Copper Transfer Research Articles

Direct electron transfer of copper–zinc superoxide dismutase (SOD) on crystallographically oriented Au nanoparticles

The direct electron transfer (ET) of copper–zinc superoxide dismutase (SOD) has been realized, for the first time, at Au nanoparticles electrodeposited onto GC (nano-Au/GC) electrodes. Electrodeposition of Au nanoparticles in the presence of some additive (typically cysteine) resulted in the fabrication of Au nanoparticles with a high roughness morphology (enriched in the Au(1 0 0) orientation) and thus providing a favorable adsorption orientation of SOD ( key–lock interaction) suitable for a facilitated direct ET without the use of mediators or so-called ET promoters. The redox reaction of the SOD confined on the nano-Au/GC electrode was found to have a formal potential of +0.02 V vs. Ag/AgCl/KCl(sat). Whereas, round-shape spherical plumbs of Au nanoparticles were electrodeposited in the presence of iodide ions (as additive) which lack for the favorable rough surface and consequently the suitable interaction with the SOD is missing and hence the ET is not realized at this surface.

Solution Structure and Intermolecular Interactions of the Third Metal-binding Domain of ATP7A, the Menkes Disease Protein

The third metal-binding domain of the human Menkes protein (MNK3), a copper(I)-transporting ATPase, has been expressed in Escherichia coli and characterized in solution. The solution structure of MNK3, its copper(I)-binding properties, and its interaction with the physiological partner, HAH1, have been studied. MNK3 is the domain most dissimilar in structure from the other domains of the Menkes protein. This is reflected in a significant rearrangement of the last strand of the four-stranded beta-sheet when compared with the other known homologous proteins or protein domains. MNK3 is also peculiar with respect to its interaction with the copper(I) ion, as it was found to be a comparatively weak binder. Copper(I) transfer from metal-loaded HAH1 was observed experimentally, but the metal distribution was shifted toward binding by HAH1. This is at variance with what is observed for the other Menkes domains.

An experimental study of mass transfer of platinum-group elements, gold, nickel and copper in sulfur-dominated vapor at magmatic temperatures

We report results of an experimental study on platinum-group elements (PGE) and Au mass transfer by an S-vapor in the Fe–Ni–Cu sulfide system at magmatic temperatures. Using the tube-in-tube technique, we have examined the quantity of PGE, Au and base–metals (BM) transferred via the vapor from the PGE donor [S-rich (Fe,Ni,Cu) 1− x S doped with about 2000 ppm of each PGE and Au] to a S-poor PGE-free pyrrhotite (Po) used as the PGE receiver. At the end of the experiments, the receiver Po contained significant quantities of Ni, Cu, Au, Pt and Pd, but little Ir, Ru and Rh. The most important factors influencing the vapor mobility of Ni, Cu, Au and PGE are the phase assemblage present in the donor, which in turn is controlled by temperature, S, Ni and Cu content of the system, and the sulfur fugacity ( fS 2). In experiments containing only Au-alloy and monosulfide solid-solution (Mss) in the donor, Cu is transferred more than Ni. Gold is transferred 10 times more efficiently than Pd and Pt, and Pd and Pt are transferred 10 times more efficiently than Rh, Ru and Ir. At the lowest S contents, when Pt-alloys form in the donor system, Pt is transferred less than Pd. In experiments containing Mss and sulfide liquid, the amount of all PGE transferred is higher, although the order remains the same Pd–Pt > Rh–Ru–Ir. The amount of Ni transferred is higher than in the Mss-alloy system. In contrast, the amount of Au transferred is lower where the sulfide liquid is present. The amount of S in the receiver Po may be used as a proxy for fS 2. Within each phase assemblage the amount of Ni and PGE transferred increases with the amount of S in the receiver Po suggesting that these elements are transferred as sulfide complexes. In contrast, Cu and Au show no correlation with fS 2 suggesting that these elements are transferred as metals. These results are not directly applicable to natural systems because the simplicity of the experiments. Nevertheless three geological systems where these results could be relevant are be considered. Firstly, in the case of PGE deposits some are enriched in Pd, Cu and Au, while some are rich in Pt. According to our experiments this could occur if a magmatic sulfide underwent S devolatilization to form S-poor Mss plus Pt-alloy (to form the Pt-rich deposits) and the vapor transported Cu, Au and Pd to a lower pressure and temperature site and deposited these metals there to form the Pd-rich deposits. Secondly, in the case of magmatic Ni–Cu sulfide deposits, which in many cases are enriched in Cu and Pd; S, Cu and Pd could be transferred from the country rock as a vapor leaving Mss and Pt-alloy in the country rock. Finally, during S devolatilization of mantle nodules, Pd, Cu and Au could be removed by the vapor.

Changes in Copper Speciation and Geochemical Fate in Freshwaters Following Sewage Discharges

The main factors determining the geochemical fate of copper in urban freshwaters affected by raw sewage discharges were investigated in this work. Water samples from the Irai and Iguacu rivers were collected monthly during a 1-year period at two points located upstream and downstream from the city of Curitiba, in Brazil. Results revealed that raw sewage discharges from the heavily urbanized area caused an enhancement of humic-coated suspended solids in the Iguacu River. In these waters copper is predominantly associated with the humic-coated particles whereas in the Irai River copper was found primarily in the aqueous phase. The transfer of copper from the aqueous to the solid phase changed its physical speciation along the watercourse. Thus, aspects related to the overall transport of trace metals in watercourses become an important issue to be considered in further studies concerning the effect of sewage discharges on the geochemical speciation and fate of trace metals in urban rivers.

The P174L Mutation in Human Sco1 Severely Compromises Cox17-dependent Metallation but Does Not Impair Copper Binding

Sco1 is a metallochaperone that is required for copper delivery to the Cu(A) site in the CoxII subunit of cytochrome c oxidase. The only known missense mutation in human Sco1, a P174L substitution in the copper-binding domain, is associated with a fatal neonatal hepatopathy; however, the molecular basis for dysfunction of the protein is unknown. Immortalized fibroblasts from a SCO1 patient show a severe deficiency in cytochrome c oxidase activity that was partially rescued by overexpression of P174L Sco1. The mutant protein retained the ability to bind Cu(I) and Cu(II) normally when expressed in bacteria, but Cox17-mediated copper transfer was severely compromised both in vitro and in a yeast cytoplasmic assay. The corresponding P153L substitution in yeast Sco1 was impaired in suppressing the phenotype of cells harboring the weakly functional C57Y allele of Cox17; however, it was functional in sco1delta yeast when the wild-type COX17 gene was present. Pulse-chase labeling of mitochondrial translation products in SCO1 patient fibroblasts showed no change in the rate of CoxII translation, but there was a specific and rapid turnover of CoxII protein in the chase. These data indicate that the P174L mutation attenuates a transient interaction with Cox17 that is necessary for copper transfer. They further suggest that defective Cox17-mediated copper metallation of Sco1, as well as the subsequent failure of Cu(A) site maturation, is the basis for the inefficient assembly of the cytochrome c oxidase complex in SCO1 patients.

Structure of human Wilson protein domains 5 and 6 and their interplay with domain 4 and the copper chaperone HAH1 in copper uptake.

Human Wilson protein is a copper-transporting ATPase located in the secretory pathway possessing six N-terminal metal-binding domains. Here we focus on the function of the metal-binding domains closest to the vesicular portion of the copper pump, i.e., domain 4 (WLN4), and a construct of domains 5 and 6 (WLN5-6). For comparison purposes, some experiments were also performed with domain 2 (WLN2). The solution structure of apoWLN5-6 consists of two ferredoxin folds connected by a short linker, and (15)N relaxation rate measurements show that it behaves as a unit in solution. An NMR titration of apoWLN5-6 with the metallochaperone Cu(I)HAH1 reveals no complex formation and no copper exchange between the two proteins, whereas titration of Cu(I)HAH1 with WLN4 shows the formation of an adduct that is in fast exchange on the NMR time scale with the isolated protein species as confirmed by (15)N relaxation data. A similar interaction is also observed between Cu(I)HAH1 and WLN2; however, the relative amount of the adduct in the protein mixture is lower. An NMR titration of apoWLN5-6 with Cu(I)WLN4 shows copper transfer, first to WLN6 then to WLN5, without the formation of an adduct. Therefore, we suggest that WLN4 and WLN2 are two acceptors of Cu(I) from HAH1, which then somehow route copper to WLN5-6, before the ATP-driven transport of copper across the vesicular membrane.

Intermolecular Transfer of Copper Ions from the CopC Protein of Pseudomonas syringae. Crystal Structures of Fully Loaded CuICuII Forms

CopC is a small soluble protein expressed in the periplasm of Pseudomonas syringae pathovar tomato as part of its copper resistance response (cop operon). Equilibrium competition reactions confirmed two separated binding sites with high affinities for Cu(I) (10(-7) > or = K(D) > or = 10(-13) M) and Cu(II) (K(D) = 10(-13(1)) M), respectively. While Cu(I)-CopC was converted cleanly by O2 to Cu(II)-CopC, the fully loaded form Cu(I)Cu(II)-CopC was stable in air. Variant forms H1F and H91F exhibited a lower affinity for Cu(II) than does the wild-type protein while variant E27G exhibited a higher affinity. Cation exchange chromatography detected each of the four different types of intermolecular copper transfer reactions possible between wild type and variant forms: Cu(I) site to Cu(II) site; Cu(II) site to Cu(I) site; Cu(I) site to Cu(I) site; Cu(II) site to Cu(II) site. The availability of an unoccupied site of higher affinity induced intermolecular transfer of either Cu(I) or Cu(II) in the presence of O2 while buffering concentrations of cupric ion at sub-picomolar levels. Crystal structures of two crystal forms of wild-type Cu(I)Cu(II)-CopC and of the apo-H91F variant demonstrate that the core structures of the molecules in the three crystal forms are conserved. However, the conformations of the amino terminus (a Cu(II) ligand) and the two copper-binding loops (at each end of the molecule) differ significantly, providing the structural lability needed to allow transfer of copper between partners, with or without change of oxidation state. CopC has the potential to interact directly with each of the four cop proteins coexpressed to the periplasm.

Copper transfer studies between the N-terminal copper binding domains one and four of human Wilson protein

Human Wilson protein functions in the secretory pathway to insert copper ultimately into the multicopper oxidase ceruloplasmin and also plays a role in the excretion of excess copper to the bile. This copper-transporting P-type ATPase possesses six N-terminal cytosolic copper-binding domains contained within an ∼72 amino acid consensus motif and the first four of these domains, denoted WLN1–4, are implicated in copper acquisition from the metallochaperone HAH1, whereas the domains closest to the membrane portion of the enzyme, WLN5–6, are essential for copper transport across the membrane. In order to test our hypothesis that copper transfer occurs between domains in the N-terminus of Wilson protein, we expressed and purified to homogeneity copper-binding domains 1, 3, 4, 5–6, and 6, denoted by WLN1, WLN3, WLN4, WLN5–6, and WLN6, respectively. Since we determined WLN1 and WLN4 to have the highest and lowest isoelectric points (6.77 and 3.85, respectively) and thus are readily separated via ion exchange chromatography, we developed a copper transfer assay between these domains. We anaerobically incubated either Cu(I)-WLN1 with apo-WLN4 or apo-WLN1 with Cu(I)-WLN4, then separated these domains and quantified the amount of copper that migrates from one domain to another by ICP-MS. Regardless of whether we start with Cu(I)-WLN1 or Cu(I)-WLN4 as the initial copper donor, we demonstrate facile copper transfer between WLN1 and WLN4, thereby demonstrating the feasibility of copper transfer between these domains in vivo.

Relationships between CTR1 activity and copper status in different rat organs

The putative product of CTR1 (SLC31A1 according to the Entrez data base) is regarded as the main candidate for an eukaryotic transmembrane copper importer. The tissue-specific function of mammalian CTR1 is still unknown. A quasi-steady-state level of the CTR1 mRNA was assayed by semiquantitative RT-PCR and compared with the copper status in rat organs (liver, cerebellum, choroid plexus, and mammary gland), which differed in copper metabolism during development and differentiation. The CTR1 activity correlated with production of intracellular and extracellular cuproenzymes and deceased in nuclei of mesenchymal cells at high copper concentrations when copper metabolism followed the embryonic pattern. According to phylogenetic analysis, the most conserved regions of CTR1 are transmembrane domains (TMD) 2 and 3, which together contain seven amino acid residues capable of coordinating a copper atom. A model of the cuprophylic channel formed by TMD2 and TMD3 of the CTR1 homotrimer was proposed. In this model, copper is transported through the channel to the cytosolic C-terminal motif His-Cys-His. The ability of His-Cys-His to coordinate Cu(I) was evaluated by molecular modeling (MM+, ZINDO/1). Potential copper transfer from His-Cys-His to the Cys-X-X-Cys Cu (I) motif, present in all cytosol Cu-chaperones, was shown. The role of CTR1 as a donor and an acceptor of copper in higher eukaryotes is discussed.

Thermochemical characteristics of transfer of copper(II) and nickel(II) nitrates from water to water-formamide mixtures

The enthalpies of mixing of aqueous solutions of copper(II) and nickel(II) nitrates with water-formamide mixtures were measured calorimetrically at 298.15 K in the entire composition range. The standard enthalpies of transfer of the electrolytes from water to water-formamide mixtures were calculated from these data. The shape of the isotherms of transfer of the electrolytes is discussed on the basis of the calculated enthalpies of transfer of individual ions and contributions to these quantities made by universal, chemical, and electrostatic interactions of ions with the solvents.

Significance of physicochemical forms of storage in microalgae in predicting copper transfer to filter-feeding oysters (Crassostrea gigas)

Copper distribution has been examined in two microalgae (Haslea ostrearia, Diatom; Tetraselmis suecica, Prasinophyceae) exposed to Cu at 30 microg/L(-1). Exchangeable copper linked at the cell surface was desorbed using 8-hydroxyquinoline-5-sulfonate as complexing agent. Then, incorporated copper was separated between soluble and insoluble fractions. In addition, algae were resuspended in acid solutions, the pHs of which covered the range existing in the digestive tract of bivalves. Considering that the soluble fraction is the most easily transferred in the food chain and that exchangeable Cu is easily desorbed, the percentages of Cu potentially available in microalgae have been assessed. These percentages have been compared with those retained in oysters Crassostrea gigas fed with contaminated microalgae in previous studies. In H. ostrearia, the potentially available fraction of Cu (90%) was very similar to the percentage retained by oysters (93%) when the bivalves were acclimated to this food for 3 weeks. Only half (21%) of the potentially available Cu of T. suecica (42%) was readily assimilated in oysters after 3 weeks. This is in agreement with the results of the desorption tests at physiological pHs which showed that only 15-25% of Cu was lost, despite solubilization of other constituents of T. suecica as demonstrated by the decrease in their dry weight. Bioavailability determined from metal speciation in food allows a relevant prediction of the trophic transfer in the case of H. ostrearia, but caution is recommended in generalizing this mode of assessment as shown in the case of T. suecica.

Cadmium-binding protein components of flaxseed: Influence of cultivar and location

The distribution of cadmium-(Cd)-binding components from five flaxseed cultivars grown at three locations in southern Manitoba was investigated to examine genotypic and environmental effects. Three protein fractions with different electrostatic properties, eluting at 0.10, 0.25 and 0.50 M NaCl by ion-exchange chromatography DEAE-Sephacel, represented 12%, 66% and 7% of the bound (extracted) protein, respectively, while 15% of the protein remained unbound. Cadmium and other divalent metal (zinc, copper and calcium) contents of protein fractions were strongly influenced by location. Cultivar differences in protein and cadmium contents of the protein fractions were highly significant. Cadmium and zinc accumulated similarly in the 0.10 and 0.25 M protein fractions at 51% and 40–43%, respectively. Transfer of copper occurred prominently in the 0.50 M fraction while most of the calcium (55%) remained unbound. The distribution of cadmium, zinc, copper and calcium in fractions of flaxseed proteins was strongly influenced by cultivar and location, indicating differences in their accumulation, migration and transfer.

Copper accumulation by bacteria and transfer to scallop larvae

A bacterial copper-resistant strain was isolated from a hatchery-conditioned adult of the scallop Argopecten purpuratus and was identified as Vibrio sp. according to its physiological characteristics. The lowest concentration of Cu 2+ required for its complete inhibition in VNSS medium was 50 μg ml −1. The Vibrio strain was found to accumulate copper, exhibiting cellular and loosely bound copper levels of 201.14 and 493.21 μg g −1 dry weight, respectively, after 24 h of incubation in VNSS medium supplemented with 15 μg ml −1 of Cu 2+, with cellular concentration factors of 10.17 and 14.35 after 12 and 24 h of exposure. When a scallop larvae culture was exposed to a concentration of 6.46 × 10 2 CFU ml −1 of Cu-enriched Vibrio, they accumulated 20.42 ± 1.12 and 30.96 ± 1.85 μg Cu 2+ g −1 dry weight after 12 and 24 h, respectively. This study suggests that bacterial copper accumulation could be very active in marine environments increasing the occurrence of copper transfer to marine food chains.

X-ray Absorption Investigation of a Unique Protein Domain Able To Bind both Copper(I) and Copper(II) at Adjacent Sites of the N-Terminus of Haemophilus ducreyi Cu,Zn Superoxide Dismutase

The N-terminal metal binding extension of the Cu,Zn superoxide dismutase from Haemophilus ducreyi is constituted by a histidine-rich region followed by a methione-rich sequence which shows high similarity with protein motifs involved in the binding of Cu(I). X-ray absorption spectroscopy experiments selectively carried out with peptides corresponding to the two metal binding regions indicate that both sequences can bind either Cu(II) or Cu(I). However, competition experiments demonstrate that Cu(II) is preferred by histidine residues belonging to the first half of the motif, while the methionine-rich region preferentially binds Cu(I) via the interaction with three methionine sulfur atoms. Moreover, we have observed that the rate of copper transfer from the peptides to the active site of a copper-free form of the Cu,Zn superoxide dismutase mutant lacking the N-terminal extension depends on the copper oxidation state and on the residues involved in metal binding, histidine residues being critically important for the efficient transfer. Differences in the enzyme reactivation rates in the presence of mixtures of the two peptides when compared to those obtained with the single peptides suggest that the two halves of the N-terminal domain functionally interact during the process of copper transfer, possibly through subtle modifications of the copper coordination environment.

Cysteine-to-Serine Mutants of the Human Copper Chaperone for Superoxide Dismutase Reveal a Copper Cluster at a Domain III Dimer Interface

Cysteine-to-serine mutants of a maltose binding protein fusion with the human copper chaperone for superoxide dismutase (hCCS) were studied with respect to (i) their ability to transfer Cu to E,Zn superoxide dismutase (SOD) and (ii) their Zn and Cu binding and X-ray absorption spectroscopic (XAS) properties. Previous work has established that Cu(I) binds to four cysteine residues, two of which, C22 and C25, reside within an Atox1-like N-terminal domain (DI) and two of which, C244 and C246, reside in a short unstructured polypeptide chain at the C-terminus (DIII). The wild-type (WT) protein shows an extended X-ray absorption fine structure (EXAFS) spectrum characteristic of cluster formation, but it is not known how such a cluster is formed. Cys to Ser mutagenesis was used to investigate the Cu binding in more detail. Single Cys to Ser mutations, as represented by C22S and C244S, did little to affect the metal binding ratios of hCCS. Both mutants still showed approximately 2 Cu(I) ions and 1 Zn ion per protein. The double mutants C22/24S and C244/246S, on the other hand, showed Cu binding stoichiometries close to 1:1. The Zn-EXAFS of WT CCS showed a 3-4 histidine ligand environment that is consistent with Zn binding in the SOD-like domain II of CCS. The Zn environment remained unchanged between wild type and all of the mutant CCS proteins. Single Cys to Ser mutations displayed lower activity than WT protein, although close to full activity could be rescued by increasing the CCS:SOD ratios to 8:1 in the assay mixture. The structure of the Cu centers of the single mutants as revealed by EXAFS was also similar to that of WT protein, with clear indications of a Cu cluster. On the other hand, the double mutants showed a greater degree of perturbation. The DI C22/25S mutant was 70% active and formed a cluster with a more intense Cu-Cu interaction. The DIII C244/246S mutant retained only a fraction (16%) of activity and did not form a cluster. The results suggest the formation of a DIII-DIII cluster within a dimeric or tetrameric protein and further suggest that this cluster may be an important element of the copper transfer machinery.

Identification of a Disulfide Switch in BsSco, a Member of the Sco Family of CytochromecOxidase Assembly Proteins,

BsSco is a membrane-associated protein from Bacillus subtilis characterized by the sequence CXXXCP, which is conserved in yeast and human mitochondrial Sco proteins, and their bacterial homologues. BsSco is involved in the assembly of the Cu(A) center in cytochrome c oxidase and may play a role in the transfer of copper to this site. We have characterized the soluble domain of BsSco by biochemical, spectroscopic, and structural approaches. Soluble BsSco is monomeric in solution, and the two conserved cysteines are involved in an intramolecular cystine bridge. The cystine bridge is easily reduced, and circular dichroism spectroscopy shows no large-scale changes in BsSco's secondary structure upon reduction. The crystal structure of soluble BsSco, determined at 1.7 A resolution, reveals typical elements of a thioredoxin fold. The CXXXCP motif, in which Cys45 and Cys49 are conserved, is located in a turn structure on the surface of the protein. In various native and His135Ala mutant structures, both disulfide-bonded and non-disulfide-bonded forms of CXXXCP are observed. However, despite extensive attempts, copper has not been found near or beyond the CXXXCP motif, a presumptive copper-binding site. Another potential copper binding residue, His135, is located in a highly flexible loop parallel to the CXXXCP loop but is more than 10 A from Cys45 and Cys49. If these three residues are to coordinate copper, a conformational change is necessary. The structural identification of a disulfide switch demonstrates that BsSco has the capability to fill a redox role in Cu(A) assembly.

An NMR study of the interaction between the human copper(I) chaperone and the second and fifth metal‐binding domains of the Menkes protein

The interaction between the human copper(I) chaperone, HAH1, and one of its two physiological partners, the Menkes disease protein (ATP7A), was investigated in solution using heteronuclear NMR. The study was carried out through titrations involving HAH1 and either the second or the fifth soluble domains of ATP7A (MNK2 and MNK5, respectively), in the presence of copper(I). The copper-transfer properties of MNK2 and MNK5 are similar, and differ significantly from those previously observed for the yeast homologous system. In particular, no stable adduct is formed between either of the MNK domains and HAH1. The copper(I) transfer reaction is slow on the time scale of the NMR chemical shift, and the equilibrium is significantly shifted towards the formation of copper(I)-MNK2/MNK5. The solution structures of both apo- and copper(I)-MNK5, which were not available, are also reported. The results are discussed in comparison with the data available in the literature for the interaction between HAH1 and its partners from other spectroscopic techniques.

Yeast Cox17 Solution Structure and Copper(I) Binding

Cox17 is a 69-residue cysteine-rich, copper-binding protein that has been implicated in the delivery of copper to the Cu(A) and Cu(B) centers of cytochrome c oxidase via the copper-binding proteins Sco1 and Cox11, respectively. According to isothermal titration calorimetry experiments, fully reduced Cox17 binds one Cu(I) ion with a K(a) of (6.15 +/- 5.83) x 10(6) M(-1). The solution structures of both apo and Cu(I)-loaded Cox17 reveal two alpha helices preceded by an extensive, unstructured N-terminal region. This region is reminiscent of intrinsically unfolded proteins. The two structures are very similar overall with residues in the copper-binding region becoming more ordered in Cu(I)-loaded Cox17. Based on the NMR data, the Cu(I) ion has been modeled as two-coordinate with ligation by conserved residues Cys(23) and Cys(26). This site is similar to those observed for the Atx1 family of copper chaperones and is consistent with reported mutagenesis studies. A number of conserved, positively charged residues may interact with complementary surfaces on Sco1 and Cox11, facilitating docking and copper transfer. Taken together, these data suggest that Cox17 is not only well suited to a copper chaperone function but is specifically designed to interact with two different target proteins.

Multiple Metal Resistant Transferable Phenotypes in Bacteria as Indicators of Soil Contamination with Heavy Metals (6 pp)

Environmental contamination by heavy metals affects microbial communities. The number of single and multiple heavy metal resistant bacteria may be an indictor of the level of contamination. This paper details the isolation and characterisation of metal resistant microorganisms isolated from rhizosphere/soil samples obtained from an abandoned zinc, lead and copper mine and a local unaffected site. This data was compared to the level of heavy metal in the soils to establish the effect of metals on the microbial community and to determine the relationship between pollutant levels and resistant strains. This paper outlines the diversity of transferable resistance determinants between both sites and details the levels of heavy metal resistant bacteria and those expressing transferable multiple heavy metal tolerance. The sample sites were located in Co. Galway, Ireland. The first sample site (site A) was a former lead, zinc and copper mine, which was closed in 1961 due to exhaustion of ore. The second site (site B) was located two and a half kilometres from the mining site and was not affected by the mining operations. Composite soil samples were characterised for general soil matrix composition, organic content, pH and general chemical parameters. The soil was also enumerated for the total viable heterotrophic counts and tested on Pseudomonas selective agar (PSA) for total Pseudomonas counts and Sucrose Asparagine (SA), which is semi-selective for fluorescent Pseudomonas. Samples from both site A and site B were analysed by atomic absorption spectrophotometry for the presence of heavy metals. In the case of copper, which has a Dutch list recommended minimum permissible level of 190 µg/Kg dry weight, the levels detected at site A were 1270 µg/Kg dry weight while site B was detected at 36 µg/Kg. The arsenic levels detected at site A were eight times the permissible level (416 µg/Kg) while only half the permissible level was found at site B (13 µg/Kg). Zinc concentrations were also high at site A (4460 µg/Kg) while at site B (553 µg/Kg) they were well below the Dutchlist guidelines (720 µg/Kg). A large number of heavy metal tolerant strains were isolated from both sites. 270 isolates (site A (170) and site B (100)) were screened against 8 metals to examine the extent of multiple resistance. 82% of the strains from site A were found to be resistant to 5 metals. A total of 18% showed resistance to all 8 metals and of those examined only 4% were resistant to only one metal. In contrast isolates from site B showed no multiple resistance to more than 5 metals, while 62% showed resistance to individual metals only. Site A had a higher level of multiple heavy metal resistance strains. Stains isolated from site A had 23 (14%) isolates resistant to zinc, copper, nickel, arsenic and cobalt and site B had no bacteria resistant to all five of these selected metals. The transferability of heavy metal resistance was investigated in the case of 60 multiple heavy metal resistant isolates taken from site A and 50 multiple resistance isolates from site B. Transfer was only detected in isolates from site A, 13% showed transfer and expression of copper, zinc and arsenic resistance determinants. In most cases the transconjugants only expressed resistance to copper, zinc and arsenic, which were the metals, used for selection. This co-transfer of all three determinants suggests a genetic link between these resistance determinants. Heavy metal resistant bacteria are present in both sites, however, the number and presence of multiple transferable resistance phenotypes are confined to the isolates from the heavy metal contaminated site. The presence of high levels of heavy metals selects these multiple resistance phenotypes. Within these communities there seems to be little diversity between the microorganisms, which provides a hugely preferable environment for gene transfer of such metal resistant determinants. The experiments have shown a microbes ability to mobilize heavy metal determinants and a relationship between heavy metal resistance and metal contamination has been identified These multiple heavy metal resistant bacteria could eventually be used for detection and qualification of the level of heavy metal-polluted soil/water environments.

SILICATE MELT INCLUSIONS IN PORPHYRY COPPER DEPOSITS: IDENTIFICATION AND HOMOGENIZATION BEHAVIOR

Silicate melt inclusions are ubiquitous in phenocrysts in porphyry copper deposits. Compared to melt inclusions in other igneous environments, those in porphyry copper deposits are more difficult to recognize and analyze, for a variety of reasons. The inclusions are usually partially to completely crystallized or altered by hydrothermal fluids, resulting in a dark, granular appearance. In this study, a protocol for identifying and homogenizing crystallized melt-inclusions from porphyry copper deposits is described. The protocol has been used to obtain preliminary data from melt inclusions in the Red Mountain, Arizona, and the Tyrone, New Mexico, porphyry copper deposits. Many melt inclusions in syn- and post-mineralization samples show evidence of alteration by magmatic-hydrothermal fluids that produced alteration and mineralization in the plutons, and many inclusions trapped crystals or fluid (or both) along with the melt. Elevated concentrations of metal in pre-mineralization (H2O-undersaturated) melt inclusions, and lower concentrations of metal in later syn- and post-mineralization (H2O-saturated) melt inclusions are consistent with models that invoke quantitative transfer of copper and other metals from the melt into the magmatic aqueous phase when the melt reaches H2O saturation. Results from Red Mountain and Tyrone are compared to melt inclusions from the White Island, New Zealand, active volcano.