Binding Sites For Cu Research Articles

EPR study of a copper impurity center in a single crystal of 6-chlorouracil

Single crystals of 6-chlorouracil containing traces of copper were gamma-irradiated at room temperature and studied using EPR spectroscopy. The ionizing radiation induced the conversion of copper impurities, initially present in the host lattice as Copper(I) complexes, into paramagnetic copper(II) complexes. These complexes exhibited intricate EPR spectra characteristic of Copper(II) complex with two ligated nitrogen atoms. Using EPR spectroscopy, the binding site of Cu(II) ions in a single crystal of 6-chlorouracil was determined. It was proposed that planar coordination bonding to the cupric Cu(II) ion, having two pairs of oxygen and nitrogen atoms as ligands, served to bridge neighboring 6-chlorouracil molecules, thereby replacing a pair of hydrogen bonds with stronger Cu-coordination bonding. The spectroscopic parameters, including g tensor, A(63Cu), and A(14N) tensors, closely resemble those previously reported for copper(II) planar centers in similar systems. However, a small deviation in the principal axes of the g and A(63Cu) tensors from axial symmetry suggests a subtle distortion from the ideal planar geometry of the complex. The theoretically derived spectroscopic parameters further corroborate the proposed structure of the complex.

Stabilization of a Cu-binding site by a highly conserved tryptophan residue

Copper serves as an essential cofactor for nearly all living organisms. There are still many gaps remaining in our knowledge of how Gram-positive bacteria import copper and maintain homeostasis. To obtain a better understanding of how these processes work, here we focus on the ycnKJI operon responsible for regulating copper levels in the Gram-positive bacterium Bacillus subtilis. This operon encodes three Cu-related proteins: a copper-dependent transcriptional repressor (YcnK), a putative copper importer (YcnJ), and a copper-binding protein of unknown function (YcnI). We previously found that YcnI's extracellular Domain of Unknown Function 1775 (DUF1775) houses a monohistidine brace motif that coordinates a single Cu(II) ion. The Cu(II) binding site includes a highly conserved tryptophan residue. Here, we investigate the role of that tryptophan and the ability of the protein to interact with other oxidation states of Cu. We find that YcnI exhibits strong preference for binding Cu in the oxidized Cu(II) state, and that the conserved tryptophan residue is not essential for the interaction. We determine the structure of a tryptophan variant to 1.95 Å resolution that indicates that the tryptophan is needed to stabilize the metal binding interaction, and find that this variant has weaker affinity for Cu(II) than the wild-type protein. Together, these data provide further insights into the DUF1775 domain family and reveal the role of the conserved tryptophan residue.

Human serum albumin as a copper source for anticancer thiosemicarbazones.

Thiosemicarbazones (TSCs) are a class of biologically active compounds with promising anticancer activity. Their typical mechanism, especially of the clinically far developed representative Triapine, is chelation of iron (Fe), with the Fe-containing enzyme ribonucleotide reductase as primary intracellular target. However, for the subclass of terminally disubstituted, nanomolar-active derivatives like Dp44mT and Me2NNMe2, recent findings suggest that the chelation, stability, and reduction properties of the copper(II) (Cu) complexes are essential for their modes of action. Consequently, it is important to elucidate whether blood serum Cu(II) is a potential metal source for these TSCs. To gain more insights, the interaction of Triapine, Dp44mT or Me2NNMe2 with purified human serum albumin (HSA) as the main pool of labile Cu(II) was investigated by UV-vis and electron paramagnetic resonance measurements. Subsequently, a size-exclusion chromatography inductively coupled plasma mass spectrometry method for the differentiation of Cu species in serum was developed, especially separating the non-labile Cu enzyme ceruloplasmin from HSA. The results indicate that the TSCs specifically chelate copper from the N-terminal Cu-binding site of HSA. Furthermore, the Cu(II)-TSC complexes were shown to form ternary HSA conjugates, most likely via histidine. Noteworthy, Fe-chelation from transferrin was not overserved, even not for Triapine. In summary, the labile Cu pool of HSA is a potential source for Cu-TSC complex formation and, consequently, distinctly influences the anticancer activity and pharmacological behavior of TSCs.

Revisit the E2 Domain of Amyloid Precursor Protein: Ferroxidase, Superoxide and Peroxynitrite Scavenging Activities.

Amyloid precursor protein (APP) is the biological precursor of β-amyloids, a known histopathological hallmark associated with Alzheimer's disease (AD). The function of APP is of great interest yet remains elusive. One of the extracellular domains of APP, the E2 domain, has been proposed to possess ferroxidase activity and affect neuronal iron homeostasis. However, contradicting evidence has been reported, and its precise role remains inconclusive. Here, we studied the Cu-binding site of the E2 domain using extended X-ray absorption fine structure (EXAFS), UV-vis, and electron paramagnetic resonance (EPR) and discovered that a new labile water ligand coordinates to the Cu(II) cofactor in addition to the four known histidines. We explored the proposed ferroxidase activity of the Cu(II)-E2 domain through reactions with ferrous iron and observed single-turnover ferrous oxidation activity with a rate up to 1.0 × 102 M-1 s-1. Cu(I)-E2 reacted with molecular oxygen at a rate of only 5.3 M-1 s-1, which would restrict any potential multiturnover ferroxidase activity to this slow rate and prevents observation of activity under multiturnover conditions. The positive electrostatic potential surface of the protein indicates possible reactivity with negatively charged small substrates such as superoxide radicals (O2•-) and peroxynitrite (ONOO-) that are major contributors to the oxidative stress prevalent in the extracellular environment. Our assays showed that Cu(I)-E2 can remove O2•- at a rate of 1.6 × 105 M-1 s-1, which is slower than the rates of native SODs. However, the reaction between Cu(I)-E2 and ONOO- achieved a rate of 1.1 × 105 M-1 s-1, comparable to native ONOO- scavenger peroxiredoxins (105-107 M-1 s-1). Therefore, the E2 domain of APP can serve as an enzymatic site that may function as a ferroxidase under substrate-limiting conditions, a supplemental O2•- scavenger, and an ONOO- remover in the vicinity of the cellular iron efflux channel and protect neuron cells from reactive oxygen species (ROS) and reactive nitrogen species (RNS) damage.

Differences in the spectral characteristics of dissolved organic matter binding to Cu(II) in wetland soils with moisture gradients

The environmental behavior of heavy metals in soil is significantly regulated by their binding with dissolved organic matter (DOM), which is affected by soil moisture contents. However, the mechanism of this interaction in soils with varying moisture is still not well understood. Using a combination of ultrafiltration, Cu(II) titration, and multispectral (ultraviolet-visible absorption, 3D fluorescence, Fourier transform infrared) analysis techniques, we studied the differences in the spectral characteristics and Cu(II) binding properties of soil dissolved organic matter (DOM) and its different molecular weight (MW) fractions with moisture gradients. We found that the abundance and spectral characters of soil DOM changed with increasing soil moisture, i.e., the increase in abundance while the decrease in aromaticity and humification index. The components of DOM, shown by Fluorescence region-integration (FRI) analysis, also changed, with an increase in the proportion of protein-like substances and a decrease of humic-like and fulvic-like substances. The overall Cu(II) binding potential of soil DOM diminished with increasing soil moisture, as indicated by the fluorescence parallel factor (PARAFAC) analysis. This is aligns with the changes in DOM composition, as the humic-like and fulvic-like fractions exhibited higher Cu(II) binding potential compared to the protein-like fractions. The low MW fraction of the MW-fractionated samples showed a stronger binding potential for Cu(II) compared to the high MW fraction. Finally, the active binding site of Cu(II) in DOM, as revealed by UV-difference spectroscopy and 2D-FTIR-COS analysis, decreased with increasing soil moisture, with the order of preferentially functional groups shifting from OH, NH, and CO to CN and CO. This study emphasizes the impact of moisture variations on the characteristics of DOM and its interaction with Cu(II), providing insight into the environmental fate of heavy metal contaminants in soil in areas with alternating land and water conditions.

Identification and Analysis of Fungal-Specific Regions in the Aspergillus fumigatus Cu Exporter CrpA That Are Essential for Cu Resistance but Not for Virulence.

The opportunistic fungus Aspergillus fumigatus is the primary invasive mold pathogen in humans, and is responsible for an estimated 200,000 yearly deaths worldwide. Most fatalities occur in immunocompromised patients who lack the cellular and humoral defenses necessary to halt the pathogen's advance, primarily in the lungs. One of the cellular responses used by macrophages to counteract fungal infection is the accumulation of high phagolysosomal Cu levels to destroy ingested pathogens. A. fumigatus responds by activating high expression levels of crpA, which encodes a Cu+ P-type ATPase that actively transports excess Cu from the cytoplasm to the extracellular environment. In this study, we used a bioinformatics approach to identify two fungal-unique regions in CrpA that we studied by deletion/replacement, subcellular localization, Cu sensitivity in vitro, killing by mouse alveolar macrophages, and virulence in a mouse model of invasive pulmonary aspergillosis. Deletion of CrpA fungal-unique amino acids 1-211 containing two N-terminal Cu-binding sites, moderately increased Cu-sensitivity but did not affect expression or localization to the endoplasmic reticulum (ER) and cell surface. Replacement of CrpA fungal-unique amino acids 542-556 consisting of an intracellular loop between the second and third transmembrane helices resulted in ER retention of the protein and strongly increased Cu-sensitivity. Deleting CrpA N-terminal amino acids 1-211 or replacing amino acids 542-556 also increased sensitivity to killing by mouse alveolar macrophages. Surprisingly, the two mutations did not affect virulence in a mouse model of infection, suggesting that even weak Cu-efflux activity by mutated CrpA preserves fungal virulence.

An Amine-Functionalized Ultramicroporous Metal–Organic Framework for Postcombustion CO2 Capture

Among the most promising methods by which to capture CO2 from flue gas, the emission of which has accelerated global warming, is energy-efficient physisorption using metal-organic framework (MOF) adsorbents. Here, we present a novel cuprous-based ultramicroporous MOF, Cu(adci)-2 (adci- = 2-amino-4,5-dicyanoimidazolate), which was rationally synthesized by combining two strategies to design MOF physisorbents for enhanced CO2 capturing, i.e., aromatic amine functionalization and the introduction of ultramicroporosity (pore size <7 Å). Synchrotron powder X-ray diffraction and a Rietveld analysis reveal that the Cu(adci)-2 structure has one-dimensional square-shaped channels, in each of which all affiliated ligands, specifically NH2 groups at the 2-position of the imidazolate ring, have the same orientation, with a pair of NH2 groups therefore facing each other on opposite sides of the channel walls. While Cu(adci)-2 exhibits a high CO2 adsorption capacity (2.01 mmol g-1 at 298 K and 15 kPa) but a low zero-coverage isosteric heat of adsorption (27.5 kJ mol-1), breakthrough experiments under dry and 60% relative humidity conditions show that its CO2 capture ability is retained even in the presence of high amounts of moisture. In a Monte Carlo simulation and a radial distribution analysis, the preferential CO2 binding site of Cu(adci)-2 was predicted to be between two ligands, forming a sandwich-like structure and implying that its CO2 adsorption properties originate from the enhancement of Lewis base-acid and London dispersion interactions due to the amino groups and ultramicroporosity, respectively.

Azo-hydrazone tautomerism in a simple coumarin azo dye and its contribution to the naked-eye detection of Cu2+ and other potential applications

A new tailor-made azo dye of coumarin connected to phenolic derivative is presented herein. Azo-hydrazone tautomerism in aqueous solution of the dye was observed and studied using spectroscopic assays such as 1H NMR, absorption and emission assays, and theoretical studies. Tautomerism was attributed to the presence of a labile phenolic hydrogen in the ortho position to the azo functionality and the hydrazone was found to be the more dominant tautomer. Influence of metal ions on the azo-hydrazone chemical equilibrium and how the accompanying colour and spectroscopic changes can be exploited for various functions, especially the detection and quantification of Cu2+ in aqueous environments was explored. The presence of Cu2+ affects the azo-hydrazone equilibrium resulting in visual appearance and spectroscopic changes and the likely binding sites for Cu2+ were evaluated. Cu2+ pushes the azo-hydrazone equilibrium towards the more conjugated form and the presence of other metal ions does not have any perceivable impact on this mechanism. The dye showed potential applications as a sensor in colorimetric and spectroscopic detection and quantification of Cu2+ in domestic and environmental water samples, photo-imprinting and as a logic gate. The limits of detection (LOD) and quantification (LOQ) for Cu2+ were found to be 0.0779 mg/L and 0.236 mg/L, respectively, much lower than the World Health Organization (WHO) guideline limit for Cu2+ levels in drinking water.

Copper Binding and Oligomerization Studies of the Metal Resistance Determinant CrdA from Helicobacter pylori.

Within this research, the CrdA protein from Helicobacter pylori (HpCrdA), a putative copper-binding protein important for the survival of bacterium, was biophysically characterized in a solution, and its binding affinity toward copper was experimentally determined. Incubation of HpCrdA with Cu(II) ions favors the formation of the monomeric species in the solution. The modeled HpCrdA structure shows a conserved methionine-rich region, a potential binding site for Cu(I), as in the structures of similar copper-binding proteins, CopC and PcoC, from Pseudomonas syringae and from Escherichia coli, respectively. Within the conserved amino acid motif, HpCrdA contains two additional methionines and two glutamic acid residues (MMXEMPGMXXMXEM) in comparison to CopC and PcoC but lacks the canonical Cu(II) binding site (two His) since the sequence has no His residues. The methionine-rich site is in a flexible loop and can adopt different geometries for the two copper oxidation states. It could bind copper in both oxidation states (I and II), but with different binding affinities, micromolar was found for Cu(II), and less than nanomolar is proposed for Cu(I). Considering that CrdA is a periplasmic protein involved in chaperoning copper export and delivery in the H. pylori cell and that the affinity of the interaction corresponds to a middle or strong metal–protein interaction depending on the copper oxidation state, we conclude that the interaction also occurs in vivo and is physiologically relevant for H. pylori.

H101G Mutation in Rat Lens αB-Crystallin Alters Chaperone Activity and Divalent Metal Ion Binding.

The molecular chaperone function of αB-crystallins is heavily involved in maintaining lens transparency and the development of cataracts. The aim of the study was to investigate whether divalent metal ion binding improves the stability and αB-crystallin chaperone activity. In this study, we have developed an H101G αB-crystallin mutant and compared the surface hydrophobicity, chaperone activity, and secondary and tertiary structure with the wild type in the presence and absence of metal ions. Substitution of His101 with glycine resulted in structural and functional changes. Spectral analysis and chaperone-like activity assays showed that substitution of glycine resulted in a higher percentage of random coils, increased hydrophobicity, and 22±2% higher chaperone-like activity. Whereas in the presence of the Cu2+ ion, H101G exhibited 32±1% less chaperone-like activity compared to the wild type. Cu2+ has been reported to enhance the chaperone-like activity of lens α-crystallin. Our results indicate that H101 is the predominant Cu2+ binding site, and the mutation resulted in a partial unfolding that impaired the binding of Cu2+ to H101 residue. In conclusion, this study further helps to understand the important binding site for Cu2+ to αB-crystallin.

Serum levels of copper and zinc in diabetic retinopathy: Potential new therapeutic targets (Review).

Diabetic retinopathy (DR) is a microvascular disorder occurring due to the long-term effects of diabetes, leading to vision-threatening damage to the retina. The human body has an elaborate system for managing and regulating the number of key trace metals circulating in the blood and stored cells. Inadequate zinc (Zn) and concurrent excess of copper (Cu) levels are associated with an increased level of oxidative stress, which may aggravate the microvascular lesions in diabetes mellitus. Several studies have revealed a significantly lower serum Zn concentration and increased Cu levels in DR when compared with diabetic patients without retinopathy and normal controls. These abnormalities are correlated with the duration of diabetes and higher levels of HbA1C. Multiple pathological mechanisms are proposed to explain these changes including hyperzincuria associated with polyuria, glycosuria, and proteinuria in diabetic patients, as well as impaired absorption of Zn at the gastrointestinal level. Increased levels of free Cu ions may be attributed to glycation and the release of Cu ions from the Cu-binding sites of proteins. Zn supplements and selective Cu chelators may be useful to alleviate oxidative stress and prevent DR progression.

Amyloid β Perturbs Cu(II) Binding to the Prion Protein in a Site-Specific Manner: Insights into Its Potential Neurotoxic Mechanisms.

Amyloid β (Aβ) is a Cu-binding peptide that plays a key role in the pathology of Alzheimer's disease. A recent report demonstrated that Aβ disrupts the Cu-dependent interaction between cellular prion protein (PrPC) and N-methyl-d-aspartate receptor (NMDAR), inducing overactivation of NMDAR and neurotoxicity. In this context, it has been proposed that Aβ competes for Cu with PrPC; however, there is no spectroscopic evidence to support this hypothesis. Prion protein (PrP) can bind up to six Cu(II) ions: from one to four at the octarepeat (OR) region, producing low- and high-occupancy modes, and two at the His96 and His111 sites. Additionally, PrPC is cleaved by α-secretases at Lys110/His111, yielding a new Cu(II)-binding site at the α-cleaved His111. In this study, the competition for Cu(II) between Aβ(1-16) and peptide models for each Cu-binding site of PrP was evaluated using circular dichroism and electron paramagnetic resonance. Our results show that the impact of Aβ(1-16) on Cu(II) coordination to PrP is highly site-specific: Aβ(1-16) cannot effectively compete with the low-occupancy mode at the OR region, whereas it partially removes the metal ion from the high-occupancy modes and forms a ternary OR-Cu(II)-Aβ(1-16) complex. In contrast, Aβ(1-16) removes all Cu(II) ions from the His96 and His111 sites without formation of ternary species. Finally, at the α-cleaved His111 site, Aβ(1-16) yields at least two different ternary complexes depending on the ratio of PrP/Cu(II)/Aβ. Altogether, our spectroscopic results indicate that only the low-occupancy mode at the OR region resists the effect of Aβ, while Cu(II) coordination to the high-occupancy modes and all other tested sites of PrP is perturbed, by either removal of the metal ion or formation of ternary complexes. These results provide important insights into the intricate effect of Aβ on Cu(II) binding to PrP and the potential neurotoxic mechanisms through which Aβ might affect Cu-dependent functions of PrPC, such as NMDAR modulation.

The N-terminal domain of Helicobacter pylori's Hpn protein: The role of multiple histidine residues

Helicobacter pylori is a gram-negative bacterium with gastric localization that can cause many gastrointestinal disorders. Its survival in the host environment strictly requires an efficient regulation of its metal homeostasis, in particular of Ni(II) ions, crucial for the synthesis of some essential enzymes. Hpn is a protein of 60 amino acids, 47% of which are histidines, expressed by H. pylori and avid for nickel, characterized by the presence of an ATCUN (Amino Terminal Cu(II)- and Ni(II)-binding) motif and by two further histidine residues which can act as additional metal anchoring sites. We decided to deepen the following aspects: (i) understanding the role of each histidine in the coordination of metal ions; (ii) comparing the binding affinities for Cu(II), Ni(II) and Zn(II) ions, which are potentially competing metals in vivo; (iii) understanding the Hpn ability of forming ternary and poly-nuclear complexes. For these purposes, we synthesized the Hpn N-terminal “wild-type” sequence (MAHHEEQHG-Am) and the following peptide analogues: MAAHEEQHG-Am, MAHAEEQHG-Am, MAHHEEQAG-Am and MAHAEEQAG-Am. Our results highlight that the histidines in position 4 and 8 lead to the formation of Cu(II) binuclear complexes. The ATCUN motif is by far the most efficient binding site for Cu(II) and Ni(II), while macrochelate Zn(II) complexes are formed thanks to the presence of several suitable anchoring sites (His and Glu). The metal binding affinities follow the order Zn(II) < Ni(II) < < Cu(II). In solutions containing equimolar amount of wild-type ligand, Cu(II) and Ni(II), the major species above pH 5.5 are hetero-binuclear complexes.

A new asymmetric salamo-based chemical sensor for dual channel detection of Cu2+ and B4O72−

A new type of asymmetric salamo-based colorimetric and fluorescent chemical sensor H2L was synthesized and characterized. The chemical sensor H2L has an efficient dual channel for Cu2+ and B4O72− under the solvent conditions of DMF : H2O = 9 : 1 identify the effect. The oxime nitrogen and phenolic oxygen atoms in the chemical sensor H2L can be used as the binding sites for Cu2+. Further DFT theoretical calculations confirmed that the chemical sensor H2L can combine with Cu2+ to form a 1 : 1 complex, which in turn leads to quenching of fluorescence. Meanwhile, through ESP analysis, the N and O atoms in the chemical sensor H2L have an electron donor capability, due to the introduction of B4O72−, the density of the electron cloud around the N and O atoms was changed, prompting the fluorophore of the chemical sensor H2L to produce stronger fluorescence. The minimum detection limit of chemical sensor H2L for Cu2+ is 1.99 × 10−7 M, and the minimum detection limit for B4O72− is 7.57 × 10−7 M. In addition, the chemical sensor H2L was also used to detect the Cu2+ content in different water samples in life.

Comparison of copper binding properties of DOM derived from fresh and pyrolyzed biomaterials: Insights from multi-spectroscopic investigation

The binding of dissolved organic matter (DOM) with metals affects the latter's biogeochemical processing in the environment. This study used multi-spectroscopic analyses to compare the heterogeneities of the Cu(II) binding properties of DOM derived from fresh and pyrolyzed biomaterials. The results showed that the DOM derived from fresh macrophyte (MDOM) and their corresponding biochar (BDOM) consisted mostly of protein-like and humic-like substances, respectively. The stability constant (log KM) of protein-like matter in the MDOM was 5.27, and the values of humic-like components in the BDOM were 4.32–5.15. Compared with the MDOM, the BDOM exhibited lower affinities and active binding sites for Cu(II). In addition, the BDOM contents decreased after pyrolysis. Therefore, the pyrolysis of fresh biomaterials into biochar is a promising method for reducing the potential migration risk posed by Cu(II) due to the MDOM being a positive carrier for Cu(II) contamination. Polysaccharide was the only functional group that participated in the binding of Cu(II) in both MDOM and BDOM. Aliphatic groups and amides associated with protein-like matter were responsible for the Cu(II) binding to MDOM, whereas phenolic and aromatic groups mainly participated in the complexation of BDOM-Cu(II). The CO group of amide I in the MDOM, and polysaccharide in the BDOM, showed the fastest response to Cu(II). This study was helpful for elucidating the effects of fresh and pyrolyzed biomaterials (biochars) on the environmental behavior of Cu(II) at the molecular level.

Enhancement of Oxidative Reaction by the Intramolecular Electron Transfer between the Coordinated Redox-Active Metal Ions in SOD1

The denatured Cu, Zn superoxide dismutase (SOD1) has the pro-oxidant activity that is suggested to be related with the pathogenesis of amyotrophic lateral sclerosis (ALS). We showed from the changes in the coordinated metal ions that the Cu ion in the Cu-binding site is the catalytic site of the pro-oxidant activity, and a redox-active metal ion in the Zn-binding site has the auxiliary function to enhance the pro-oxidant activity. The auxiliary function is suggested to arise from the intramolecular electron transfer between the coordinated metal ions in the denatured SOD1. The oxidation/reduction cycle of Cu in the Cu-binding site is assisted with changing the oxidation state of a metal ion in the Zn-binding site. The magnitude of the toxicity of the denatured SOD1 is discussed based on the ability of the auxiliary function.

Influence of nitrogen forms and application rates on the phytoextraction of copper by castor bean (Ricinus communis L.).

Fertilization is an important agricultural strategy for enhancing the efficiency of phytoremediation in copper (Cu)-contaminated soils. In this study, the effects of nitrogen (N) forms, including ammonium (NH4+-N) and nitrate (NO3--N), on the growth, translocation, and accumulation of Cu in the tissues of Ricinus communis L. were investigated in pot and hydroponic experiments. The results demonstrated that higher biomass and N contents in plants were obtained when N was supplied as NO3--N rather than NH4+-N. Application of N increased the Cu content in the roots of R. communis, with a higher content after NH4+-N (53.10-64.20 mg kg-1) than NO3--N (37.62-53.75 mg kg-1) treatment. On the contrary, the levels of Cu translocation factors were much higher in NO3--fed plants (0.34-0.45) than in NH4+-fed plants (0.28-0.38). The suggested amount of N for fertilizer application is 225 kg hm-2, which resulted in the highest Cu content in R. communis and optimal plant growth. As the main Cu-binding site, root cell walls accumulated less Cu in plants treated with NH4+-N compared with NO3--N. Additionally, NH4+-N induced a higher malondialdehyde content and more severe root damage compared with NO3--N. In the leaf, a larger number of black granules, which could be protein and starch grains involved in the detoxification of Cu in R. communis, were present after NH4+-N than NO3--N treatment. These results illustrate that N forms are especially important for Cu translocation and accumulation and that immobilization and transformation of Cu in roots were improved more by NH4+-N than NO3--N. In conclusion, N fertilizers containing the appropriate forms applied at suitable rates may enhance the biomass and Cu accumulation of R. communis and thereby the remediation efficiency of Cu-contaminated soils.

Contributions of root cell wall polysaccharides to Cu sequestration in castor (Ricinus communis L.) exposed to different Cu stresses

Cell wall polysaccharides play a vital role in binding with toxic metals such as copper (Cu) ions. However, it is still unclear whether the major binding site of Cu in the cell wall varies with different degrees of Cu stresses. Moreover, the contribution of each cell wall polysaccharide fraction to Cu sequestration with different degrees of Cu stresses also remains to be verified. The distribution of Cu in cell wall polysaccharide fractions of castor (Ricinus communis L.) root was investigated with various Cu concentrations in the hydroponic experiment. The results showed that the hemicellulose1 (HC1) fraction fixed 44.9%–67.8% of the total cell wall Cu under Cu stress. In addition, the pectin fraction and hemicelluloses2 (HC2) fraction also contributed to the Cu binding in root cell wall, accounting for 11.0%–25.9% and 14.1%–26.6% of the total cell wall Cu under Cu treatments, respectively. When the Cu levels were ≤25 μmol/L, pectin and HC2 contributed equally to Cu storage in root cell wall. However, when the Cu level was higher than 25 μmol/L, the ability of the pectin to bind Cu was easy to reach saturation. Much more Cu ions were bound on HC1 and HC2 fractions, and the HC2 played a much more important role in Cu binding than pectin. Combining fourier transform infrared (FT-IR) and two-dimensional correlation analysis (2D-COS) techniques, the hemicellulose components were showed not only to accumulate most of Cu in cell wall, but also respond fastest to Cu stress.

Dissolved organic matter characteristics of urban stormwater runoff from different functional regions during grassy swale treatment

Abstract In this study, the water purification performance of grassy swales for treating stormwater runoff was evaluated using a simulated experimental device. The binding properties of dissolved organic matter (DOM) in stormwater runoff samples from Beijing were measured by ultraviolet visible absorbance spectroscopy and excitation–emission matrices, combined with parallel factor analysis (PARAFAC). The concentrations of total nitrogen and phosphorus were significantly decreased after treatment by grassy swales: 50%–95.23% of total suspended solids and total organic carbon were removed, respectively. Additionally, the removal of heavy metal ions was 30.95%–97.39%. The spectral parameters indicated that humification and aromaticity of DOM would be greatly increased by grassy swale treatment. Two humic-like components and one protein-like component were identified in the runoff DOM using excitation–emission matrices coupled to PARAFAC. With progressive grassy swale treatment, the content of humic-like components increased and protein-like substances gradually decreased. Two-dimensional correlation spectroscopy demonstrated that the effluent DOM of grassy swale samples displayed different binding sites for Cu (II). Thus, the binding sequencing of Cu (II) with DOM fluorophores can be greatly influenced by grassy swale treatment. The results may provide a solid foundation for the treatment of urban runoff pollution.

Inter-species difference of copper accumulation in three species of marine mussels: Implication for biomonitoring.

Marine mussels have been used widely as biomonitors of coastal contamination in many countries. Due to the restrain of their geographical distributions, it is often necessary to employ more than one species of mussels within a large-scale biomonitoring program. In the present study, we compared the differences of copper (Cu) bioaccumulation in three species of marine mussels (green mussel Perna viridis, blue mussel Mytilus edulis, and hard-shelled mussel Mytilus coruscus) widely distributing along the Chinese coastal waters, under identical Cu exposure conditions. Over the 21-days exposure to dissolved Cu, the green mussels and blue mussels exhibited comparable newly accumulated Cu concentrations, possibly due to their comparable Cu uptake rate constant ku (blue mussel, 0.573 L g-1 d-1; green mussel, 0.530 L g-1 d-1) and efflux rate constant ke (blue mussel, 0.053 d-1; green mussel, 0.065 d-1). In contrast, there was no net Cu accumulation in the hard-shell mussels, which may be accounted by the lower ku (0.394 L g-1 d-1) but higher ke (0.081 d-1) than the other two mussel species. Further subcellular distribution analyses showed that the cellular debris and metallothionein-like protein (MTLP) fraction were the key binding sites for Cu, and the MTLP fraction may act as a main contributor in Cu regulation and elimination in the blue mussels and hard-shell mussels. There was no strong evidence that the subcellular partitioning and dynamics of Cu in the mussels were responsible for the difference underlying the Cu accumulation in the three species of mussels. Our comparative study thereby suggested that it may be feasible to directly compare the Cu bioavailability in the green mussels and blue mussels based on their Cu biomonitoring data. Cu biomonitoring data from the hard-shell mussels may underestimate the actual Cu bioavailability of the sampling area given its much stronger regulation of Cu bioaccumulation as compared to the other two mussel species.