'Venus trapped, Mars transits': Cu and Fe redox chemistry, cellular topography and in situ ligand binding in terrestrial isopod hepatopancreas.

Woodlice efficiently sequester copper (Cu) in 'cuprosomes' within hepatopancreatic 'S' cells. Binuclear 'B' cells in the hepatopancreas form iron (Fe) deposits; these cells apparently undergo an apocrine secretory diurnal cycle linked to nocturnal feeding. Synchrotron-based µ-focus X-ray spectroscopy undertaken on thin sections was used to characterize the ligands binding Cu and Fe in S and B cells of Oniscus asellus (Isopoda). Main findings were: (i) morphometry confirmed a diurnal B-cell apocrine cycle; (ii) X-ray fluorescence (XRF) mapping indicated that Cu was co-distributed with sulfur (mainly in S cells), and Fe was co-distributed with phosphate (mainly in B cells); (iii) XRF mapping revealed an intimate morphological relationship between the basal regions of adjacent S and B cells; (iv) molecular modelling and Fourier transform analyses indicated that Cu in the reduced Cu(+) state is mainly coordinated to thiol-rich ligands (Cu-S bond length 2.3 Å) in both cell types, while Fe in the oxidized Fe(3+) state is predominantly oxygen coordinated (estimated Fe-O bond length of approx. 2 Å), with an outer shell of Fe scatterers at approximately 3.05 Å; and (v) no significant differences occur in Cu or Fe speciation at key nodes in the apocrine cycle. Findings imply that S and B cells form integrated unit-pairs; a functional role for secretions from these cellular units in the digestion of recalcitrant dietary components is hypothesized.

Bacterially produced calcium phosphate nanobiominerals: sorption capacity, site preferences, and stability of captured radionuclides.

A Serratia sp. bacterium manufactures amorphous calcium phosphate nanominerals (BHAP); this material has shown increased sorption capacity for divalent radionuclide capture. When heat-treated (≥450 °C) the cell biomass is removed and the biominerals are transformed to hydroxyapatite (HAP). Using a multimethod approach, we have elucidated both the site preferences and stability of analogue radionuclide incorporation for Sr, Co, Eu, and U. Strontium incorporates within the bulk amorphous inorganic phase of BHAP; however, once temperature modified to crystalline HAP, bonding was consistent with Sr substitution at the Ca(1) and/or Ca(2) sites. Cobalt incorporation occurs within the bulk inorganic amorphous phase of BHAP and within the amorphous grain boundaries of HAP. Europium (an analogue for trivalent actinides) substituted at the Ca(2) and/or the Ca(3) position of tricalcium phosphate, a known component of HAP grain boundaries. Uranium was surface complexed with no secondary minerals detected. With multiple sites for targeted radionuclide incorporation, high loadings, and good stability against remobilization, BHAP is shown to be a potential material for the remediation of aqueous radionuclide in groundwater.

Ex situ formation of metal selenide quantum dots using bacterially derived selenide precursors.

Luminescent quantum dots were synthesized using bacterially derived selenide (Se(II-)) as the precursor. Biogenic Se(II-) was produced by the reduction of Se(IV) by Veillonella atypica and compared directly against borohydride-reduced Se(IV) for the production of glutathione-stabilized CdSe and ß-mercaptoethanol-stabilized ZnSe nanoparticles by aqueous synthesis. Biological Se(II-) formed smaller, narrower size distributed QDs under the same conditions. The growth kinetics of biologically sourced CdSe phases were slower. The proteins isolated from filter sterilized biogenic Se(II-) included a methylmalonyl-CoA decarboxylase previously characterized in the closely related Veillonella parvula. XAS analysis of the glutathione-capped CdSe at the S K-edge suggested that sulfur from the glutathione was structurally incorporated within the CdSe. A novel synchrotron based XAS technique was also developed to follow the nucleation of biological and inorganic selenide phases, and showed that biogenic Se(II-) is more stable and more resistant to beam-induced oxidative damage than its inorganic counterpart. The bacterial production of quantum dot precursors offers an alternative, 'green' synthesis technique that negates the requirement of expensive, toxic chemicals and suggests a possible link to the exploitation of selenium contaminated waste streams.

Pb and Zn imaging and in situ speciation at the geogenic/biogenic interface in sentinel earthworms using electron microprobe and synchrotron micro-focus X-ray spectroscopy.

UNLABELLED: Using synchrotron- and electron microscope-based X-ray microanalyses, the distribution and speciation of Zn and Pb were examined in situ in two earthworm species (Dendrodrilus rubidus and Lumbricus rubellus) living in heavily-polluted soils. MAIN FINDINGS: (i) Zn spectra in ingested soil and in tissues more closely resembled Zn(3)(PO(4))(2) than ZnS; (ii) Zn speciation in tissues gave a best fit for Zn to the inner shell of 4 oxygens at 1.94 Å (or nitrogens at 1.96 Å); (iii) the best fit for Pb in tissue was with a shell of oxygens at 2.18 Å and a shell of sulphurs at 2.67 Å; (iv) a component of the Zn and much of the Pb detectable in gut contents was co-distributed with S; (v) Zn and Pb display 'soft' acid affinities in soil, but 'hard' acid affinities in tissue. This is the first metal characterisation study conducted on an invertebrate quench-frozen in the field.

In situ metal imaging and Zn ligand-speciation in a soil-dwelling sentinel: complementary electron microprobe and synchrotron microbeam X-ray analyses.

Understanding the relationships between accumulated metal speciation in cells and tissues of ecologically significant taxa such as earthworms will improve risk assessments. Synchrotron-based µ-focus X-ray spectroscopy was used to detect, localize, and determine ligand-speciation of Zn and Pb in thin sections of two epigeic earthworm species collected from a Pb/Zn-mine soil. The findings indicated that Zn and Pb partition predominantly as typical hard acids (i.e., strong affinities for O-donors) within liverlike chloragocytes. Moreover, Zn speciation was very similar in the chloragog and intestinal epithelia but differed subtly in the kidneylike nephridial tubules; neither Zn nor Pb was detectable in the ventral nerve cord. High resolution X-ray mapping of high pressure-frozen, ultrathin, freeze-substituted sections in a transmission electron microscope (TEM), combined with conventional TEM structural analysis, identified a new cell type packed with highly organized rough endoplasmic reticulum and containing deposits of Cd (codistributed with S); there was no evidence that these cells are major depositories of Zn or Pb. These data may be used in a systems biology approach to assist in the interpretation of metal-evoked perturbations in whole-worm transcriptome and metabolome profiles.

As-resistance in laboratory-reared F1, F2 and F3 generation offspring of the earthworm Lumbricus rubellus inhabiting an As-contaminated mine soil.

Previous studies provided no unequivocal evidence demonstrating that field populations of Lumbricus rubellus Hoffmeister (1843), exhibit genetically inherited resistance to As-toxicity. In this study F1, F2 and F3 generation offspring derived from adults inhabiting As-contaminated field soil were resistant when exposed to 2000 mg kg(-1) sodium arsenate. The offspring of uncontaminated adults were not As-resistant. Cocoon viability was 80% for F1 and 82% for F2 offspring from As-contaminated adults and 59% in the F1 control population. High energy synchrotron analysis was used to determine whether ligand complexation of As differed in samples of: resistant mine-site adults, the resistant F1 and F2 offspring of the mine-site earthworms exposed to the LC(25) sodium arsenate (700 mg kg(-1)) of the F1 parental generation; and adult L. rubellus from an uncontaminated site exposed to LC(25) concentrations of sodium arsenate (50 mg kg(-1)). XANES and EXAFS indicated that As was present as a sulfur-coordinated species.

Arsenic mobilization from iron oxyhydroxides is regulated by organic matter carbon to nitrogen (C:N) ratio.

Arsenic (As) is mobilized from delta and floodplain aquifer sediments throughout S.E. Asia via reductive dissolution of As bound to iron (Fe) oxyhydroxides. The reductive driving force is organic carbon, but its source and constitution is uncertain. Here batch incubation experiments were conducted to investigate the role of organic matter (OM) carbon:nitrogen (C:N) ratio on the mobilization of arsenic, Fe and N from As dosed, Fe oxyhydroxide coated sands. As mobilization into pore waters from the sand was strongly regulated by the C:N ratio of the OM, and also the concentration of OM present. The lower the C:N, the more As released. Fe and ammonium release were similarly dependent on the quality and quantity of OM, but Fe mobilization was more rapid and ammonium release slower than As suggesting that the mobilization of these 3 moieties although interdependent, were not directly linked. It was concluded that low C:N ratios for OM responsible for reducing aquifers were As in groundwater is observed were likely.

Arsenic rich iron plaque on macrophyte roots--an ecotoxicological risk?

Arsenic is known to accumulate with iron plaque on macrophyte roots. Three to four years after the Aznalcóllar mine spill (Spain), residual arsenic contamination left in seasonal wetland habitats has been identified in this form by scanning electron microscopy. Total digestion has determined arsenic concentrations in thoroughly washed 'root+plaque' material in excess of 1000 mg kg(-1), and further analysis using X-ray absorption spectroscopy suggests arsenic exists as both arsenate and arsenite. Certain herbivorous species feed on rhizomes and bulbs of macrophytes in a wide range of global environments, and the ecotoxicological impact of consuming arsenic rich iron plaque associated with such food items remains to be quantified. Here, greylag geese which feed on Scirpus maritimus rhizome and bulb material in areas affected by the Aznalcóllar spill are shown to have elevated levels of arsenic in their feces, which may originate from arsenic rich iron plaque.

Probing the biogeochemistry of arsenic: response of two contrasting aquifer sediments from Cambodia to stimulation by arsenate and ferric iron.

Many millions of people worldwide are at risk of severe poisoning through exposure to groundwater contaminated with sediment-derived arsenic. An ever-increasing body of work is reinforcing the link between microbially-mediated redox cycling in aquifer sediments and the mobilisation of sorbed As(V) into groundwaters as the potentially more mobile and toxic As(III) anion. However, to date, few studies have examined the biogeochemical cycling of Fe and As species by microbes indigenous to Cambodian sediments. In this study two contrasting sediments, taken from a shallow As-rich reducing aquifer in the Kien Svay district of Cambodia, were used in a laboratory microcosm study. We present evidence to show that microbes present in these sediments are able to reduce Fe(III) and As(V) when provided with an electron donor, and that the two sediments respond differently to stimulation with Fe(III) and As(V). Shifts in the community composition of the two sediments after stimulation with As(V) suggest a potential role for members of the beta-Proteobacteria in As(V) reduction, a phylogenetic grouping known to contain microorganisms capable of As(III) oxidation, but not previously implicated in As(V) reduction. PCR-based analysis of the sediment microbial DNA using primers specific to the arrA gene, (a gene essential for microbial As(V) respiration), indicates the presence of microorganisms capable of dissimilatory As(V) reduction.

Fungal transformations of uranium oxides.

The biogeochemical activities of free-living and symbiotic fungi must be acknowledged in attempts to understand uranium cycling and dispersal in the environment. Although the near-surface geochemistry of uranium is very complex and a wide variety of mineral phases is known, uranium trioxide (UO3) and triuranium octaoxide (U(3)O(8)) can be used as well characterized models in the study of biotransformations. We have used a complex methodological approach involving advanced solid state speciation and scanning electron microscopy to study the ability of saprotrophic, ericoid and ectomycorrhizal fungi to transform these model oxides. This study has revealed that fungi exhibit a high uranium oxide tolerance, and possess the ability to solubilize UO3 and U(3)O(8) and to accumulate uranium within the mycelium to over 80 mg (g dry weight)(-1) biomass. X-ray absorption spectroscopy of uranium speciation within the biomass showed that in most of the fungi the uranyl ion was coordinated to phosphate ligands, but in ectomycorrhizal fungi mixed phosphate/carboxylate coordination was observed. Abundant uranium precipitates associated with phosphorus were found in the mycelium and encrusted the hyphae. Some of the fungi caused the biomineralization of well-crystallized uranyl phosphate minerals of the meta-autunite group. This is the first experimental evidence for fungal transformations of uranium solids and the production of secondary mycogenic uranium minerals.

Cation occupancies in Mg, Co, Ni, Zn, Al ferrite spinels: a multi-element EXAFS study.

The distribution of cations between tetrahedral (A) sites and octahedral (B) sites in ferrite spinels has been studied using K-edge x-ray absorption spectroscopy. The samples include natural and synthetic end-member magnetites (Fe3O4), a natural Mn- and Zn-rich magnetite (franklinite) and synthetic binary, ternary and quaternary ferrites of stoichiometry M(²+)M2(³+)O4, where M(²+) = Mg, Co, Ni, Zn and M(³+) = Fe, Al. XAS data were obtained for all metals. Complete, unfiltered, EXAFS spectra were refined to determine the percentage distribution of each element over the A and B sites and these data were combined with microprobe analyses to quantify the tetrahedral occupancy for each element in each sample. Measured site occupancies and an internally consistent set of (M-O)(A) and (M-O)(B) bond lengths were used to calculate unit-cell parameters, which show excellent agreement with measured values, pointing to the reliability of the measured occupancy factors. The average occupancies determined for the tetrahedral sites in ferrites are (atoms per formula unit) Mg 0.44, Co 0.24, Ni 0.11, Zn 0.76, Al 0.11 and Fe(³+) 0.92-0.19. The wide range found for Fe(³+) is consistent with it playing a relatively passive role by making good any A-site deficit left by the other competing cations.

Arsenic sequestration in iron plaque, its accumulation and speciation in mature rice plants (Oryza sativa L.).

A compartmented soil-glass bead culture system was used to investigate characteristics of iron plaque and arsenic accumulation and speciation in mature rice plants with different capacities of forming iron plaque on their roots. X-ray absorption near-edge structure spectra and extended X-ray absorption fine structure were utilized to identify the mineralogical characteristics of iron plaque and arsenic sequestration in plaque on the rice roots. Iron plaque was dominated by (oxyhydr)oxides, which were composed of ferrihydrite (81-100%), with a minor amount of goethite (19%) fitted in one of the samples. Sequential extraction and XANES data showed that arsenic in iron plaque was sequestered mainly with amorphous and crystalline iron (oxyhydr)oxides, and that arsenate was the predominant species. There was significant variation in iron plaque formation between genotypes, and the distribution of arsenic in different components of mature rice plants followed the following order: iron plaque > root > straw > husk > grain for all genotypes. Arsenic accumulation in grain differed significantly among genotypes. Inorganic arsenic and dimethylarsinic acid (DMA) were the main arsenic species in rice grain for six genotypes, and there were large genotypic differences in levels of DMA and inorganic arsenic in grain.

XAS and XMCD evidence for species-dependent partitioning of arsenic during microbial reduction of ferrihydrite to magnetite.

Poorly crystalline Fe(III) oxyhydroxides, ubiquitously distributed as mineral coatings and discrete particles in aquifer sediments, are well-known hosts of sedimentary As. Microbial reduction of these phases is widely thought to be responsible for the genesis of As-rich reducing groundwaters found in many parts of the world, most notably in Bangladesh and West Bengal, India. As such, it is important to understand the behavior of As associated with ferric oxyhydroxides during the early stages of Fe(lll) reduction. We have used X-ray absorption spectroscopy (XAS) and X-ray magnetic circular dichroism (XMCD) to elucidate the changes in the bonding mechanism of As(III) and As(V) as their host Fe(III) oxyhydroxide undergoes bacterially induced reductive transformation to magnetite. Two-line ferrihydrite, with adsorbed As(III) or As(V), was incubated under anaerobic conditions in the presence of acetate as an electron donor, and Geobacter sulfurreducens, a subsurface bacterium capable of respiring on Fe(lll), but not As(V). In both experiments, no increase in dissolved As was observed during reduction to magnetite (complete upon 5 days incubation), consistent with our earlier observation of As sequestration by the formation of biogenic Fe(III)-bearing minerals. XAS data suggested that the As bonding environment of the As(III)-magnetite product is indistinguishable from that obtained from simple adsorption of As(lll) on the surface of biogenic magnetite. In contrast, reduction of As(V)-sorbed ferrihydrite to magnetite caused incorporation of As5+ within the magnetite structure. XMCD analysis provided further evidence of structural partitioning of As5+ as the small size of the As5+ cation caused a distortion of the spinel structure compared to standard biogenic magnetite. These results may have implications regarding the species-dependent mobility of As undergoing anoxic biogeochemical transformations, e.g., during early sedimentary diagenesis.

Interactions between the Fe(III)-reducing bacterium Geobacter sulfurreducens and arsenate, and capture of the metalloid by biogenic Fe(II).

Previous work has shown that microbial communities in As-mobilizing sediments from West Bengal were dominated by Geobacter species. Thus, the potential of Geobacter sulfurreducens to mobilize arsenic via direct enzymatic reduction and indirect mechanisms linked to Fe(III) reduction was analyzed. G. sulfurreducens was unable to conserve energy for growth via the dissimilatory reduction of As(V), although it was able to grow in medium containing fumarate as the terminal electron acceptor in the presence of 500 muM As(V). There was also no evidence of As(III) in culture supernatants, suggesting that resistance to 500 muM As(V) was not mediated by a classical arsenic resistance operon, which would rely on the intracellular reduction of As(V) and the efflux of As(III). When the cells were grown using soluble Fe(III) as an electron acceptor in the presence of As(V), the Fe(II)-bearing mineral vivianite was formed. This was accompanied by the removal of As, predominantly as As(V), from solution. Biogenic siderite (ferrous carbonate) was also able to remove As from solution. When the organism was grown using insoluble ferrihydrite as an electron acceptor, Fe(III) reduction resulted in the formation of magnetite, again accompanied by the nearly quantitative sorption of As(V). These results demonstrate that G. sulfurreducens, a model Fe(III)-reducing bacterium, did not reduce As(V) enzymatically, despite the apparent genetic potential to mediate this transformation. However, the reduction of Fe(III) led to the formation of Fe(II)-bearing phases that are able to capture arsenic species and could act as sinks for arsenic in sediments.

Ligand arsenic complexation and immunoperoxidase detection of metallothionein in the earthworm Lumbricus rubellus inhabiting arsenic-rich soil.

Although earthworms have been found to inhabit arsenic-rich soils in the U.K., the mode of arsenic detoxification is currently unknown. Biochemical analyses and subcellular localization studies have indicated that As3+-thiol complexes may be involved; however, it is not known whether arsenic is capable of inducing the expression of metallothionein (MT) in earthworms. The specific aims of this paper were (a) to detect and gain an atomic characterization of ligand complexing by X-ray absorption spectrometry (XAS), and (b) to employ a polyclonal antibody raised against an earthworm MT isoform (w-MT2) to detect and localize the metalloprotein by immunoperoxidase histochemistry in the tissues of earthworms sampled from arsenic-rich soil. Data suggested that the proportion of arsenate to sulfur-bound species varies within specific earthworm tissues. Although some arsenic appeared to be in the form of arsenobetaine, the arsenic within the chlorogogenous tissue was predominantly coordinated with S in the form of -SH groups. This suggests the presence of an As::MT complex. Indeed, MT was detectable with a distinctly localized tissue and cellular distribution. While MT was not detectable in the surface epithelium or in the body wall musculature, immunoperoxidase histochemistry identified the presence of MT in chloragocytes around blood vessels, within the typhlosolar fold, and in the peri-intestinal region. Focal immunostaining was also detectable in a cohort of cells in the intestinal wall. The results of this study support the hypothesis that arsenic induces MT expression and is sequestered by the metalloprotein in certain target cells and tissues.

Role of oxalic acid overexcretion in transformations of toxic metal minerals by Beauveria caledonica.

The fungus Beauveria caledonica was highly tolerant to toxic metals and solubilized cadmium, copper, lead, and zinc minerals, converting them into oxalates. This fungus was found to overexcrete organic acids with strong metal-chelating properties (oxalic and citric acids), suggesting that a ligand-promoted mechanism was the main mechanism of mineral dissolution. Our data also suggested that oxalic acid was the main mineral-transforming agent. Cadmium, copper, and zinc oxalates were precipitated by the fungus in the local environment and also in association with the mycelium. The presence of toxic metal minerals often led to the formation of mycelial cords, and in the presence of copper-containing minerals, these cords exhibited enhanced excretion of oxalic acid, which resulted in considerable encrustation of the cords by copper oxalate hydrate (moolooite). It was found that B. caledonica hyphae and cords were covered by a thick hydrated mucilaginous sheath which provided a microenvironment for chemical reactions, crystal deposition, and growth. Cryo-scanning electron microscopy revealed that mycogenic metal oxalates overgrew parental fungal hyphae, leaving a labyrinth of fungal tunnels within the newly formed mineral matter. X-ray absorption spectroscopy revealed that oxygen ligands played a major role in metal coordination within the fungal biomass during the accumulation of mobilized toxic metals by B. caledonica mycelium; these ligands were carboxylic groups in copper phosphate-containing medium and phosphate groups in pyromorphite-containing medium.

Solution chemistry of uranyl ion with iminodiacetate and oxydiacetate: A combined NMR/EXAFS and potentiometry/calorimetry study.

The solution chemistry of uranyl ion with iminodiacetate (IDA) and oxydiacetate (ODA) was investigated using NMR and EXAFS spectroscopies, potentiometry, and calorimetry. From the NMR and EXAFS data and depending on stoichiometry and pH, three types of metal:ligand complex were identified in solution in the pH range 3-7: 1:1 and 1:2 monomers; a 2:2 dimer. From NMR and EXAFS data for the IDA system and previous studies, we propose the three complex types are [UO(2)(IDA)(H(2)O)(2)], [UO(2)(IDA)(2)](2)(-), and [(UO(2))(2)(IDA)(2)(mu-OH)(2)](2)(-). From EXAFS spectroscopy, similar 1:1, 2:2, and 1:2 complexes are found for the ODA system, although (13)C NMR spectroscopy was not a useful probe in this system. For the 1:1 and 1:2 complexes in solution, EXAFS spectroscopy is ambiguous because the data can be fitted with either a long U-N/O(ether) value (ca. 2.9 A) suggesting 1,7-coordination of the ligand or a U-C interaction at a similar distance, consistent with terminal bidentate coordination. However, the NMR data of the IDA system suggest that 1,7-coordination is the more likely. The stability constants of the three complexes were determined by potentiometric titrations; the log beta values are 9.90 +/-, 16.42 +/-, and 10.80 +/- for the 1:1, 1:2, and 2:2 uranyl-IDA complexes, respectively, and 5.77 +/-, 7.84 +/-, and 4.29 +/- for the 1:1, 1:2, and 2:2 uranyl-ODA complexes, respectively. The thermodynamic constants for the complexes were calculated from calorimetric titrations; the enthalpy changes (kJ mol(-)(1)) and entropy changes (J K(-)(1) mol(-)(1)) of complexation for the 1:1, 1:2, and 2:2 complexes respectively are the following. IDA: 12 +/- 2, 230 +/- 8; 8 +/- 2, 151 +/- 9; -33 +/- 3, -283 +/- 11. ODA: 26 +/- 2, 198 +/- 12; 20 +/- 2, 106 +/- 8; -24 +/- 2; -219 +/- 8.

Synthesis and characterization of uranyl compounds with iminodiacetate and oxydiacetate displaying variable denticity.

Eight uranyl compounds containing the dicarboxylate ligands iminodiacetate (IDA) or oxydiacetate (ODA) have been characterized in the solid state. The published polymeric structures for [UO(2)(C(4)H(6)NO(4))(2)] and [UO(2)(C(4)H(4)O(5))](n) have been confirmed, while Ba[UO(2)(C(4)H(5)NO(4))(2)] x 3H(2)O, [(CH(3))(2)NH(CH(2))(2)NH(CH(3))(2)][UO(2)(C(4)H(4)O(5))(2)] [orthorhombic space group Pnma, a = 10.996(5) A, b = 21.42(1) A, c = 8.700(3) A, Z = 4], and [C(2)H(5)NH(2)(CH(2))(2)NH(2)C(2)H(5)][UO(2)(C(4)H(4)O(5))(2)] [monoclinic space group P2(1)/n, a = 6.857(3) A, b = 9.209(5) A, c = 16.410(7) A, beta = 91.69(3), Z = 2] contain monomeric anions. The distance from the uranium atom to the central heteroatom (O or N) in the ligand varies. Crystallographic study shows that U-heteroatom (O/N) distances fall into two groups, one 2.6-2.7 A in length and one 3.1-3.2 A, the latter implying no bonding interaction. By contrast, EXAFS analysis of bulk samples suggests that either a long U-heteroatom (O/N) distance (2.9 A) or a range of distances may be present. Three possible structural types, two symmetric and one asymmetric, are identified on the basis of these results and on solid-state (13)C NMR spectroscopy. The two ligands in the complex can be 1,4,7-tridentate, giving five-membered rings, or 1,7-bidentate, to form an eight-membered ring. (C(4)H(12)N(2))[(UO(2))(2)(C(4)H(5)NO(4))(2)(OH)(2)] x 8H(2)O [monoclinic space group P2(1)/a, a = 7.955(9) A, b = 24.050(8) A, c = 8.223(6) A, beta = 112.24(6), Z = 2], (C(2)H(10)N(2))[(UO(2))(2)(C(4)H(5)NO(4))(2)(OH)(2)] x 4H(2)O, and (C(6)H(13)N(4))(2)[(UO(2))(2)(C(4)H(4)O(5))(2)(OH)(2)] x 2H(2)O [monoclinic space group C2/m, a = 19.024(9) A, b = 7.462(4) A, c = 2.467(6) A, beta = 107.75(4), Z = 4] have a dimeric structure with two capping tridentate ligands and two mu(2)-hydroxo bridges, giving edge-sharing pentagonal bipyramids.

Structural and functional characterization of the Zn(II) site in dimethylargininase-1 (DDAH-1) from bovine brain. Zn(II) release activates DDAH-1.

L-N(omega),N(omega)-dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is a Zn(II)-containing enzyme that, through hydrolysis of side-chain methylated l-arginines, regulates the activity of nitric-oxide synthase. Herein we report the structural and functional properties of the Zn(II)-binding site in DDAH-1 from bovine brain. Activity measurements of the native and metal-free enzyme have revealed that the endogenously bound Zn(II) inhibits the enzyme. Native DDAH-1 could be fully or partially activated using various concentrations of phosphate, imidazole, histidine, and histamine, a process that is paralleled by the release of Zn(II). The slow activation of the enzyme by the bulky complexing agents EDTA and 1,10-phenantroline suggests that the Zn(II)-binding site is partially buried in the protein structure. The apparent Zn(II)-dissociation constant of 4.2 nm, determined by 19F NMR using the chelator 5F-BAPTA (1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid), lies in the range of intracellular free Zn(II) concentrations. These results suggest a regulatory role for the Zn(II)-binding site. The coordination environment of the Zn(II) in DDAH-1 has been examined by Zn K-edge x-ray absorption spectroscopy. The extended x-ray absorption fine structure observed is consistent with Zn(II) being coordinated by 2 S and 2 N (or O) atoms. The biological implications of these findings are discussed.

Thiocyanate binding to the molybdenum centre of the periplasmic nitrate reductase from Paracoccus pantotrophus.

The periplasmic nitrate reductase (NAP) from Paracoccus pantotrophus is a soluble two-subunit enzyme (NapAB) that binds two haem groups, a [4Fe-4S] cluster and a bis(molybdopterin guanine dinucleotide) (MGD) cofactor that catalyses the reduction of nitrate to nitrite. In the present study the effect of KSCN (potassium thiocyanate) as an inhibitor and Mo ligand has been investigated. Results are presented that show NAP is sensitive to SCN(-) (thiocyanate) inhibition, with SCN(-) acting as a competitive inhibitor of nitrate (K(i) approximately 4.0 mM). The formation of a novel EPR Mo(V) species with an elevated g(av) value (g(av) approximately 1.994) compared to the Mo(V) High-g (resting) species was observed upon redox cycling in the presence of SCN(-). Mo K-edge EXAFS analysis of the dithionite-reduced NAP was best fitted as a mono-oxo Mo(IV) species with three Mo-S ligands at 2.35 A (1 A=0.1 nm) and a Mo-O ligand at 2.14 A. The addition of SCN(-) to the reduced Mo(IV) NAP generated a sample that was best fitted as a mono-oxo (1.70 A) Mo(IV) species with four Mo-S ligands at 2.34 A. Taken together, the competitive nature of SCN(-) inhibition of periplasmic nitrate reductase activity, the elevated Mo(V) EPR g(av) value following redox cycling in the presence of SCN(-) and the increase in sulphur co-ordination of Mo(IV) upon SCN(-) binding, provide strong evidence for the direct binding of SCN(-) via a sulphur atom to Mo.