Enrichment in Trace Metals (Al,Mn, Co,Cu, Mo,Cd, Fe,Zn, Pb and Hg) of Macro-Invertebrate Habitats at Hydrothermal Vents Along the Mid-Atlantic Ridge

The present study describes several features of the aquatic environment with the emphasis on the total vs. filter-passing fraction (FP) of heavy metals in microhabitats of two typical deep-sea vent organisms: the filter-feeder, symbiont-bearing Bathymodiolus and the grazer shrimps Rimicaris/Mirocaris from the Mid-Atlantic Ridge (MAR). The concentration of 10 trace elements: Al, Mn, Co, Cu, Mo, Cd, Fe, Zn, Pb and Hg was explored highlighting common and distinctive features among the five hydrothermal vent sites of the MAR: Menez Gwen, Lucky Strike, Rainbow, Saldanha, and Menez Hom that are all geo-chemically different when looking at the undiluted hydrothermal fluid composition. The drop off in the percentage of FP from total metal concentration in mussel and/or shrimp inhabited water samples (in mussel beds at Rainbow, for instance, FP fraction of Fe was below 23%, Zn 24 %, Al 65%, Cu 70%, and Mn 89%) as compared to non-inhabited areas (where 94% of the Fe, 90% of the Zn, 100% of the other metals was in the FP fraction) may indicate an influence of vent organisms on their habitat’s chemistry, which in turn may determine adaptational strategies to elevated levels of toxic heavy metals. Predominance of particulate fraction over the soluble metals, jointly with the morphological structure and elemental composition of typical particles in these vent habitats suggest a more limited metal bioavailability to vent organisms as previously thought. In addition, it is evoked that vent invertebrates may have developed highly efficient metal-handling strategies targeting particulate phase of various metals present in the mixing zones that enables their survival under these extreme conditions.     

Trace Metals (Cd,Co, Cr,Cu, Hg,Ni, Pb,and Zn) in Food Supplements of Marine Origin

We determined the concentrations of Cd, Co, Cr, Cu, Hg, Ni, Pb, and Zn in dietary supplements of marine origin. Four supplement categories were studied; algae, coral, krill, and shark cartilage. A direct mercury analyzer was used for Hg determinations while acid digestions and ICP-AES were used for Cr analysis and ICP-MS for the other trace metals. Algae are the supplements showing the highest concentrations of Pb, Cr, and Ni with respective means of 1.6 mg Pb/kg dry weight (d.w.), 3.2 Cr mg/kg d.w., and 8.0 mg Ni/kg d.w. Krill supplements have the highest levels of Cd, Cu, and Zn with 0.65 mg Cd/kg d.w., 63 mg Cu/kg d.w., and 50 mg Zn/kg d.w., respectively. Shark cartilage supplements show the highest levels of Hg and Co with mean concentrations of 160 μg Hg/kg d.w. and 73 ± 51 μg Co/kg d.w., respectively. No samples in our study exceeded the provisional tolerable daily intakes set by Health Canada, the joint committee of the World Health Organization/Food and Agricultural Organization, or the U.S. Environmental Protection Agency. Nevertheless, Ni and Pb in algae and Hg in shark cartilage may end up contributing to a very significant portion of the allowable daily intake—leaving little room for normal intake through food consumption and other exposure pathways.     

Distribution of Metals (Cu,Zn, Pb,and Cd) in Sediments of the Anzali Lagoon,North Iran

Concentrations of four metals (Cu, Zn, Pb, and Cd) in the sediments of the Anzali Lagoon in the northern part of Iran were determined to evaluate the level of contamination and spatial distribution. The sediments were collected from 21 locations in the lagoon. At each lagoon site a core, 60 cm long, was taken. The ranges of the measured concentrations in the sediments are as follows: 17–140 mg kg^?1 for Cu, 20–113 mg kg^?1 for Zn, 1–37 mg kg^?1 for Pb and 0.1–3.5 mg kg^?1 for Cd in surficial (0-20 cm) and 16–87 mg kg^?1 for Cu, 28.5–118 mg kg^?1 for Zn, 3–20 mg kg^?1 for Pb and 0.1–3.5 mg kg^?1 for Cd in deep (40–60 cm) sediments. The results of the geoaccumulation index (I_geo) show that Cd causes moderate to heavy pollution in most of the study area. Environmental risk evaluation showed that the pollution in the Anzali Lagoon is moderate to considerable and the ranking of the contaminants followed the order: Cd > Cu > Pb > Zn. Some locations present severe pollution by metals depending on the sources, of which sewage outlets and phosphate fertilizers are the main sources of contaminants to the area.     

Tolerance and hyperaccumulation of a mixture of heavy metals (Cu,Pb, Hg,and Zn) by four aquatic macrophytes

In the present investigation, four macrophytes, namely Typha latifolia (L.), Lemna minor (L.), Eichhornia crassipes (Mart.) Solms-Laubach, and Myriophyllum aquaticum (Vell.) Verdc, were evaluated for their heavy metal (Cu, Pb, Hg, and Zn) hyperaccumulation potential under laboratory conditions. Tolerance analyses were performed for 7 days of exposure at five different treatments of the metals mixture (Cu⁺², Hg⁺², Pb⁺², and Zn⁺²). The production of chlorophyll and carotenoids was determined at the end of each treatment. L. minor revealed to be sensitive, because it did not survive in all the tested concentrations after 72 hours of exposure. E. crassipes and M. aquaticum displayed the highest tolerance to the metals mixture. For the most tolerant species of aquatic macrophytes, The removal kinetics of E. crassipes and M. aquaticum was carried out, using the following mixture of metals: Cu (0.5 mg/L) and Hg, Pb, and Zn 0.25 mg/L. The obtained results revealed that E. crassipes can remove 99.80% of Cu, 97.88% of Pb, 99.53% of Hg, and 94.37% of Zn. M. aquaticum withdraws 95.2% of Cu, 94.28% of Pb, 99.19% of Hg, and 91.91% of Zn. The obtained results suggest that these two species of macrophytes could be used for the phytoremediation of this mixture of heavy metals from the polluted water bodies.     

Heavy Metals (Cd,Cu, Ni,Pb, and Zn) Fractionation in River Sediments,Hamedan, Western Iran

Five heavy metals (Cd, Cu, Ni, Pb, and Zn) in river sediments from Abshineh River, Hamedan, western Iran, were fractionated by a sequential extraction procedure. Cu, Ni, Pb, and Zn existed in sediments mainly in residual fraction (mean 92%, 86%, 77%, and 65%, respectively), whereas Cd occurred mostly as organic matter (mean 41%) and exchangeable (mean 25%) fractions. The mean percent of mobile fraction of Cd, Cu, Ni, Pb, and Zn in contaminated sediments was 25, 13, 4, 24, and 10, respectively, which suggests that the mobility and bioavailability of the five metals in sediments probably decline in the following order: Cd = Pb > Cu > Zn > Ni. The metal levels were also evaluated according to the contamination factor, which revealed significant anthropogenic pollution of Cd and Pb.     

Bioaccumulation of Key Metals and Other Contaminants by Seaweeds from the Egyptian Mediterranean Sea Coast in Relation to Human Health Risk

This study examined the bioaccumulation of key metals and other contaminants (SO₄, F, Na, K, Ca, Mg, P, Si, Fe, Mn, Zn, Ni, Co, Cu, and Cd) by seaweeds from the Egyptian Mediterranean Sea coast and their risk to human health. Bioaccumulation factor calculations indicated that S, F, Na, K, Zn, Cu, and Cd had high seaweed uptake and this referred to the potential pollution of the coastal area. Multivariate analysis of the studied contaminants confirmed the bioaccumulation factor results as well as the physiological processes in seaweeds. Human health risk assessment of F, Mn, Zn, Ni, Cu, and Cd was conducted using hazard quotient (HQ) and incremental lifetime cancer risk (ILCR) analyses. The carcinogenic assessment of sediments and ingestion of seaweeds using ILCR calculations revealed that Mn, Ni, and Cd in sediments and F and Zn in seaweeds require remedial action in order to reduce their human health risks. Additionally, HQ calculations for the contaminants in seaweeds and reference to toxicological references values from different agencies revealed that F and Co present high human health risk effects. Consequently, the potential public health risks from dietary exposure to hazardous contaminants in seaweeds must be continually subjected to research, regulation, and debate.     

Effects of Total Soft Tissue and Shell Thickness on the Accumulation of Heavy Metals (Cd,Cu, Pb,and Zn) in the Green-Lipped Mussel Perna viridis

The effects of the total soft tissue dry weight and shell thickness and on the accumulation of Cd, Cu, Pb, and Zn were determined in the green-lipped mussel Perna viridis. In agreement with Boyden's formula (1977), our results showed that the plotting of metal concentrations against the total soft tissue dry weight and shell thickness of the mussel on a double logarithmic basis gave negative coefficients especially for Cd, Pb, and Zn. Therefore, the smaller mussels (lower total soft tissue dry weight) had higher concentrations of Cd, Pb, and Zn than the larger ones. Since shell thickness could be considered to estimate of the age of the mussel, it was also found that the younger mussels accumulated more Cd, Pb, and Zn than the older ones. This indicated that P. viridis has a different metabolic strategy for each of the metals studied which may be related to age. However, the accumulation of Cu was hardly affected by the sizes and ages of the mussel. This indicated that the accumulation pathways of Cu and the processes affecting the bioavailability of Cu to the mussel are different from those for Cd, Pb, and Zn.     

Interactions between toxic (As, Cd, Hg and Pb) and nutritional essential (Ca, Co, Cr, Cu, Fe, Mn, Mo, Ni, Se, Zn) elements in the tissues of cattle from NW Spain

Since the toxicity of one metal or metalloid can be dramatically modulated by the interaction with other toxic or essential metals, studies addressing the chemical interactions between trace elements are increasingly important. In this study correlations between the main toxic (As, Cd, Hg and Pb) and nutritional essential (Ca, Co, Cr, Cu, Fe, Mn, Mo, Ni, Se, Zn) elements were evaluated in the tissues (liver, kidney and muscle) of 120 cattle from NW Spain, using Spearman rank correlation analysis based on analytical data obtained by ICP-AES. Although accumulation of toxic elements in cattle in this study is very low and trace essential metals are generally within the adequate ranges, there were significant associations between toxic and essential metals. Cd was positively correlated with most of the essential metals in the kidney, and with Ca, Co and Zn in the liver. Pb was significantly correlated with Co and Cu in the liver. A large number of significant associations between essential metals were found in the different tissues, these correlations being very strong between Ca, Cu, Fe, Mn, Mo and Zn in the kidney. Co was moderately correlated with most of the essential metals in the liver. In general, interactions between trace elements in this study were similar to those found in polluted areas or in experimental studies in animals receiving diets containing high levels of toxic metals or inadequate levels of nutritional essential elements. These interactions probably indicate that mineral balance in the body is regulated by important homeostatic mechanisms in which toxic elements compete with the essential metals, even at low levels of metal exposure. The knowledge of these correlations may be essential to understand the kinetic interactions of metals and their implications in the trace metal metabolism.     

210Po Activity and Concentrations of Selected Trace Elements (As, Cd, Cu, Hg, Pb, Zn) in the Muscle Tissue of Tunas Thunnus albacares and Katsuwonus pelamis from the Eastern Pacific Ocean

Daily mineral intake (DMI) of Cu and Zn, percentage weekly intake (PWI) of As, Cd, Hg, Pb, and doses of ²¹⁰Po were estimated by using their elemental concentration in muscle of two tuna species and the average tuna consumption in Mexico. Skipjack tuna Katsuwonus pelamis had significantly (p?<?0.05) higher levels of As (1.38???g?g^?1 dw) and Cu (1.85???g?g^?1 dw) than yellowfin tuna Thunnus albacares, whereas Pb concentrations (0.18???g?g^?1 dw) were significantly (p?<?0.05) higher in T. albacares. The sequence of elemental concentrations in both species was Zn?>?Cu?>?As?>?Hg?>?Pb?>?Cd. In T. albacares, concentrations of Cd and Pb in muscle tissue were positively correlated (p?<?0.05) with weight of specimens, while Cu was negatively correlated. DMI values were below 10?%. PWI figures (<2?%) are not potentially harmful to human health. ²¹⁰Po concentration in T. albacares and K. pelamis accounts for 13.5 to 89.7?% of the median individual annual dose (7.1???Sv) from consumption of marine fish and shellfish for the world population.     

Changes of Structure and Stock in Exploited Fish Communities in Estuaries of the Southern Baltic Coast (Mecklenburg-Vorpommern,Germany)

By means of analyses of catch values of selected fish species possible effects of eutrophication on the estuaries are described. Although the registered stock changes are not only a result of eutrophication a stock increase in pikeperch could be verified at the end of the first half of this century. With progressive eutrophication a great decline of migratory fish stocks took place at the end of the sixties/beginning of the seventies. The decline of eel stocks is not caused by eutrophication. Afterwards pike stocks show an aggravating decline in hypertrophic estuaries which are related to the decline of macrophytes. Baltic whitefish has still kept up its stock level under these conditions. Symptoms for a growing danger of an unexpected disaster are increasing to the whole fish community of extremely polluted waters.     

The Effect of Mustard Gas on Salivary Trace Metals (Zn,Mn, Cu,Mg, Mo,Sr, Cd,Ca, Pb,Rb)

We have determined and compared trace metals concentration in saliva taken from chemical warfare injures who were under the exposure of mustard gas and healthy subjects by means of inductively coupled plasma optical emission spectroscopy (ICP-OES) for the first time. The influence of preliminary operations on the accuracy of ICP-OES analysis, blood contamination, the number of restored teeth in the mouth, salivary flow rate, and daily variations in trace metals concentration in saliva were also considered. Unstimulated saliva was collected at 10:00–11:00 a.m. from 45 subjects in three equal groups. The first group was composed of 15 healthy subjects (group 1); the second group consisted of 15 subjects who, upon chemical warfare injuries, did not use Salbutamol spray, which they would have normally used on a regular basis (group 2); and the third group contained the same number of patients as the second group, but they had taken their regular medicine (Salbutamol spray; group 3). Our results showed that the concentration of Cu in saliva was significantly increased in the chemical warfare injures compared to healthy subjects, as follows: healthy subjects 15.3± 5.45(p.p.b.), patients (group 2) 45.77±13.65, and patients (Salbutamol spray; group 3) 29 ±8.51 (P <0.02). In contrast, zinc was significantly decreased in the patients, as follows: healthy subjects 37 ± 9.03(p.p.b.), patients (group 2) 12.2 ± 3.56, and patients (Salbutamol spray; group 3) 20.6 ±10.01 (P < 0.01). It is important to note that direct dilution of saliva samples with ultrapure nitric acid showed the optimum ICP-OES outputs.     

The action of divalen Zn,Cd, Hg,Cu and Pb ions on the ATPase activity of a plasma membrane fraction isolated from roots ofZea mays

The effects of the divalent metal ions Zn, Cd, Hg, Cu and Pb on the ATPase activity of a plasma membrane fraction isolated from roots ofZea mays have been investigated. When Mg-ions (3 mM), with or without K-ions (50mM) are included in the reaction medium, inhibition of ATPase activity was found in all cases, the relative order of the inhibitors over the concentration range 10 to 100M, being Hg>>CuCd>ZnPb. Below 1.0M only Hg caused substantial inhibition. In the absence of Mg ions, Zn and to a lesser extent Cd, activated the enzyme up to a concentration of 1 mM, activity being further stimulated in the presence of K-ions (50mM). No activation of ATPase activity was found for Hg, Cu or Pb. It was concluded that Zn-ATP and Cd-ATP are both alternative substrates for the enzyme. Further experiments showed that both K_m and V_max for the substrates Zn-ATP and Cd-ATP are very much lower than for the usual substrate Mg-ATP.These present results are discussed in relation to the known actions of these divalent cations on the trans-root potential and H-ion efflux in excised maize roots (Kennedy and Gonsalves, 1987).     

Accumulation of Ag, Cd, Co, Cu, Hg, Ni and Pb in Pavlova viridis Tseng (Haptophyceae)

The flagellate alga Pavlova viridis Tseng was investigated in the laboratory for accumulation of the heavy metals, silver, cadmium, cobalt, copper, mercury, nickel and lead. The cultures were grown in an artificial seawater medium mixed with the individual metals at different concentrations. Based on data from the controls, the baseline metal concentrations in P. viridis were shown to be in an order of Cu > Pb > Co > Cd > Ni > Ag > Hg. In the experimental groups, the seven metals displayed different isotherm equilibrium patterns and the metal uptake capacity of the alga was Ni > Pb > Co > Hg > Cu > Cd > Ag at equilibrium. When assessed using the bioconcentration factors, metal accumulation by P. viridis was demonstrated to be the most efficient at a concentration of 0.001 mg L-1 for Ag, Cd and Co, and at 0.01 mg L-1 for Cu, Hg, Ni and Pb. This study suggests that P. viridis can be a source of mineral supplements in mariculture. The alga is not, however, recognized as an effective agent for removing heavy metals from wastewater. This revised version was published online in June 2006 with corrections to the Cover Date.     

Heavy metals (Cu,Zn, Pb,Cd) in sediment,zooplankton and epibenthic invertebrates from the area of the continental slope of the Banc d'Arguin (Mauritania)

The concentrations of copper, zinc, lead and cadmium in the surface sediment (upper 5 mm) were generally higher in the silt fraction than in the bulk sediment. No significant geographical trend in the metal concentrations of the surface sediments was found, nor a correlation between concentrations in bulk sediment as well as in the silt fraction and the % silt could be established. In general, the metal concentrations in both bulk sediment and silt are lower, when compared to marine environments in other climatological regions.In zooplankton, the metal concentrations were relatively high: expressed in µg g^–1 on a dry weight (D.W.) basis, they ranged from 15–90 for copper, 70–580 for zinc, 12–55 for lead and 4–10 for cadmium.In epibenthic invertebrate species, both in crustaceans and bivalve molluscs, the concentrations of copper, zinc, and lead were in the same order of magnitude as compared to corresponding species from other geographical latitudes. Cadmium concentrations were relatively low, ranging from 0.13–0.42 µg g^–1 D.W. in the bivalve molluscs Pitaria tumens and from 0.04–0.27 µg g^–1 D.W. in the shrimp Processa elegantula. Also in the crab species Ilia spinosa, Inachus sp. and Pagurus sp., the cadmium concentrations were low, varying between 0.1 and 0.2 µg g^–1 D.W.No significant relation between the metal concentration in whole-body samples and sediment (either bulk or silt) was present. Also no gradient was apparent in concentrations in organisms sampled at different depths (5 to 200 m) along two off-shore transects perpendicular to the Banc d'Arguin. Data indicated lower metal concentration in epibenthic organisms from sampling stations along a northern transect (southwest of Cap Blanc) than in organisms from the southern transect, off Cap Timiris.Evidence was obtained for a considerable atmospheric input of heavy metals, in particular zinc and lead, in a certain area along the continental slope of the Banc d'Arguin.     

Element content (Cu, Fe, Mn, Ni, Pb, and Zn) of the ruderal plant Verbascum olympicum Boiss. from East Mediterranean

In this study, heavy metal content (Cu, Fe, Mn, Ni, Pb, Zn) was determined in soils and different organs of Verbascum olympicum Boiss. This species is endemic to Uluda? and spreads on destroyed areas such as: roadsides, developed building areas, ski lift stations and sheep folds. Soils and different organs (roots, stems, leaves and flowers) of plant samples were analyzed using an atomic absorption spectrophotometer for determining the element content. Heavy metal contents in soils and different organs in this species were highly correlated (P < 0.05). However, the contribution of plant organs to the accumulation capacity varied according to the metal. These results suggest that this species may be useful as a bioindicator for heavy metals.     

Comparison of Concentrations of Selected Metals and Organics in Fish Tissue and Sediment in the Grand River,Ohio, and the Southern Lake Erie Drainage Basin

The purpose of this paper is to develop a database of fish tissue and sediment concentrations of selected chemicals in the southern Lake Erie drainage basin, to compare contaminant concentrations in fish tissue and sediment collected from the Grand River, Ohio, in the vicinity of a former soda ash and chromate plant with that for other waterbodies in the drainage basin, and to evaluate the potential human health risks posed by these chemicals, with special focus on chromium. The results of this study indicate that chemical concentrations in fish tissue and sediment from the Grand River are generally lower than that of other waterbodies in the southern Lake Erie drainage basin. Although arsenic and beryllium concentrations in Grand River sediment are above their respective human health-based concentrations, these concentrations are comparable with local and regional background and are not expected to pose a significant incremental risk to human health. In addition, sediment concentrations do not exceed ecological screening criteria. Although PCB and several pesticides were found to have concentrations in fish tissue from the Grand River in excess of human health based concentrations, these concentrations are among the lowest found in waterbodies in the southern Lake Erie drainage basin.     

Biosorption of Cd, Cu, Pb, and Zn from aqueous solutions by the fruiting bodies of jelly fungi (Tremella fuciformis and Auricularia polytricha)

In this study, dried and humid fruiting bodies of Tremella fuciformis and Auricularia polytricha were examined as cost-effective biosorbents in treatment of heavy metals (Cd²⁺, Cu²⁺, Pb²⁺, and Zn²⁺) in aqueous solution. The humid T. fuciformis showed the highest capacity to adsorb the four metals in the multi-metal solutions. The Pb²⁺ adsorption rates were 85.5%, 97.8%, 84.8%, and 91.0% by dried T. fuciformis, humid T. fuciformis, dried A. polytricha, and humid A. polytricha, respectively. The adsorption amount of Pb²⁺ by dried and humid T. fuciformis in Cd²⁺ + Pb²⁺, Cu²⁺ + Pb²⁺, Pb²⁺ + Zn²⁺, Cd²⁺ + Cu²⁺ + Pb²⁺, and Cd²⁺ + Zn²⁺ + Pb²⁺ solutions were not lower than that in Pb²⁺ solutions. The results suggested that in humid T. fuciformis, Cd²⁺, Cu²⁺, and Zn²⁺ promoted the Pb²⁺ adsorption by the biomass. In the multi-metal solutions of Cd²⁺ + Cu²⁺ + Pb²⁺ + Zn²⁺, the adsorption amount and rates of the metals by all the test biosorbents were in the order of Pb²⁺ > Cu²⁺ > Zn²⁺ > Cd²⁺. Compared with the pseudo first-order model, the pseudo second-order model described the adsorption kinetics much better, indicating a two-step biosorption process. The present study confirmed that fruiting bodies of the jelly fungi should be useful for the treatment of wastewater containing Cd²⁺, Cu²⁺, Pb²⁺, and Zn²⁺.     

Leaching potential of heavy metals (Cd, Ni, Pb, Cu and Zn) from acidic sandy soil amended with dolomite phosphate rock (DPR) fertilizers

There is an increasing concern on heavy metal leaching from the soils amended with sewage sludge. A column study was conducted to examine the extent of leaching of five important heavy metals (Cd, Ni, Pb, Cu and Zn) from an acidic sandy soil amended with different dolomite phosphate rock (DPR) fertilizers (an application rate of 1% fertilizers) developed from DPR and N-Viro (consisting of biosolids and fly ash) at 0%, 10%, 20%, 30%, 40%, 50% and 100% DPR. Ten leaching events were carried out with each event done at an interval of 7 days and with total leaching volume of 1183mm, which is equivalent to the mean annual rainfall of this region during the period of 2001-2003. Leachate was collected after each leaching event and analyzed for heavy metals. The maximum leachate concentrations of Cd, Ni, Pb, Cu and Zn were all below drinking water quality guidance limits set by Florida Department of Environmental Protection and World Health Organization, suggesting that the application of DPR fertilizers may not pose a threat to water quality by leaching. Most of leachate concentrations of Cd, Ni and Pb were below their detection limits and there were no significant differences between the control and the treatments with different DPR fertilizers. By contrast, there were higher leachate concentrations of Cu and Zn (ranging from 0.7 to 37.1mug Cu/l and 5.1 to 205.6mug Zn/l for all treatments) due to their higher contents in both the soil and different DPR fertilizers compared with Cd, Ni and Pb. The leachate concentrations of Cu and Zn for each treatment decreased with increasing leaching events. The differences in leachate concentrations of Cu and Zn between the control and the treatments with different DPR fertilizers containing N-Viro were significant, especially in the first several leaching events and, moreover, they increased with increasing proportion of N-Viro in the DPR fertilizers. There were similar trends in total losses of Cu and Zn after ten leaching events. Greater differences in both leachate concentrations and total losses of Zn between the control and the treatments containing N-Viro were noted. Total losses of Zn for the treatments containing N-Viro were 3.0-5.1 times higher than those for the control compared with 1.4-2.2 times higher for total losses of Cu, suggesting that greater proportions of Zn losses came from the DPR fertilizers due to the greater mobility of Zn in the DPR fertilizers compared with Cu.     

Preparation and characterization of a (Cu,Zn)-pMMO from Methylococcus capsulatus (Bath)

We report the preparation of a (Cu,Zn)-particulate methane monooxygenase (pMMO) in which the bulk of the copper ions of the electron-transfer clusters (E-clusters) has been replaced by divalent Zn ions. The Cu and Zn contents in the (Cu,Zn)-pMMO were determined by both inductively coupled plasma mass spectroscopy (ICP-MS) and X-ray absorption K-edge spectroscopy. Further characterization of the (Cu,Zn)-pMMO was provided by pMMO-activity assays as well as low-temperature electron paramagnetic resonance (EPR) spectroscopy following reductive titration and incubation in air or air/propylene mixtures. The pMMO-activity assays indicated that the (Cu,Zn)-pMMO was no longer capable of supporting catalytic turnover of hydrocarbon substrates. However, the EPR studies revealed that the catalytic cluster (C-cluster) copper ions in the (Cu,Zn)-pMMO were still capable of supporting the activation of dioxygen when reduced, and that the 14N-superhyperfine features associated with one of the type 2 Cu(II) centers in the hydroxylation C-cluster remained unperturbed. The replacement of the E-cluster copper ions by Zn ions did compromise the ability of the protein to mediate the transfer of reducing equivalents from exogenous reductants to the C-clusters. These observations provide strong support for the electron transfer and catalytic roles for the E-cluster and C-cluster copper ions, respectively.