Heavy Metal Binding Sites Research Articles

Ionic liquid catalyzed low-temperature hydrothermal carbonization of sewage sludge to produce hydrochar with low heavy metal content and positive energy recovery

An ionic liquid (IL, [DMAPA]HSO4) was prepared to facilitate the removal of heavy metals by hydrothermal carbonization (HTC) in sewage sludge (SS) and to obtain a positive energy recovery (ER, (Energyoutput/Energyinput − 1) > 0). The results found that the removal efficiencies of the Fe, Mn, Zn, Co, and Cd from SS exceeded 75 % with positive ER (6 %) at 20 wt% IL dosage (IL:SS). IL promoted the HTC reactions of proteins and polysaccharides to produce fixed carbon and small molecule polymers. The process mainly relies on IL to catalyze the dehydration and graphitization of SS and to destroy the heavy metal binding sites such as carboxyl and hydroxyl groups. Additionally, IL aids in constructing the macropore structures in hydrochar, thereby facilitating the release of heavy metals and water during the HTC process. This discovery holds promise for removing heavy metals from SS by one-pot HTC processes with positive energy recovery.

Molecular Cloning and Expression Analysis of the Typical Class III Chitinase Genes from Three Mangrove Species under Heavy Metal Stress.

Chitinases are considered to act as defense proteins when plants are exposed to heavy metal stresses. Typical class III chitinase genes were cloned from Kandelia obovate, Bruguiera gymnorrhiza, and Rhizophora stylosa by using RT-PCR and RACE and named KoCHI III, BgCHI III, and RsCHI III. Bioinformatics analysis revealed that the three genes encoding proteins were all typical class III chitinases with the characteristic catalytic structure belonging to the family GH18 and located outside the cell. In addition, there are heavy metal binding sites in the three-dimensional spatial structure of the type III chitinase gene. Phylogenetic tree analysis indicated that CHI had the closest relationship with chitinase in Rhizophora apiculata. In mangrove plants, the balance of the oxidative system in the body is disrupted under heavy metal stress, resulting in increased H2O2 content. Real-time PCR illustrated that the expression level under heavy metal stress was significantly higher than that in the control group. Expression levels of CHI III were higher in K. obovate than in B. gymnorrhiza and R. stylosa. With the increase in heavy metal stress time, the expression level increased continuously. These results suggest that chitinase plays an important role in improving the heavy metal tolerance of mangrove plants.

Cloning, Expression, and Bioinformatics Analysis of Heavy Metal Resistance Gene afe_1862 from Acidithiobacillus ferrooxidans L1 in Escherichia coli.

Molecular studies of copper and cadmium resistances in acidophilic bacteria are significant in biomining. In this study, afe_1862, which encodes a heavy metal-binding protein in Acidithiobacillus ferrooxidans L1, was amplified using PCR, cloned into the pET32a plasmid, and sequenced. Following SDS-PAGE analysis, optimization of the expression conditions and heterologous overexpression of afe_1862 in Escherichia coli BL21 in the presence of Cu2+ and Cd2+ were studied as well. The results indicated that AFE_1862 has higher resistance to Cu2+ than Cd2+. Bioinformatics analysis illustrated that AFE_1862 has a conserved HMA domain containing heavy metal-binding sites, which may play a role in transporting or detoxifying heavy metals.

Cadmium-induced conformational changes in type 2 metallothionein of medicinal plant Coptis japonica: insights from molecular dynamics studies of apo, partially and fully metalated forms

Plants play an important role in the removal of excess heavy metals from soil and water. Medicinal plants can also have non-traditional use in phytoremediation technologies. Among the heavy metals, Cadmium (Cd) is the most abundant and readily taken up by the crop plants. Plant metallothioneins (MTs) are small proteins having cysteine-rich residues and appear to play key roles in metal homoeostasis. Plant metallothionein 2 (MT 2) from Coptis japonica (Gold-thread; CjMT 2) is a typical member of this subfamily and features two cysteine-rich regions containing eight and six cysteine residues, respectively, separated by 42 amino acids long linker region. In-silico analysis of MT 2 protein sequences of C. japonica was performed. In this study, ab initio methods were utilised for the prediction of three-dimensional structure of CjMT 2. After structure validation, heavy metal-binding sites were predicted for the selected modelled structures of CjMT 2. To obtain Cdi-CjMT 2 (i = 1–7), metalated complex individual docking experiments were performed. The stability of the metalated docked structures was assessed by molecular dynamics (MD) simulation studies. Our study showed that CjMT 2 binds up to 4 Cd2+ ions in two distinct domains: a N-terminal β-domain that binds to 2 Cd2+ ions and a C-terminal α-domain that binds with 2 Cd2+ ions. Our analysis revealed that Cys residues of alpha and beta domain and some residues of spacer region of CjMT 2 protein might be important for the cadmium interaction. MD simulation studies provided insight into metal-induced conformational changes and mechanism of metalation of CjMT 2, an intrinsically disordered protein. This study provides useful insights into mechanism of cadmium-type 2 metallothionein interaction.

Removal of Multiple Contaminants from Water by Polyoxometalate Supported Ionic Liquid Phases (POM-SILPs).

The simultaneous removal of organic, inorganic, and microbial contaminants from water by one material offers significant advantages when fast, facile, and robust water purification is required. Herein, we present a supported ionic liquid phase (SILP) composite where each component targets a specific type of water contaminant: a polyoxometalate-ionic liquid (POM-IL) is immobilized on porous silica, giving the heterogeneous SILP. The water-insoluble POM-IL is composed of antimicrobial alkylammonium cations and lacunary polyoxometalate anions with heavy-metal binding sites. The lipophilicity of the POM-IL enables adsorption of organic contaminants. The silica support can bind radionuclides. Using the POM-SILP in filtration columns enables one-step multi-contaminant water purification. The results show how multi-functional POM-SILPs can be designed for advanced purification applications.

Characterization and Expression of a Phytochelatin Synthase Gene in Birch-leaf Pear (Pyrus betulaefolia Bunge)

Phytochelatins play a major role in metal detoxification in plants. However, the molecular mechanisms of heavy metal detoxification remain unknown, and birch-leaf pear genes related to metal detoxification have not yet been identified. Here, we report the isolation of cDNA and genomic DNA sequences encoding a phytochelatin synthase (PCS) from birch-leaf pear (Pyrus betulaefolia Bunge). The PbPCS1 cDNA sequence contained 1,965 nucleotides of a 1,494 bp open reading frame (ORF), which encodes a 497-amino-acid protein with a molecular weight of 55.00 kDa. Its corresponding genomic DNA sequence consists of 3,820 nucleotides and eight exons separated by seven introns. The deduced amino acid sequence of PbPCS1 contains five Cys residues (56, 90, 91, 109 and 113 amino acids) that are highly conserved in the plant PCS1 family, and 12 cysteine residues putatively involved in heavy metal binding sites, which included three adjacent Cys–Cys components (331–332, 351–352 and 369–370 amino acids) in the C-terminal variable domain. Homology analysis of the deduced PbPCS1 amino acid sequence revealed that it shares a high sequence similarity amongst N-terminal amino acids and low similarity with C-terminal amino acids with plant PCS1 proteins deposited with NCBI. PbPCS1 was most related to PCS1 from legume plants Lotus japonicus (GenBank accession no. AAT80342) and soybean (Glycine max L.; AAL78384) as they were clustered into the same clade in a neighbor-joining phylogenetic tree. Quantitative real-time PCR (qPCR) expression analysis revealed that PbPCS1 had a very low basal expression level in untreated whole seedlings, and levels were higher in roots than in leaves and stems. After 24 h of exposure to 20 μM CdSO4, CuSO4 or ZnSO4, PbPCS1 expression increased significantly in different organs. In addition, L-buthionine-sulfo-ximine (BSO) can inhibit PbPCS1 expression in roots, stems and leaves, while L-glutathionereduced (GSH) stimulates PbPCS1 expression in different organs of birch-leaf pear.

Regeneration and reuse of spent NaOH-treated oil palm frond for copper and zinc removal from wastewater

The use of NaOH-treated oil palm frond (OPF) sorbent for Cu and Zn removal and its subsequent regeneration process are reported. The regeneration of the spent sorbent was achieved by desorbing the metals in 0.1 M of sodium hydroxide (NaOH), ethylene diamine tetraacetic acid (EDTA), hydrochloric acid (HCl) and nitric acid (HNO 3) solutions. The reusability study of the sorbent was conducted using 100 mg/l of Cu and Zn at a pH of 4.5 and 5.5, respectively for 1 h. The results were to be correlated with the mechanism of the metal uptake. Freundlich isotherm fitted the data to indicate the presence of heterogeneous metal sorption sites. Zn showed better regeneration efficiency of up to 88% and HCl was the best regeneration agent. The results confirmed that ion exchange was the main mechanism for the metal uptake. The desorption efficiency dropped by merely about 20% while the sorption capacity experienced a drastic drop after reuse for the fourth cycle. The damage occurred on the heavy metal binding sites by the strong acid was responsible for this drop.

Differential Protein Mobility of the γ-Aminobutyric Acid, Type A, Receptor α and β Subunit Channel-lining Segments

The gamma-aminobutyric acid, type A (GABAA), receptor ion channel is lined by the second membrane-spanning (M2) segments from each of five homologous subunits that assemble to form the receptor. Gating presumably involves movement of the M2 segments. We assayed protein mobility near the M2 segment extracellular ends by measuring the ability of engineered cysteines to form disulfide bonds and high affinity Zn(2+)-binding sites. Disulfide bonds formed in alpha1beta1E270Cgamma2 but not in alpha1N275Cbeta1gamma2 or alpha1beta1gamma2K285C. Diazepam potentiation and Zn2+ inhibition demonstrated that expressed receptors contained a gamma subunit. Therefore, the disulfide bond in alpha1beta1E270Cgamma2 formed between non-adjacent subunits. In the homologous acetylcholine receptor 4-A resolution structure, the distance between alpha carbon atoms of 20' aligned positions in non-adjacent subunits is approximately 19 A. Because disulfide trapping involves covalent bond formation, it indicates the extent of movement but does not provide an indication of the energetics of protein deformation. Pairs of cysteines can form high affinity Zn(2+)-binding sites whose affinity depends on the energetics of forming a bidentate-binding site. The Zn2+ inhibition IC50 for alpha1beta1E270Cgamma2 was 34 nm. In contrast, it was greater than 100 microM in alpha1N275Cbeta1gamma2 and alpha1beta1gamma2K285C receptors. The high Zn2+ affinity in alpha1beta1E270Cgamma2 implies that this region in the beta subunit has a high protein mobility with a low energy barrier to translational motions that bring the positions into close proximity. The differential mobility of the extracellular ends of the beta and alpha M2 segments may have important implications for GABA-induced conformational changes during channel gating.

Identification of the Transmembrane Metal Binding Site in Cu+-transporting PIB-type ATPases

P(IB)-type ATPases have an essential role maintaining copper homeostasis. Metal transport by these membrane proteins requires the presence of a transmembrane metal occlusion/binding site. Previous studies showed that Cys residues in the H6 transmembrane segment are required for metal transport. In this study, the participation in metal binding of conserved residues located in transmembrane segments H7 and H8 was tested using CopA, a model Cu(+)-ATPase from Archaeoglobus fulgidus. Four invariant amino acids in the central portion of H7 (Tyr(682) and Asn(683)) and H8 (Met(711) and Ser(715)) were identified as required for Cu(+) binding. Replacement of these residues abolished enzyme activity. These proteins did not undergo Cu(+)-dependent phosphorylation by ATP but were phosphorylated by P(i) in the absence of Cu(+). Moreover, the presence of Cu(+) could not prevent the enzyme phosphorylation by P(i). Other conserved residues in the H7-H8 region were not required for metal binding. Mutation of two invariant Pro residues had little effect on enzyme function. Replacement of residues located close to the cytoplasmic end of H7-H8 led to inactive enzymes. However, these were able to interact with Cu(+) and undergo phosphorylation. This suggests that the integrity of this region is necessary for conformational transitions but not for ligand binding. These data support the presence of a unique transmembrane Cu(+) binding/translocation site constituted by Tyr-Asn in H7, Met and Ser in H8, and two Cys in H6 of Cu(+)-ATPases. The likely Cu(+) coordination during transport appears distinct from that observed in Cu(+) chaperone proteins or catalytic/redox metal binding sites.

NMR Identification of Heavy Metal-Binding Sites in a Synthetic Zinc Finger Peptide: Toxicological Implications for the Interactions of Xenobiotic Metals with Zinc Finger Proteins

Lead (Pb), mercury (Hg), and cadmium (Cd) are toxic and interfere with protein metal-binding sites. The Cys2/His2 zinc finger is a structural motif required for sequence-specific DNA binding and is present in zinc finger transcription factors (ZFP): Sp1, Egr-1, and TFIIIA. Neurotoxic studies have shown that heavy metals directly inhibit the DNA binding of ZFP and result in adverse cellular effects. Recently, we demonstrated the ability of heavy metals to alter the DNA binding of a synthetic Cys2/His2 finger peptide (Razmiafshari and Zawia, Toxicol. Appl. Pharmacol. 166, 1–12, 2000). To determine the precise site of interactions between heavy metals and this protein domain, Pb, Hg, Cd, and Ca were reconstituted with the synthetic apopeptide and studied by one- and two-dimensional NMR spectroscopy. In the presence of Zn, Cd, Hg, and Pb, but not Ca, distinct peptide NMR signal changes in the aliphatic region were observed and attributed to metal–cystiene interactions. However, chemical shifts indicative of metal–histidine binding were elicited by all the metals in the peptide's aromatic region. Chemical shift assignments and sequential connectivity were established in the presence and absence of Zn, Pb, and Ca through TOCSY and NOESY spectra. Cysteine and histidine residues showed a distinct change in their amide and beta resonances in the presence of Zn and Pb, suggesting the metal-ligand binding sites were near these residues. However, Ca led to no significant spectral changes in these regions, suggesting that it is not actively involved in the binding site. These studies reveal this structure as a mediator of metal-induced alterations in protein function.

Identification, expression, and characterization of Escherichia coli guanine deaminase.

Using the human cDNA sequence corresponding to guanine deaminase, the Escherichia coli genome was scanned using the Basic Local Alignment Search Tool (BLAST), and a corresponding 439-residue open reading frame of unknown function was identified as having 36% identity to the human protein. The putative gene was amplified, subcloned into the pMAL-c2 vector, expressed, purified, and characterized enzymatically. The 50.2-kDa protein catalyzed the conversion of guanine to xanthine, having a K(m) of 15 microM with guanine and a k(cat) of 3.2 s(-1). The bacterial enzyme shares a nine-residue heavy metal binding site with human guanine deaminase, PG[FL]VDTHIH, and was found to contain approximately 1 mol of zinc per mol of subunit of protein. The E. coli guanine deaminase locus is 3' from an open reading frame which shows homology to a bacterial purine base permease.

Contribution of carboxyl groups to heavy metal binding sites in fungal wall

Abstract Carboxyl groups contained in the cell walls of four different fungal species (Rhizopus arrhizus, Mucor miehei. Penicillium chrysogenum and Trichoderma reesei) were esterified with acidic methanol. The extent of esterification was monitored by measuring (gas chromatography) the methanol released after hydrolysis. The effect of biomass modification on metal binding was determined by zinc binding experiments at pH 5.5. A good correlation was observed between carboxyl blocking and reduction of zinc binding. Controls were also performed to examine the possible influence of methanol or acidic treatment alone. A good specificity was observed and the results demonstrate that carboxyl groups are responsible for at least 30% of R. arrhizus and M. miehei zinc binding properties. This contribution is higher with P. chrysogenum and T. reesei, with 55% and 70% of zinc binding, respectively. In addition, a non specific effect of methanol was observed with R. arrhizus, that strongly suggests that lipids contribu...

Heavy metal binding to heparin disaccharides. II. First evidence for zinc chelation.

To map out the heavy metal binding sites of iduronic acid containing oligosaccharides isolated from human kidneys, we studied Zn(II) binding by nuclear magnetic resonance (NMR) and molecular modeling to two disaccharides isolated after nitrous acid depolymerization of heparin and two synthetic disaccharides representative of the heparin structure, namely, IdopA2S (alpha 1,4)AnManOH, 1 alpha, IdopA2S (alpha 1,4)AnManOH6S, 1b, IdopA2S-(alpha 1,4)GlcNS alpha Me, 2a, and IdopA2S (alpha 1,4)GlcNS6S alpha Me, 2b (see previous article in this series). A conformational analysis of the metal free and metal bound solutions was made by comparing calculated [(NOE)]s, [T1]s, and [J]s to experimental values. The 1C4, 4C1, and 2S0 conformations of the L-idopyranosiduronate ring and the 4E and 4T3 of the anhydro-D-mannitol ring are evaluated as are rotations about the C5-C6 hydroxymethylene of the AnManOH(6S) or GlcNS (6S) residues. The NOE between IdopA2S H1 and H3 and the known NOE between H2 and H5, as well as the T1 of IdopA2S H3, are introduced as NMR observables sensitive to the IdopA2S ring conformation. Similarly, a NOE between IdopA2S H5 and AnManOH(6S) or GlcNS(6S) H3 was observed that directly restricts the allowed interglycosidic conformational space. For all disaccharides, the Zn(II) bound spectral data are consistent with models in which these motions are partially "frozen" such that the 1C4 conformation of the IdopA2S is stabilized along with the 4T3 conformation of the AnManOH(6S) ring. The interglycosidic conformation is also stabilized in one of two minima. Electrostatic potential energy calculations gave the best overall agreement with experiment and suggest metal binding conformations with the carboxylate and ring oxygen of the IdopA2S residues (1C4 conformation) and either O3 of the GlcNS(6S) residues or the sulfate oxygens of the 6-sulphate for 2b providing additional chelating sites. These chelation models concur with the observation of marked 13C and 1H NMR chemical shifts for the IdopA2S resonances and of GlcNS H3 for 2 alpha and GlcNS6S C6 for 2b. This study of model compounds implicates the IdopA2S(alpha 1,4)GlcNS6S group as part of the heavy metal binding site in biologically important acidic oligosaccharides such as heparin.

Mechanism of inhibition of human neutrophil collagenase by Gold(I) chrysotherapeutic compounds: Interaction at a heavy metal binding site

Abstract The mechanism of inhibition of two forms of human neutrophil collagenase (HNC) by six Au(I) compounds, some of which are used as chrysotherapeutic agents, has been investigated. The two forms of enzyme studied are active and latent HNC, the latter of which is activated by p-chloromercuribenzoate (PCMB). The effects of PCMB and Zn(II), which are normally included in the assays, on the activity of both forms of HNC and on their inhibition by these Au(I) compounds have also been studied. Zn(II) stimulates the activity of both the active and PCMB-activated latent forms of HNC up to a concentration of 50-100 microM, after which it inhibits markedly. PCMB activates latent HNC up to a concentration of 100 microM followed by inhibition at higher concentrations. Active HNC is not stimulated at PCMB concentrations below 100 microM, but is inhibited at higher concentrations. The stimulatory effects of Zn(II) and PCMB on HNC and its inhibition by PCMB are all attributable to binding at distinct sites. The inhibition of both active and PCMB-activated latent HNC by the Au(I) compounds is noncompetitive and is reversed by Zn(II). The inhibition of both forms of HNC by SKF 80544 and SKF 36914, which do not contain thiol ligands, is weak to moderate and is not influenced by the PCMB concentration. In contrast, PCMB markedly enhances the inhibition by Myocrisin, Sanocrisin, and Solganol by complexing to their thiol ligands to facilitate release of the Au(I) atom for binding to HNC. Cd(II) and Cu(II) also inhibit HNC noncompetitively, and inhibition is also reversed by Zn(II). Collectively, these data indicate that latent HNC contains a heavy metal binding site distinct from the active site at which Au(I), Cd(II), and Cu(II) bind to cause noncompetitive inhibition. Occupancy of this site by Zn(II) is characterized by retention of activity.

Site-directed mutagenesis of the α subunit of tryptophan synthase from salmonella typhimurium

Site-specific mutagenesis has been used to prepare two mutant forms of the alpha subunit of tryptophan synthase from Salmonella typhimurium in which either cysteine-81 or cysteine-118 is replaced by a serine residue. These mutant proteins are potentially useful for x-ray crystallographic studies since a heavy metal binding site is specifically eliminated in each mutant. The purified mutant proteins are fully active in four reactions catalyzed by the wild type alpha 2 beta 2 complex of tryptophan synthase. However, the mutant alpha 2 beta 2 complexes dissociate more readily and are less heat-stable than the wild type alpha 2 beta 2 complex. Thus, cysteine-81 and cysteine-118 of the alpha subunit serve structural but not functional roles.

Heavy metal binding sites in river water

THE transport of heavy metals (HMs) and their effects on biological systems in natural waters are greatly influenced by their interactions with soluble and insoluble water components1,2.