp residues of FptA to Pch and from Pch to Tb3+. The fluorescence intensity at 546 nm (Fig. 4A) and at 338 nm (Fig. 4B) were plotted against the amount of Pch–Tb added. Both curves have two parts with different slopes: at low Pch–Tb concentrations the slope is steeper and at higher concentrations the slope is less steep. This indicates that in the early part of the titration, 1296 B. Yang et al. / Journal of Inorganic Biochemistry 105 (2011) 1293–1298 Fig. 3. Fluorescence emission spectra of Pch–Tb binding to purified FptA. The excitation wavelength was set at 355 nm (panel A) or 295 nm (panel B) and the emission of fluorescence monitored for copurified FptA–Pch in solution at 3.95 μM in 50 mM Tris– HCl, pH 8.0 and 0.1% OPOE (black spectrum). 0.9, 2.9, 5.7, 9.5 and 11.7 μM of Pch–Tb were added and the fluorescence monitored 2 min after addition (red to blue spectra). The spectra of Pch–Tb alone at 4 μM in 50 mM Tris–HCl, pH 8.0 and 0.1% OPOE is shown as a control (spectra in orange in panels A and B). Fig. 4. Titration curves. The experimental conditions are as described in the legend to Fig. 3. Fluorescence intensity at 546 nm (panel A) and at 338 nm (panel B) are plotted against the amount of Pch–Tb added. essentially all of the added Pch–Tb binds to FptA. The breakpoint between the two slopes is near a [Pch2–Tb] to [FptA] ratio of 1, indicating a 1:1 stoichiometry for FptA and Pch–Tb in the FptA–Pch– Tb complex. In the same experimental conditions, there was no evidence of Tb3+ binding directly to FptA in the absence of the siderophore. To determine the binding constant (Kd) of Pch–Tb to FptA, purified FptA was incubated in the presence of increasing concentrations of Pch–Tb and formation of FptA–Pch–Tb was followed by monitoring the fluorescence at 546 nm (excitation at 290 nm). A Kd of 4.6 μM was determined (see Experimental Procedures). Equivalent data were obtained in either TrisHCl (pH 8.0) or Pipes (pH 7.0) buffer. The ability of Pch–Tb to bind to FptA was also investigated in vivo with the Pvd- and Pch-deficient P. aeruginosa strain PAD07. After overnight culture, the cells were pelleted, resuspended in 50 mM Tris–HCl, pH 8.0 and incubated at 4 °C; at this temperature TonB transporters are not able to transport ferrisiderophores [25,38]. The PAD07 cells were incubated in the presence of Pch–Tb and excited at 295 nm (Fig. 5A). Emission of fluorescence was observed at 337 nm corresponding to the fluorescence of Trp in various proteins. The presence of Pch–Tb increased fluorescence emission at 435 nm and 546 nm, corresponding to the transition from π* to π of Pch and 5D4 to 7 F5 of Tb3+, respectively, (Fig. 5A). This indicates that Pch–Tb3+ binds to FptA in vivo. These increases of fluorescence were not observed for Pch–Tb3+ in solution in the absence of cells. As in vitro, the formation of FptA–Pch–Tb could also be followed in vivo by exciting the cells at 355 nm. For cells alone without addition of Pch–Tb, fluorescence emission was observed at 500 nm. After addition of Pch–Tb, the doublet at 546 nm characteristic of Pch–Tb and the peak at 450 nm characteristic of Pch appeared (Fig. 5B). These experiments clearly show that Pch–Tb is able to bind to FptA both in vivo and in vitro. Fig. 5. Fluorescence emission spectra of FptA–Pch–Tb formed in vivo. A. PAD07 cells were washed and resuspended at an OD at 600 nm of 1.56, in 50 mM Tris–HCl, pH 8.0, at 4 °C and the emission of fluorescence monitored (excited wavelength set at 295 nm) (□). Fluorescence emission was monitored after addition of 77 μM Pch–Tb (△), and for 100 μM Pch–Tb in solution in 50 mM Tris–HCl, pH 8.0, at 4 °C, without cells (○). B. The experiment described in A was repeated with the excitation wavelength set at 355 nm: PAD07 cells (□), and PAD07 cells in the presence of 9 μM Pch2–Tb (△). B. Yang et al. / Journal of Inorganic Biochemistry 105 (2011) 1293–1298 1297 3.3. Pch–Tb transport by FptA To test whether FptA is able to transport Pch–Tb across the bacterial outer membrane, cells expressing FptA (PAD07) and cells deleted for fptA (PAO6541) were incubated in the presence of Pch–Tb. To detect Pch–Tb in various cell compartments, the cells were broken, and the cell compartments separated and studied for the emission of fluorescence (excitation at 355 nm, Fig. 6). To detect siderophore-free Tb3+, 200 μM Pch was added to the samples before measurement of fluorescence emission (Fig. 6). No trace of Pch–Tb or free Tb3+ was detected in the periplasmic, cytoplasmic or inner membrane fractions of PAD07 cells expressing FptA. Only the outer membrane fractions of PAD07 cells contained Tb 3+. Thus, Pch–Tb seems not to be transported by FptA or if transport does occur, Pch–Tb does not accumulate in the bacteria and is immediately re-excreted. 3.4. Ability of Pch–Tb to inhibit Pch–Fe transport As Pch–Tb is able to bind to FptA, we tested whether Pch–Tb inhibited Pch–Fe uptake. 55Fe uptake assays were carried out in the absence and in the presence of a series of concentrations of Pch–Tb (Fig. 7). No clear inhibition of Pch–55Fe uptake could be demonstrated in the presence of Pch–Tb. Presumably, the binding constant of Pch–Tb for FptA (Kd = 4 μM) is insufficient for effective competition with Pch–Fe uptake by FptA. 4. Discussion In solution at neutral pH the number of water molecules coordinated to Tb3+ is 9 [39]. Due to a radiationless path for deexcitation via coupling Fig. 6. Cellular distribution of Tb3+ and Pch–Tb in P. aeruginosa cells. PAD07 cells were prepared in 50 mM Tris–HCl pH 8.0 at an OD at 600 nm of 2.6. Four aliquots of 50 μM Pch–Tb were added (at time intervals of 10 min) and the samples incubated for 30 min at room temperature. The cells were harvested and various cell compartments separated. The emission of fluorescence in each cell compartment was monitored after excitation at 355 nm before (PAD07, ○; PAO6541, Δ) and after addition (PAD07, ●; PAO6541, ▲) of 200 μM Pch. (Panel A, outer membranes; panel B, periplasm; data not shown for the inner membranes and cytoplasm). Fluorescence emission spectrum of Pch excited at 355 nm, black lines in both panels A and B. Fig. 7. Time-dependent Pch–55Fe uptake in the presence of a series of concentrations of Pch–Tb. PAD07 cells at an OD600 of 1 were incubated for 15 minutes in 50 mM Tris–HCl (pH 8.0) at 37 °C, before the initiation of transport assays by the addition of 10 nM Pch– 55 Fe (●). Samples (100 μL) of the suspension were removed at various times and pelleted, and the radioactivity in the bacterial pellet was counted. The experiment was repeated with the protonophore CCCP at a concentration of 200 μM (○) and in the presence of 1(□), 10 (◊) and 100 μM (Δ) Pch–Tb. to OH vibrational overtones, the Tb3+ aqueous ion is only weakly luminescent. The replacement of coordinated water molecules by a multidentate ligand in most cases leads to an enhancement of the fluorescence intensity of this metal ion. Complexation with the diphenolate ligand, N,N’-di(2-hydroxybenzyl)ethylenediamine-N,N’-diacetic acid, results in about 104 more fluorescence than for the Tb3+ aqueous ion [31]. The fluorescence emission intensities for the 5D4 → 7F6, 7 F5, 7F4, and 7F3 transitions of Tb3+ were substantially enhanced in the presence of Pch (Fig. 2), but the fluorescence of Pch at 450 nm was quenched. The 5D4 → 7F5 transition generated two major peaks, whereas the 5D4→7 F6 transition gave a single peak (Fig. 2). There are two causes of the enhancement of Tb3+ fluorescence: the water molecules coordinated to this metal are replaced by Pch, and the Forster dipoledipole non-radiative energy transfer between Pch and Tb3+. Pch titration with Tb3+ conforms reasonably to the ideal case of a sharp inflection after the addition of 0.5 equivalent of Tb3+ (Fig. 2B), indicating that a stable Pch–Tb complex can be formed in 50 mM Pipes, pH 7.0, with a 2:1 (Pch:Tb3+) stoichiometry with an affinity of K = 2 ± 1 · 1011 M− 2. Pch also chelates Fe3+ with a 2:1 (Pch:Fe3+) stoichiometry [13,20,21], with one molecule of Pch tetradentately coordinated to Fe3+ and the second molecule bound bidentately to complete an octahedral geometry [22,23]. As the coordination number of Tb3+ is 9, the two molecules of Pch are most likely involved equally in the coordination of this metal ion and possibly one water molecule remains coordinated to Tb3+. The high resolution structure of FptA [23] revealed that the Pch binding pocket is mainly composed of hydrophobic (Leu116, Leu 117 and Met271), and aromatic (Phe114, Tyr334 and Trp702) residues, a feature consistent with the hydrophobic characteristics of Pch. When Pch–Tb was incubated in the presence of purified FptA and excited at 355 nm or at 295 nm, the fluorescence corresponding to 5D4 → 7F5 transition displayed two major peaks at 457 nm and 546 nm (Fig. 3). When the experiment was repeated with Pch–free Tb3+, no increase of fluorescence at 546 nm was detected, indicating that this metal was not able to interact with FptA in the absence of Pch. This shows that Pch–Tb binds to FptA and that the interaction can be visualized by the emission of fluorescence at 546 nm (excitation at 295 nm) due to FRET from Trp residues of FptA to Pch, and subsequently from Pch to bound Tb3+ (Fig. 3B). By measuring the fluorescence at 338 nm or 546 nm a 1:1 (FptA–Pch2–Tb or FptA–Pch–Tb) stoichiometry was confirmed (Fig. 5) and a Kd of 4.6 μM. This dissociation constant is clearly lower compared to the one determined between FptA and Pch–Fe (Kd = 2.5 nM) [25,26]. The FptA structure shows clearly enough space in the siderophore-binding pocket for a metal ion, a molecule of Pch and either a second molecule of Pch or another 1298 B. Yang et al. / Journal of Inorganic Biochemistry 105 (2011) 1293–1298 chelator, like cepabactin [40]. Therefore, we suggest that FptA mostly binds Tb3+ coordinated by two molecules of Pch. FptA contains 17 Trp residues, but only Trp702 is in the Pch-binding site and is likely to be involved in FRET with Pch–Tb. Two other Trp residues (Trp295 and Trp413) are present in the extracellular loops and may be close enough to Pch for FRET if binding of the siderophore involves appropriate conformational changes of these loops. Iron-siderophore transport across the outer membrane is an energy consuming process. Energy is provided by the protonmotive force from the cytoplasmic membrane and involves interactions between the siderophore receptor and the TonB–ExbB–ExbD complex of the inner membrane. At 4 °C, this active transport is stopped and the iron-siderophore can only bind to the receptor on the surface of cells but not be imported. In the presence of Pch–Tb, the fluorescence of FptA at 338 nm was quenched (excitation wavelength: 295 nm), the fluorescence at 435 nm and at 546 nm, corresponding to Pch and Tb3+, respectively, were enhanced (Fig. 6), indicating that Pch–Tb binds to FptA on the surface of the cells. The same experiment was repeated with cells incubated at 37 °C, and cell fractions were tested: Tb3+ ions were present only in the fractions containing outer membranes (Fig. 6). No trace of the metal was found in the periplasmic or cytoplasmic fractions (Fig. 6). This may indicate that either FptA binds Pch–Tb but is not able to transport it across the outer membrane or that transport occurs via FptA, but the Pch–Tb complex does not accumulate within the cells and is immediately excreted by an efflux system. Further studies are necessary to clarify this point. For the moment, nothing is known about Pch secretion or efflux systems which could excrete unwanted Pch–metal complexes. Work using metal detection by ICP-AES has also shown that Tb3+ does not accumulate in P. aeruginosa via the Pch pathway [5]. Indeed, this previous study demonstrates that TonB-dependent metal uptake, in the presence of Pch, was only efficient for Fe3+; Co2+, Ga3+ and Ni2+ were also transported, but the uptake rates were 5- to 40-fold lower than that for Fe3+. No uptake was seen for all the other metals tested (Ag+, Al3+, Cd2+, Cr2+, Cu2+, Eu3+, Hg2+, Mn2+, Pb2+, Sn2+, Tb3+, Tl+ and Zn2+). Consistent with these observations, excess Pch–Tb was unable to inhibit Pch–Fe uptake by P. aeruginosa cells (Fig. 7). In conclusion, Pch2–Tb is an potentially valuable fluorescent complex for studies of the interaction between FptA and its siderophore, due to the FRET between the Trp residue(s) of FptA and Pch (Trp being the donor and Pch the acceptor of fluorescence) and between Pch and Tb3+ (Pch being the donor and Tb3+ the acceptor). In particular, this approach avoids the need for chemical modification of the siderophore or genetically labeling of FptA with a fluorescent tag. Abbreviation Pch Pyochelin FptA Pch outer membrane transporter FRET Fluorescence Resonance Energy Transfer Acknowledgments This work was partly funded by the Centre National de la Recherche Scientifique (CNRS) and a grant from the ANR (Agence Nationale de Recherche, ANR-08-BLAN-0315-01), by the National Natural Science Foundation of PR China (No. 20901048) and the Shanxi Scholarship Council of China (No. 201013). We thank K. Brillet for the purification of FptA. References [1] H. Boukhalfa, A.L. Crumbliss, Biometals 15 (2002) 325–339. [2] G. Winkelmann, Biochem. Soc. Trans. 30 (2002) 691–696. [3] A.M. Albrecht-Gary, S. Blanc, N. Rochel, A.Z. Ocacktan, M.A. Abdallah, Inorg. Chem. 33 (1994) 6391–6402. [4] K.N. Raymond, E.A. Dertz, S.S. Kim, Proc. Natl. Acad. Sci. U S A 100 (2003) 3584–3588. [5] A. Braud, M. Hannauer, G.L.A. Milsin, I.J. 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