Europium(III) as luminescence probe for interactions of a sulfate-reducing microorganism with potentially toxic metals.

Microorganisms show a high affinity for trivalent actinides and lanthanides, which play an important role in the safe disposal of high-level radioactive waste as well as in the mining of various rare earth elements. The interaction of the lanthanide Eu(III) with the sulfate-reducing microorganism Desulfosporosinus hippei DSM 8344T, a representative of the genus Desulfosporosinus that naturally occurs in clay rock and bentonite, was investigated. Eu(III) is often used as a non-radioactive analogue for the trivalent actinides Pu(III), Am(III), and Cm(III), which contribute to a major part of the radiotoxicity of the nuclear waste. D. hippei DSM 8344T showed a weak interaction with Eu(III), most likely due to a complexation with lactate in artificial Opalinus Clay pore water. Hence, a low removal of the lanthanide from the supernatant was observed. Scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy revealed a bioprecipitation of Eu(III) with phosphates potentially excreted from the cells. This demonstrates that the ongoing interaction mechanisms are more complex than a simple biosorption process. The bioprecipitation was also verified by luminescence spectroscopy, which showed that the formation of the Eu(III) phosphate compounds starts almost immediately after the addition of the cells. Moreover, chemical microscopy provided information on the local distribution of the different Eu(III) species in the formed cell aggregates. These results provide first insights into the interaction mechanisms of Eu(III) with sulfate-reducing bacteria and contribute to a comprehensive safety concept for a high-level radioactive waste repository, as well as to a better understanding of the fate of heavy metals (especially rare earth elements) in the environment.

Localization and chemical speciation of europium(III) in Brassica napus plants.

For the reliable safety assessment of repositories of highly radioactive waste, further development of the modelling of radionuclide migration and transfer in the environment is necessary, which requires a deeper process understanding at the molecular level. Eu(III) is a non-radioactive analogue for trivalent actinides, which contribute heavily to radiotoxicity in a repository. For in-depth study of the interaction of plants with trivalent f elements, we investigated the uptake, speciation, and localization of Eu(III) in Brassica napus plants at two concentrations, 30 and 200 µM, as a function of the incubation time up to 72 h. Eu(III) was used as luminescence probe for combined microscopy and chemical speciation analyses of it in Brassica napus plants. The localization of bioassociated Eu(III) in plant parts was explored by spatially resolved chemical microscopy. Three Eu(III) species were identified in the root tissue. Moreover, different luminescence spectroscopic techniques were applied for an improved Eu(III) species determination in solution. In addition, transmission electron microscopy combined with energy-dispersive X-ray spectroscopy was used to localize Eu(III) in the plant tissue, showing Eu-containing aggregates. By using this multi-method setup, a profound knowledge on the behavior of Eu(III) within plants and changes in its speciation could be obtained, showing that different Eu(III) species occur simultaneously within the root tissue and in solution.

A comprehensive study on the interaction of Eu(III) and U(VI) with plant cells (Daucus carota) in suspension.

Daucus carota suspension cells showed a high affinity towards Eu(III) and U(VI) based on a single-step bioassociation process with an equilibrium after 48-72 h. Cells responded with an increased metabolic activity towards heavy metal stress. Luminescence spectroscopy pointed to multiple species for both f-block elements in the culture media, providing initial hints of their interaction with cells and released metabolites. Using nuclear magnetic resonance spectroscopy, we could prove that malate, as an released metabolite in the culture medium, was found to complex with U. Luminescence spectroscopy also showed that Eu(III)-EDTA species are interacting with the cells. Furthermore, Eu(III) and U(VI) coordination is dominated by phosphate groups provided by the cells. We found that Ca ion channels of D. carota cells were involved in the uptake of U(VI), which led to a bioprecipitation of U(VI) in the vacuole of the cells, most probably as uranyl(VI) phosphates along with an intracellular sorption of U(VI) on biomembranes by lipid structures. Eu(III) could be found locally concentrated in the cell wall and in the cytoplasm with a co-localization with phosphorous and oxygen.

Curium(III) speciation in the presence of microbial cell wall components.

Trivalent actinides such as Cm(III) are able to strongly interact with microbes and especially with bacterial cell walls. However, detailed knowledge of the influence of different cell wall components is somewhat lacking. For this investigation, we studied the formation of aqueous Cm(III) complexes with cell wall components (e.g., lipopolysaccharide, peptidoglycan, and plasma membranes) using time-resolved laser-induced fluorescence spectroscopy (TRLFS). For all systems, two specific Cm(III) complexes with the biomacromolecules were observed as a function of pH. Specifically, Cm(III) was found to bind to phosphate and carboxyl groups present in the structure of the biomacromolecules. Stability constants and luminescence parameters of the specific Cm(III) complexes were determined and are presented. The pH of the surrounding aqueous solution, the plasma membrane concentration, and proteins included in the crude plasma membrane fraction were found to significantly impact the complexation of Cm(III). The Cm(III) luminescence spectra with plasma membranes, cell wall polymers, as well as Gram-negative (Sporomusa sp. MT-2.99 and Pseudomonas fluorescens) and Gram-positive (Paenibacillus sp. MT-2.2) bacteria will be explained by linear combination fitting using the investigated components.

Bioassociation of U(VI) and Eu(III) by Plant (Brassica napus) Suspension Cell Cultures-A Spectroscopic Investigation.

In this study, we investigated the interaction of U(VI) and Eu(III) with Brassica napus suspension plant cells as a model system. Concentration-dependent (0-200 µM) bioassociation experiments showed that more than 75% of U(VI) and Eu(III) were immobilized by the cells. In addition to this phenomenon, time-dependent studies for 1 to 72 h of exposure showed a multistage bioassociation process for cells that were exposed to 200 µM U(VI), where, after initial immobilization of U(VI) within 1 h of exposure, it was released back into the culture medium starting within 24 h. A remobilization to this extent has not been previously observed. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to correlate the bioassociation behavior of Eu and U with the cell vitality. Speciation studies by spectroscopy and in silico methods highlighted various U and Eu species over the course of exposure. We were able to observe a new U species, which emerged simultaneously with the remobilization of U back into the solution, which we assume to be a U(VI) phosphate species. Thus, the interaction of U(VI) and Eu(III) with released plant metabolites could be concluded.

Curium(III) and europium(III) as luminescence probes for plant cell (Brassica napus) interactions with potentially toxic metals.

We have investigated the interaction of the actinide Cm(III) and its lanthanide homologue Eu(III) with cells of Brassica napus in suspension. This study combines biochemical techniques (plant cell response) with spectroscopic experiments to determine the chemical speciation of hazardous metals in contact with the plant cells. Experiments conducted over a period of 7 d showed that B. napus cells were able to bioassociate both potentially toxic metals in significant amounts up to 0.58 µmol Eu/gfresh cells and 1.82 µmol Cm/gfresh cells at 30 µM Eu(III) and 0.68 µM Cm(III), respectively. For Cm(III), a biosorption process could be identified as soon as 5 h post-exposure with 73 ± 4% of the Cm(III) bioassociated. Luminescence spectroscopy results based on UV and site-selective excitation confirmed the existence of three Cm(III)/Eu(III) [M(III)] species in both the supernatants and cells. The findings detailed herein support that M(III) coordinates to two kinds of carboxyl groups and phosphate groups.

Molecular Binding of EuIII/CmIII by Stenotrophomonas bentonitica and Its Impact on the Safety of Future Geodisposal of Radioactive Waste.

Microbial communities occurring in reference materials for artificial barriers (e.g., bentonites) in future deep geological repositories of radioactive waste can influence the migration behavior of radionuclides such as curium (CmIII). This study investigates the molecular interactions between CmIII and its inactive analogue europium (EuIII) with the indigenous bentonite bacterium Stenotrophomonas bentonitica at environmentally relevant concentrations. Potentiometric studies showed a remarkably high concentration of phosphates at the bacterial cell wall compared to other bacteria, revealing the great potential of S. bentonitica for metal binding. Infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) confirmed the role of phosphates and carboxylate groups from the cell envelope in the bioassociation of EuIII. Additionally, time-resolved laser-induced fluorescence spectroscopy (TRLFS) identified phosphoryl and carboxyl groups from bacterial envelopes, among other released complexing agents, to be involved in the EuIII and CmIII coordination. The ability of this bacterium to form a biofilm at the surface of bentonites allows them to immobilize trivalent lanthanide and actinides in the environment.

Plant cell (Brassica napus) response to europium(III) and uranium(VI) exposure.

Experiments conducted over a period of 6 weeks using Brassica napus callus cells grown in vitro under Eu(III) or U(VI) stress showed that B. napus cells were able to bioassociate both potentially toxic metals (PTM), 628 nmol Eu/gfresh cells and 995 nmol U/gfresh cells. Most of the Eu(III) and U(VI) was found to be enriched in the cell wall fraction. Under high metal stress (200 µM), cells responded with reduced cell viability and growth. Subsequent speciation analyses using both metals as luminescence probes confirmed that B. napus callus cells provided multiple-binding environments for Eu(III) and U(VI). Moreover, two different inner-sphere Eu3+ species could be distinguished. For U(VI), a dominant binding by organic and/or inorganic phosphate groups of the plant biomass can be concluded.

Interaction of curium(III) with surface-layer proteins from Lysinibacillus sphaericus JG-A12.

Trivalent actinides such as Cm(III) are able to occupy natural Ca(II) binding sites in biological systems. For this investigation, we studied the formation of aqueous Cm(III) complexes with S-layer proteins by time-resolved laser-induced fluorescence spectroscopy (TRLFS). S-layer proteins serve as protective biointerfaces in bacteria and archaea against the surrounding solution. Experimental assays were performed at a fixed total concentration of Cm(III) (0.88 µM) using an S-layer protein (5 g/L / 39.6 µM) at varying pH levels (2.0-9.0), as well as several types of S-layer proteins of L. sphaericus JG-A12. Based on resulting luminescence spectra and lifetime data, specific and unspecific binding sites could be distinguished. Notably, specific Cm(III) binding to S-layer proteins was confirmed by the appearance of a sharp emission band at 602.5 nm, combined with a long lifetime of 310 µs. The high affinity of these specific binding sites was also verified using competing EDTA, wherein only a high EDTA concentration (40 µM) could efficiently remove Cm(III) from S-layer proteins.

Complex formation between UO22+ and α-isosaccharinic acid: insights on a molecular level.

Cellulosic materials present as tissue, paper, wood, or filter materials in low and intermediate level waste will degrade under alkaline conditions if water ingresses in a cementitious backfilled repository. The main degradation product is isosaccharinic acid. Complex formation with isosaccharinic acid may adversely affect the retention of radionuclides by the sorption or formation of solid phases. Hence, this compound is of particular concern in the context of nuclear waste disposal. Structural information of complexes is limited to spherical metal centers and little is known about the interaction of uranyl (UVIO22+) with isosaccharinic acid. Therefore, the interaction of UO22+ with α-isosaccharinate (ISA) was studied under acidic conditions focusing particularly on the structural characterization of the formed complexes. Attenuated total reflection Fourier-transform infrared (ATR-FTIR), nuclear magnetic resonance (NMR), UV-Vis, extended X-ray absorption fine structure (EXAFS) spectroscopy and electrospray-ionization mass spectrometry (ESI-MS) were combined with theoretical calculations to obtain a process understanding on the molecular level. The dominant binding motifs in the formed complexes are 5- and 6-membered rings involving the carboxylic group as well as the α- or ß-hydroxy group of ISA. Two concentration dependent complex formation mechanisms were identified involving either mono- ([UO2(ISA)(H2O)3]+) or binuclear ([(UO2)2(ISA)(H2O)6]3+) species. Furthermore, this study unveils the interaction of UO22+ with the protonated α-isosaccharinic acid (HISA) promoting its transformation to the corresponding α-isosaccharinate-1,4-lactone (ISL) and inhibiting the formation of polynuclear UO22+-ISA species. Future studies on related systems will benefit from the comprehensive knowledge concerning the behavior of ISA as a complexing agent gained in the present study.

Reversible pH-dependent curium(III) biosorption by the bentonite yeast isolate Rhodotorula mucilaginosa BII-R8.

This work describes the molecular characterization of the interaction mechanism of a bentonite yeast isolate, Rhodotorula mucilaginosa BII-R8, with curium(III) as representative of trivalent actinides and europium(III) used as inactive analogue of Cm(III). A multidisciplinary approach combining spectroscopy, microscopy and flow cytometry was applied. Time-Resolved Laser Induced Fluorescence Spectroscopy (TRLFS) analyses demonstrated that the biosorption of Cm(III) is a reversible and pH-dependent process for R. mucilaginosa BII-R8 cells. Two Cm(III)-R. mucilaginosa BII-R8 species were identified having emission maxima at 599.6 and 601.5 nm. They were assigned to Cm(III) species bound to phosphoryl and carboxyl sites from the yeast cell, respectively. Phosphate groups were involved in the sorption of this actinide, as demonstrated by the Eu(III)-phosphate accumulates at the cell membrane shown by microscopy. In addition, cell viability and metabolic potential were assessed to determine the negative effect of Eu(III) in the yeast cells. The results obtained in this work showed that the interaction of Cm(III) with the yeast R. mucilaginosa BII-R8 cells at circumneutral and alkaline pH values will make this radionuclide more mobile to reach the biosphere. Therefore, geochemical conditions in the bentonite engineering barrier need to be carefully adjusted for the safe deep geological disposal of radioactive wastes.

Association of Eu(III) and Cm(III) onto an extremely halophilic archaeon.

In addition to geological, geochemical, and geophysical aspects, also, microbial aspects have to be taken into account when considering the final storage of high-level radioactive waste in a deep geological repository. Rock salt is a potential host rock formation for such a repository. One indigenous microorganism, that is, common in rock salt, is the halophilic archaeon Halobacterium noricense DSM15987T, which was used in our study to investigate its interactions with the trivalent actinide curium and its inactive analogue europium as a function of time and concentration. Time-resolved laser-induced fluorescence spectroscopy was applied to characterize formed species in the micromolar europium concentration range. An extended evaluation of the data with parallel factor analysis revealed the association of Eu(III) to a phosphate compound released by the cells (F2/F1 ratio, 2.50) and a solid phosphate species (F2/F1 ratio, 1.80). The association with an aqueous phosphate species and a solid phosphate species was proven with site-selective TRLFS. Experiments with Cm(III) in the nanomolar concentration range showed a time- and pCH+-dependent species distribution. These species were characterized by red-shifted emission maxima, 600-602 nm, in comparison to the free Cm(III) aqueous ion, 593.8 nm. After 24 h, 40% of the luminescence intensity was measured on the cells corresponding to 0.18 µg Cm(III)/gDBM. Our results demonstrate that Halobacterium noricense DSM15987T interacts with Eu(III) by the formation of phosphate species, whereas for Cm(III), a complexation with carboxylic functional groups was also observed.

Plutonium interaction studies with the Mont Terri Opalinus Clay isolate Sporomusa sp. MT-2.99: changes in the plutonium speciation by solvent extractions.

Since plutonium could be released from nuclear waste disposal sites, the exploration of the complex interaction processes between plutonium and bacteria is necessary for an improved understanding of the fate of plutonium in the vicinity of such a nuclear waste disposal site. In this basic study, the interaction of plutonium with cells of the bacterium, Sporomusa sp. MT-2.99, isolated from Mont Terri Opalinus Clay, was investigated anaerobically (in 0.1 M NaClO4) with or without adding Na-pyruvate as an electron donor. The cells displayed a strong pH-dependent affinity for Pu. In the absence of Na-pyruvate, a strong enrichment of stable Pu(V) in the supernatants was discovered, whereas Pu(IV) polymers dominated the Pu oxidation state distribution on the biomass at pH 6.1. A pH-dependent enrichment of the lower Pu oxidation states (e.g., Pu(III) at pH 6.1 which is considered to be more mobile than Pu(IV) formed at pH 4) was observed in the presence of up to 10 mM Na-pyruvate. In all cases, the presence of bacterial cells enhanced removal of Pu from solution and accelerated Pu interaction reactions, e.g., biosorption and bioreduction.

A spectroscopic study on U(VI) biomineralization in cultivated Pseudomonas fluorescens biofilms isolated from granitic aquifers.

The interaction between the Pseudomonas fluorescens biofilm and U(VI) were studied using extended X-ray absorption fine structure spectroscopy (EXAFS), and time-resolved laser fluorescence spectroscopy (TRLFS). In EXAFS studies, the formation of a stable uranyl phosphate mineral, similar to autunite (Ca[UO2]2[PO4]2•2-6H2O) or meta-autunite (Ca[UO2]2[PO4]2•10-12H2O) was observed. This is the first time such a biomineralization process has been observed in P. fluorescens. Biomineralization occurs due to phosphate release from the cellular polyphosphate, likely as a cell's response to the added uranium. It differs significantly from the biosorption process occurring in the planktonic cells of the same strain. TRLFS studies of the uranium-contaminated nutrient medium identified aqueous Ca2UO2(CO3)3 and UO2(CO3)3 (4-) species, which in contrast to the biomineralization in the P. fluorescens biofilm, may contribute to the transport and migration of U(VI). The obtained results reveal that biofilms of P. fluorescens may play an important role in predicting the transport behavior of uranium in the environment. They will also contribute to the improvement of remediation methods in uranium-contaminated sites.

Uranium(VI) chemistry in strong alkaline solution: speciation and oxygen exchange mechanism.

The mechanism by which oxygen bound in UO2(2+) exchanges with that from water under strong alkaline conditions remains a subject of controversy. Two recent NMR studies independently revealed that the key intermediate species is a binuclear uranyl(VI) hydroxide, presumably of the stoichiometry [(UO2(OH)4(2-))(UO2(OH)5(3-))]. The presence of UO2(OH)5(3-) in highly alkaline solution was postulated in earlier experimental studies, yet the species has been little characterized. Quantum-chemical calculations (DFT and MP2) show that hydrolysis of UO2(OH)4(2-) yields UO3(OH)3(3-) preferentially over UO2(OH)5(3-). X-ray absorption spectroscopy was used to study the uranium(VI) speciation in a highly alkaline solution supporting the existence of a species with three U-O bonds, as expected for UO3(OH)3(3-). Therefore, we explored the oxygen exchange pathway through the binuclear adduct [(UO2(OH)4(2-))(UO3(OH)3(3-))] by quantum-chemical calculations. Assuming that the rate-dominating step is proton transfer between the oxygen atoms, the activation Gibbs energy for the intramolecular proton transfer within [(UO2(OH)4(2-))(UO3(OH)3(3-))] at the B3LYP level was estimated to be 64.7 kJ mol(-1). This value is in good agreement with the activation energy for "yl"-oxygen exchange in [(UO2(OH)4(2-))(UO2(OH)5(3-))] obtained from experiment by Szabó and Grenthe (Inorg. Chem. 2010, 49, 4928-4933), which is 60.8 ± 2.4 kJ mol(-1). Both the presence of UO3(OH)3(3-) and the scenario of an "yl"-oxygen exchange through a binuclear species in strong alkaline solution are supported by the present study.

The U(VI) speciation influenced by a novel Paenibacillus isolate from Mont Terri Opalinus clay.

Bacterial cell walls have a high density of ionizable functional groups available for U(VI) binding, hence have a great potential to affect the speciation of this contaminant in the environment. The studied strain of the genus Paenibacillus is a novel isolate originating from the Mont Terri Opalinus clay formations (Switzerland) which are currently investigated as a potential host rock for future nuclear waste storage. U(VI) binding to the cell surface functional groups was studied by potentiometry combined with time-resolved laser-induced fluorescence spectroscopy (TRLFS). Four bacterial U(VI) surface complexes were identified: R-COO-UO2(+), R-O-PO3-UO2, R-O-PO3H-UO2(+), and (R-O-PO3)2-UO2(2-). The corresponding complex stability constants were calculated to be 5.33 ± 0.08, 8.89 ± 0.04, 12.92 ± 0.05, and 13.62 ± 0.08, respectively. Hence UO2(2+) displays a moderate to strong interaction with the bacterial surface functional groups. In the acidic pH range (pH 3) UO2(2+) binding onto the cell envelope is governed by coordination to hydrogen phosphoryl sites. Upon increasing the pH an increasing coordination of UO2(2+) to carboxylic and deprotonated phosphoryl sites was found. At a pH greater than 7 uranyl hydroxides dominate the speciation. Additionally the bacteria-mediated release of inorganic phosphate in dependence on [U(VI)] at different pH values was studied to assess the influence of phosphate release on U(VI) mobilization.

Insights into the uranium(VI) speciation with Pseudomonas fluorescens on a molecular level.

Microorganisms have great potential to bind and thus transport actinides in the environment. Thus microbes indigenous to designated nuclear waste disposal sites have to be investigated regarding their interaction mechanisms with soluble actinyl ions when assessing the safety of a planned repository. This paper presents results on the pH-dependent sorption of U(VI) onto Pseudomonas fluorescens isolated from the granitic rock aquifers at Äspö Hard Rock Laboratory, Sweden. To characterize the U(VI) interaction on a molecular level, potentiometric titration in combination with time-resolved laser-induced fluorescence spectroscopy (TRLFS) were applied. This paper as a result is one of the very few sources which provide stability constants of U(VI) complexed by cell surface functional groups. In addition the bacteria-mediated liberation of inorganic phosphate in dependence on [U(VI)] at different pHs was studied to judge the influence of phosphate release on U(VI) mobilization. The results demonstrate that in the acidic pH range U(VI) is bound by the cells mainly via protonated phosphoryl and carboxylic sites. The complexation by carboxylic groups can be observed over a wide pH range up to around pH 7. At neutral pH fully deprotonated phosphoryl groups are mainly responsible for U(VI) binding. U(VI) can be bound by P. fluorescens with relatively high thermodynamic stability.

Coordination of uranium(VI) with functional groups of bacterial lipopolysaccharide studied by EXAFS and FT-IR spectroscopy.

The complexation of uranyl ions with lipopolysaccharide (LPS), the main component of the cell wall of Gram-negative bacteria, was investigated on a molecular level with U L(III)-edge extended X-ray absorption fine structure (EXAFS) and attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopy over a wide pH range (2.6 to 7.0). For the first time, structural determinations of uranyl complexes with cell wall compounds were extended from acidic up to neutral pH. The main functionalities responsible for uranyl binding are phosphoryl and carboxyl groups. At an excess of LPS, related to environmental conditions, the uranyl ion is mainly complexed by phosphoryl groups four-fold monodentately coordinated in the equatorial plane of the uranyl dioxo cation UO(2)(2+) showing great homologies to the uranyl mineral phase meta-autunite in the EXAFS spectra. At equimolar ratios of uranyl and functional groups of LPS, according to a slight deficit of phosphoryl groups, additional carboxyl coordination in a bidentate manner becomes important as it is shown by IR spectroscopy. From the vibrational spectra, a mixed coordination of UO(2)(2+) with both phosphoryl and carboxyl groups is derived. The coordination of uranyl ions to the LPS molecule is obviously mainly controlled by the U/LPS concentration ratio, and the influence of pH is only of minor significance at the investigated range.

Combined computational and experimental study of uranyl(VI) 1:2 complexation by aromatic acids.

The bis(salicylhydroxamato) and bis(benzohydroxamato) complexes of UO(2)(2+) in aqueous solution have been investigated in a combined experimental and computational effort using extended X-ray absorption fine structure and UV-vis spectroscopy and density functional theory (DFT) techniques, respectively. The experimentally unknown bis(benzoate) complex of UO(2)(2+) was investigated computationally for comparison. Experimental data indicate 5-fold UO(2)(2+) coordination with mean equatorial U-O distances of 2.42 and 2.40 A for the salicyl- and benzohydroxamate systems, respectively. DFT calculations on microsolvated model systems [UO(2)L(2)OH(2)] indicate UO(2)(2+) eta(2)-chelation via the hydroxamate oxygen atoms in excellent agreement with experimental data; calculated complex stabilities support that UO(2)(2+) prefers hydroxamate over carboxylate coordination. The 414 nm absorption band of UO(2)(2+) in aqueous solution is blue-shifted to 390 and 386 nm upon complexation by salicyl- and benzohydroxamate, respectively. Calculated time-dependent DFT excitation energies of [UO(2)L(2)OH(2)], however, occasionally fail to reproduce accurately experimental UV-vis spectra, which are dominated by UO(2)(2+) <-- L(-) charge-transfer contributions. We additionally show that the U(VI) large-core pseudopotential approximation recently developed by some of the authors can routinely be applied for electronic structure calculations not involving uranium 5f occupations significantly different from U(VI).

Complexation of uranium(VI) with peptidoglycan.

We investigated the interaction of UO(2)(2+) with peptidoglycan (PG), the main part of the outer membrane of Gram-positive bacteria, by potentiometric titration and time-resolved laser-induced fluorescence spectroscopy (TRLFS) over a wide pH (2.0 to 9.0) and concentration range (10(-5) to 10(-4) M U(vi), 0.01 to 0.2 g L(-1) PG). With potentiometry two different dissociation constants for the carboxyl sites of glutamic acid and diaminopimelic acid (pK(a) = 4.55 +/- 0.02 and 6.31 +/- 0.01), and one averaged pK(a) for hydroxyl and amino groups (which are not distinguishable) (9.56 +/- 0.03) and the site densities could be identified. With potentiometry three different uranyl PG complexes were ascertained: two 1 : 1 uranyl carboxyl complexes R-COO-UO(2)(+), one with the glutamic acid carboxyl group (log beta(110) = 4.02 +/- 0.03), which has a very small formation ratio, and one with the diaminopimelic acid carboxyl group (log beta(110) = 7.28 +/- 0.03), and a mixed 1 : 1 : 1 complex with additional hydroxyl or amino coordination, R-COO-UO(2)((+))-A(i)-R (A(i) = NH(2) or O(-)) (log beta(1110) = 14.95 +/- 0.02). With TRLFS, also three, but different, species could be identified: a 1 : 1 uranyl carboxyl complex R-COO-UO(2)(+) (log beta(110) = 6.9 +/- 0.2), additionally a 1 : 2 uranyl carboxyl complex (R-COO)(2)-UO(2) (log beta(120) = 12.1 +/- 0.2), both with diaminopimelic acid carboxyl groups, and the mixed species R-COO-UO(2)((+))-A(i)-R (A(i) = NH(2) or O(-)) (log beta(1110) = 14.5 +/- 0.1). The results are in accordance within the errors of determination.