Additive effects of metal excess and superoxide,a highly toxic mixture in bacteria

Heavy metal contamination is a serious environmental problem. Understanding the toxicity mechanisms may allow to lower concentration of metals in the metal-based antimicrobial treatments of crops, and reduce metal content in soil and groundwater. Here, we investigate the interplay between metal efflux systems and the superoxide dismutase (SOD) in the purple bacterium Rubrivivax gelatinosus and other bacteria through analysis of the impact of metal accumulation. Exposure of the Cd²⁺-efflux mutant ΔcadA to Cd²⁺ caused an increase in the amount and activity of the cytosolic Fe-Sod SodB, thereby suggesting a role of SodB in the protection against Cd²⁺. In support of this conclusion, inactivation of sodB gene in the ΔcadA cells alleviated detoxification of superoxide and enhanced Cd²⁺ toxicity. Similar findings were described in the Cu⁺-efflux mutant with Cu⁺. Induction of the Mn-Sod or Fe-Sod in response to metals in other bacteria, including Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Vibrio cholera and Bacillus subtilis, was also shown. Both excess Cd²⁺ or Cu⁺ and superoxide can damage [4Fe-4S] clusters. The additive effect of metal and superoxide on the [4Fe-4S] could therefore explain the hypersensitive phenotype in mutants lacking SOD and the efflux ATPase. These findings underscore that ROS defence system becomes decisive for bacterial survival under metal excess.     

Determination of optimum conditions for antibiotic production byStreptomyces galbus

The nutritional requirements and cultural conditions for optimal production of a new extracellular antifungal antibiotic byStreptomyces galbus under laboratory conditions were determined. Glycerol and glucose were found to be the best carbon sources, while as N-source nitrate was preferred. Maximum titre was reached after 7 d of incubation at 30 °C at pH 6.8. The metal ions Cu²⁺, Zn²⁺, Mn²⁺ and Fe²⁺ had some promoting effect. Casein hydrolysate improved production, but yeast extract markedly inhibited. Growth in shake flasks favoured higher yield of the antibiotic in a shorter time.     

Biosorption of Heavy Metals by Marine Algae

The ability of four different algae (three brown and one red) that have not been previously studied to adsorb Cr³⁺, Co²⁺, Ni²⁺, Cu²⁺, and Cd²⁺ ions was investigated. The metal uptake was dependent on the type of biosorbent, with different accumulation affinities towards the tested elements. The HCl-treated biomass decreased the metal biosorptive capacity particularly in the case of Cr³ adsorption with Laurencia obtusa. The extent of uptake of the different metals with the tested algae was assessed under different conditions such as pH, time of algal residence in solution with the metal, and concentration of algal biomass. The rate of uptake of the different metals was very fast in the first 2 h; thereafter the increase in metal uptake was insignificant. The amount of the metal uptake (5–15 mg range) increased steeply by increasing the weight of the biomass. An exception was L. obtusa, where a parallel increase of the uptake of different metals was observed on increasing the algal mass from 5 to 50 mg. Received: 21 December 1999 / Accepted: 24 April 2000     

The regulation of ionic nickel uptake and cytotoxicity by specific amino acids and serum components

The effects of serum components and amino acids on the uptake and cytotoxicity of NiCl₂ were examined in cultured Chinese hamster ovary (CHO) cells. CHO cells maintained in a minimal salts/glucose medium accumulated 10-fold more⁶³Ni than did cells maintained in complete medium supplemented with 10% fetal bovine serum. Cell-surface binding of⁶³Ni appeared to account for the majority of this increased accumulation of cell-associated nickel observed in the simple maintenance medium since such increases were reduced 70% by trypsin treatment. The addition of the Ni²⁺-binding amino acids cysteine or histidine to the salts/glucose medium markedly decreased⁶³Ni accumulations, an effect not observed following addition of any of several amino acids that do not bind Ni²⁺. Supplementation of the salts/glucose medium with fetal bovine serum decreased in a concentration dependent fashion both the⁶³Ni²⁺ uptake and cell detachment caused by Ni²⁺, while dialyzed (amino acid-free) serum was 3–5-fold less effective than undialyzed serum at reducing⁶³Ni²⁺ uptake and similarly exhibited only a slight protective effect against nickel-induced cytotoxicity. Supplementation of dialyzed serum with cysteine at levels approximating those in whole serum partially restored its inhibitory activity toward nickel uptake by cells and restored completely its inhibition of nickel's cytotoxicity, indicating the predominant role of specific amino acids over serum proteins in regulating the uptake and subsequent cytotoxicity of Ni²⁺. Addition of cysteine to the salts/glucose medium during a 2 h exposure of cells to either 100 μM HgCl₂ or 1 mM NiCl₂ masked the cytotoxic effects of these metal ions. These results demonstrate the importance of extracellular small molecular weight metal ion chelators in altering the biological effects of metal ions at the level of metal uptake.     

Biosorption of Heavy Metals (Cd2+, Cu2+, Co2+, and Mn2+) by Thermophilic Bacteria,Geobacillus thermantarcticus and Anoxybacillus amylolyticus: Equilibrium and Kinetic Studies

ABSTRACT

Two strains of thermophilic bacteria, Geobacillus thermantarcticus and Anoxybacillus amylolyticus, were employed to investigate the biosorption of heavy metals including Cd²⁺, Cu²⁺, Co²⁺, and Mn²⁺ ions. The effects of different biosorption parameters such as pH (2.0–10.0), initial metal concentrations (10.0–300.0 mg L^?1), amount of biomass (0.25–10 g L^?1), temperature (30–80°C), and contact time (15–120 min) were investigated. Concentrations of metal ions were determined by using an inductively coupled plasma optical emission spectrometry (ICP-OES). Optimum pHs for Cd²⁺, Cu²⁺, Co²⁺, and Mn²⁺ biosorption by Geobacillus thermantarcticus were found to be 4.0, 4.0, 5.0, and 6.0, respectively. For Anoxybacillus amylolyticus, the optimum pHs for Cd²⁺, Cu²⁺, Co²⁺, and Mn²⁺ biosorption were found to be 5.0, 4.0, 5.0, and 6.0, respectively. The Cd²⁺, Cu²⁺, Co²⁺, and Mn²⁺ removals at 50 mg L^?1 in 60 min by 50 mg dried cells of Geobacillus thermantarcticus were 85.4%, 46.3%, 43.6%, and 65.1%, respectively, whereas 74.1%, 39.8%, 35.1%, and 36.6%, respectively, for Anoxybacillus amylolyticus. The optimum temperatures for heavy metal biosorption were near the optimum growth temperatures for both strains. Scatchard plot analysis was employed to obtain more compact information about the interaction between metal ions and biosorbents. The plot results were further studied to determine if they fit Langmuir and Freundlich models.     

Isolation and Properties of Dextranases from Bacteroides oralis Ig4a

Extracellular dextranases were extracted from a dextran-degrading microorganism, Bacteroides oralis Ig4a, which had been isolated from human dental plaque, and purified. Crude enzyme preparations obtained from a broth culture supernatant by salting out with ammonium sulfate were subjected to column chromatography on DEAE-cellulose and subsequent Bio-Gel p-100, followed by isoelectric focusing. Two kinds of enzyme preparations, Enzymes I and II, with the ability to degrade soluble dextran were obtained. The optimal pHs of Enzymes I and II were 5.5 and 6.8, and the isoelectric points were pH 4.5 and 6.5, respectively. The molecular weights of Enzymes I and II were estimated by SDS-PAGE to be 44,000 and 52,000. Both enzymes were inhibited by Pb²⁺ and Fe³⁺, but not by Ca²⁺, Mg²+, Zn²⁺, or Fe²⁺. Neither the presence of EDTA nor iodoacetamide had any appreciable effect on the enzyme activity. The enzyme activity was independent of any of these metal ions. Enzyme I liberated glucose, isomaltose, maltotriose and higher oligosaccharides from dextran. In contrast, Enzyme II liberated only glucose from dextran and was assumed to be an exoglycosidase. Neither of the enzymes degraded modified insoluble glucan, which is a partially oxidized mutan of S. mutans containing predominantly α-(1, 3) linkages.     

Effect ofγ-irradiated carbohydrates on isolated synaptic membranes

The effect of-irradiated solutions of carbohydrates, mainly glucose, upon Na⁺, K⁺-ATPase and lipid peroxidation in rat brain synaptosomal membranes was studied. The membrane damage by irradiated glucose was enhanced in the presence of Fe²⁺ and was diminished when a free-radical scavenger (BHT) or metal chelators (EDTA, EGTA) were present. It is suggested that a key element in the free-radical membrane damage by irradiated carbohydrates is an Fe²⁺-complex of some species of the radiolysis products. Participation of radiotoxins of carbohydrate origin in radiobiological effects is discussed.     

Inhibition of the biosynthesis ofN-acetylneuraminic acid by metal ions and seleniumin vitro

In liver homogenate the biosynthesis ofN-acetylneuraminic acid usingN-acetylglucosamine as precursor can be followed stepwise by applying different chromatographic procedures. In this cell-free system 16 metal ions (Zn²⁺, Mn²⁺, La³⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Ce³⁺, Cd²⁺, Fe²⁺, Fe³⁺, Al³⁺, Sn²⁺, Cs⁺ and Li⁺) and the selenium compounds, selenium(IV) oxide and sodium selenite, have been checked with respect to their ability to influence a single or possible several steps of the biosynthesis ofN-acetylneuraminic acid. It could be shown that the following enzymes are sensitive to these metal ions (usually applied at a concentration of 1 mmoll^–1):N-acetylglucosamine kinase (inhibited by Zn²⁺ and vandate), UDP-N-acetylglucosamine-2-epimerase (inhibited by zn²⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Cd²⁺, Fe³⁺, Cs⁺, Li⁺, selenium(IV) oxide and selenite), andN-acetylmannosamine kinase (inhibited by Zn²⁺, Cu²⁺, Cd²⁺, and Co²⁺). Dose dependent measurements have shown that Zn²⁺, Cu²⁺ and selenite are more efficient inhibitors of UDP-N-acetylglucosamine-2-epimerase than vanadate. As for theN-acetylmannosamine kinase inhibition, a decreasing inhibitory effect exists in the following order Zn²⁺, Cd²⁺, Co²⁺ and Cu²⁺. In contrast, La³⁺, Al³⁺ and Mn²⁺ (1 mmoll^–1) did not interfere with the biosynthesis ofN-acetylneuraminic acid. Thus, the conclusion that the inhibitory effect of the metal ions investigated cannot be regarded as simply unspecific is justified.Dedicated to Professor Theodor Günther on the occasion of his 60th birthday     

Extracellular Ribonuclease Production by Rhizopus stolonifer: Influence of Metal Ions

A strain of Rhizopus stolonifer produced high levels of extracellular ribonuclease (RNase) when grown on YPG (yeast extract, peptone, glucose) medium. Influence of various medium components on the production of extracellular RNase activity showed that divalent metal ions had a marked effect on growth and enzyme production. Maximum enzyme activity (3000 U/ml) was obtained in 5 days when the culture was grown in YPG medium containing Mg²⁺ (12 mM), Mn²⁺, and Fe²⁺ (2 ppm each). Inorganic phosphate, however, repressed enzyme production. Antibodies raised against the purified extracellular RNase were then used to establish the relationship between intra- and extracellular enzymes.     

Heavy metal resistant <Emphasis Type="Italic">Distigma proteus</Emphasis> (Euglenophyta) isolated from industrial effluents and its possible role in bioremediation of contaminated wastewaters

Summary The alga, Distigma proteus, isolated from industrial wastewater showed tolerance against Cd²⁺ (8.0 μg/ml), Cr⁶⁺ (12 μg/ml), Pb²⁺ (15 μg/ml) and Cu²⁺ (10 μg/ml). The metal ions slowed down the growth of the organism after 4–5 days of exposure. The reduction in cell population was 90% for Cu²⁺, 84% for Cd²⁺, 71% for Cr⁶⁺, and 63% for Pb²⁺ after 8 days of metal stress. The order of resistance to heavy metal, in terms of reduction in the cellular population, was Cu²⁺ > Cd²⁺ > Cr⁶⁺ > Pb²⁺. Chromium- and cadmium-processing capabilities of the alga were worked out for its potential use as a bioremediator of wastewater. The reduction in the amount of Cr⁶⁺ after 2, 4, 6 and 8 days of algal culture containing 5.0 μg Cr⁶⁺ ml⁻¹ of culture medium was 77, 85, 92 and 97%, respectively. Distigma could also remove 48% Cd²⁺after 2 days, 68% after 4 days, 80% after 6 days and 90% after 8 days from the medium. The heavy metal uptake ability of Distigma can be exploited for metal detoxification and environmental clean-up operations.     

Metal cation binding by the hyperthermophilic microorganism,Archaea Methanocaldococcus Jannaschii,and its effects on silicification

Abstract: A series of experiments was conducted to determine the capacity of an archaeal strain, Methanocaldococcus jannaschii, to bind metals and to study the effects of metal binding on the subsequent silicification of the microorganisms. The results showed that M. jannaschii can rapidly bind several metal cations (Fe³⁺, Ca²⁺, Pb²⁺, Zn²⁺, Cu²⁺). Considering the lack of silicification of this strain without metal binding, these experiments demonstrate that Fe(III) ion binding to the cell wall components was of fundamental importance for successful silicification and, especially, for the excellent preservation of the cell wall. This study brings new elements to the understanding of fossilization processes, showing that the positive effect of Fe(III) on silicification, already known for Bacteria, can also apply to Archaea and that this preliminary binding can be decisive for the subsequent fossilization of these organisms. Knowledge of these mechanisms can be helpful for the search and the identification of microfossils in both terrestrial and extraterrestrials rocks, and in particular on Mars.     

Melafen action on the growth of cyanobacteria and green microalgae cultured in stress conditions

Melafen stimulating effect on cell growth of cyanobacteria Synechococcus sp. PCC 6301 cultures amounted to 30–45% at 1000 lx illumination. The melafen effect decreased when cell cultures were exposed at the illumination of the saturation range (4000 lx). Growth rate and biomass increase of Anabaena variabilis, as well as the observed melafen stimulating effect, were higher on nitrogen-free medium compared to a nitrogen-containing one by 20–25%. We conclude that melafen activates photosynthetic processes and, probably, stimulates fixation of the atmospheric nitrogen in the cells. Opposite to the stimulating effect of melafen, ions of the heavy metal Cd²⁺ inhibited both biomass increase and the average number of the cells in the cyanobacteria A. variabilis colonies. The melafen added to the medium together with the Cd²⁺ ions decreased their negative effect. The other heavy metal ions, Cu²⁺, inhibited the growth of the cyanobacteria Synechococcus sp. PCC 6301 and green microalgae Chlorella vulgaris but had a stimulation effect on carbohydrate excretion by the cell cultures. Again, the melafen decreased the toxic effect of Cu²⁺ in this case. We suppose that melafen has an antistress activity at heavy metal ions presence and reduces their toxic effect on growth of phototrophic microorganisms.     

Conformation and reactivity of DNA. IV. Base binding ability of transition metal ions to native DNA and effect on helix conformation with special reference to DNA-Zn(II) complex

The effects of metal ions of the first-row transition and of alkaline earth metals on the DNA helix conformation have been studied by uv difference spectra, circular dichroism, and sedimentation measurements. At low ionic strength (10^?3 M NaClO₄) DNA shows a maximum in the difference absorption spectra in the presence of Zn²⁺, Mn²⁺, Co²⁺, Cd²⁺, and Ni²⁺ but not with Mg²⁺ or Ca²⁺. The amplitude of this maximum is dependent on GC content as revealed by detailed studies of the DNA-Zn²⁺ complex of eight different DNA's. Pronounced changes also occur in the CD spectra of DNA transition metal complexes. A transition appears up to a total ratio of approximately 1 Zn²⁺ per DNA phosphate at 10^?3 M NaClO₄; then no further change was observed up to high concentrations. The characteristic CD changes are strongly dependent on the double-helical structure of DNA and on the GC content of DNA. Differences were also observed in hydrodynamic properties of DNA metal complexes as revealed by the greater increase of the sedimentation coefficient of native DNA in the presence of transition metal ions. Spectrophotometric acid titration experiments and CD measurements at acidic pH clearly indicate the suppression of protonation of GC base-pair regions on the addition of transition metal ions to DNA. Similar effects were not observed with DNA complexes with alkaline earth metal ions such as Mg²⁺ or Ca²⁺. The data are interpreted in terms of a preferential interaction of Zn²⁺ and of other transition metal ions with GC sites by chelation to the N-7 of guanine and to the phosphate residue. The binding of Zn²⁺ to DNA disappears between 0.5 M and 1 M NaClO₄, but complex formation with DNA is observable again in the presence of highly concentrated solutions of NaClO₄ (3?7.2 M NaClO₄) or at 0.5 to 2 M Mn²⁺. At relatively high cation concentration Mg²⁺ is also effective in changing the DNA comformation. These structural alterations probably result from both the shielding of negatively charged phosphate groups and the breakdown of the water structure along the DNA helix. Differential effects in CD are also observed between Mn²⁺, Zn²⁺ on one hand and Mg²⁺ on the other hand under these conditions. The greater sensitivity of the double-helical conformation of DNA to the action of transition metal ions is due to the affinity of the latter to electron donating sites of the bases resulting from the d electronic configuration of the metal ions. An order of the relative phosphate binding ability to base-site binding ability in native DNA is obtained as follows: Mg²⁺, Ba²⁺, < Ca²⁺ < Fe²⁺, Ni²⁺, Co²⁺ < Mn²⁺, Zn²⁺ < Cd²⁺ < Cu²⁺. The metal-ion induced conformational changes of the DNA are explained by alternation of the winding angle between base pairs as occurs in the transition from B to C conformation. These findings are used for a tentative molecular interpretation of some effects of Zn²⁺ and Mn²⁺ in DNA synthesis reported in the literature.     

Heavy metal ions affect the activity of DNA glycosylases of the Fpg family

Prokaryotic enzymes formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei) and their eukaryotic homologs NEIL1, NEIL2, and NEIL3 define the Fpg family of DNA glycosylases, which initiate the process of repair of oxidized DNA bases. The repair of oxidative DNA lesions is known to be impaired in vivo in the presence of ions of some heavy metals. We have studied the effect of salts of several alkaline earth and transition metals on the activity of Fpg-family DNA glycosylases in the reaction of excision of 5,6-dihydrouracil, a typical DNA oxidation product. The reaction catalyzed by NEIL1 was characterized by values K _m = 150 nM and k _cat = 1.2 min⁻¹, which were in the range of these constants for excision of other damaged bases by this enzyme. NEIL1 was inhibited by Al³⁺, Ni²⁺, Co²⁺, Cd²⁺, Cu²⁺, Zn²⁺, and Fe²⁺ in Tris-HCl buffer and by Cd²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ in potassium phosphate buffer. Fpg and Nei, the prokaryotic homologs of NEIL1, were inhibited by the same metal ions as NEIL1. The values of I₅₀ for NEIL1 inhibition were 7 μM for Cd²⁺, 16 μM for Zn²⁺, and 400 μM for Cu²⁺. The inhibition of NEIL1 by Cd²⁺, Zn²⁺, and Cu²⁺ was at least partly due to the formation of metal-DNA complexes. In the case of Cd²⁺ and Cu²⁺, which preferentially bind to DNA bases rather than phosphates, the presence of metal ions caused the enzyme to lose the ability for preferential binding to damaged DNA. Therefore, the inhibition of NEIL1 activity in removal of oxidative lesions by heavy metal ions may be a reason for their comutagenicity under oxidative stress.     

Interactions of DNA with divalent metal ions. I. 31P-nmr studies

³¹P-nmr has been used to investigate the specific interaction of three divalent metal ions, Mg²⁺, Mn²⁺, and Co⁺², with the phosphate groups of DNA. Mg²⁺ is found to have no significant effect on any of the ³¹P-nmr parameters (chemical shift, line-width, T₁, T₂, and NOE) over a concentration range extending from 20 to 160 mM. The two paramagnetic ions, Mn²⁺ and Co²⁺, on the other hand, significantly change the ³¹P relaxation rates even at very low levels. From an analysis of the paramagnetic contributions to the spin–lattice and spin–spin relaxation rates, the effective internuclear metal–phosphorus distances are found to be 4.5 ± 0.5 and 4.1 ± 0.5 Å for Mn²⁺ and Co²⁺, respectively, corresponding to only 15 ± 5% of the total bound Mn²⁺ and Co²⁺ being directly coordinated to the phosphate groups (inner-sphere complexes). This result is independent of any assumptions regarding the location of the remaining metal ions which may be bound either as outer-sphere complexes relative to the phosphate groups or elsewhere on the DNA, possibly to the bases. Studies of the temperature effects on the ³¹P relaxation rates of DNA in the absence and presence of Mn²⁺ and Co²⁺ yielded kinetic and thermodynamic parameters which characterize the association and dissociation of the metal ions from the phosphate groups. A two-step model was used in the analysis of the kinetic data. The lifetimes of the inner-sphere complexes are 3 × 10^?7 and 1.4 × 10^?5 s for Mn²⁺ and Co²⁺, respectively. The rates of formation of the inner-sphere complexes with the phosphate are found to be about two orders of magnitude slower than the rate of the exchange of the water of hydration of the metal ions, suggesting that expulsion of water is not the rate-determining step in the formation of the inner-sphere complexes. Competition experiments demonstrate that the binding of Mg²⁺ ions is 3–4 times weaker than the binding of either Mn²⁺ or Co²⁺. Since the contribution from direct phosphate coordination to the total binding strength of these metal ion complexes is small (～15%), the higher binding strength of Mn²⁺ and Co²⁺ may be attributed either to base binding or to formation of stronger outer-sphere metal–phosphate complexes. At high levels of divalent metal ions, and when the metal ion concentration exceeds the DNA–phosphate concentration, the fraction of inner-sphere phosphate binding increases. In the presence of very high levels of Mg²⁺ (e.g., 3.1M), the inner-sphere ? outer-sphere equilibrium is shifted toward ～100% inner-sphere binding. A comparison of our DNA results and previous results obtained with tRNA indicates that tRNA and DNA have very similar divalent metal ion binding properties. A comparison of the present results with the predictions of polyelectrolyte theories is presented.     

Uptake of heavy metals by <Emphasis Type="Italic">Stylonychia mytilus</Emphasis> and its possible use in decontamination of industrial wastewater

The heavy metal resistant ciliate, Stylonychia mytilus, isolated from industrial wastewater has been shown to be potential bioremediator of contaminated wastewater. The ciliate showed tolerance against Zn²⁺ (30 μg/mL), Hg²⁺ (16 μg/mL) and Ni²⁺ (16 μg/mL). The metal ions slowed down the growth of the ciliate as compared with the culture grown without metal stress. The reduction in cell population was 46% for Cd²⁺, 38% for Hg²⁺, 23% for Zn²⁺, 39% for Cu²⁺ and 51% for Ni²⁺ after 8 days of metal stress. S. mytilus reduced 91% of Cd²⁺, 90% of Hg²⁺ and 98% of Zn²⁺ from the medium after 96 h of incubation in a culture medium containing 10 μg/mL of the respective metal ions. Besides this, the ciliate could also remove 88% of Cu²⁺ and 73% Ni²⁺ from the medium containing 5 μg/mL of each metal after 96 h. The ability of Stylonychia to take up variety of heavy metals from the medium could be exploited for metal detoxification and environmental clean-up operations.     

Effects of metals on elastase fromPseudomonas aeruginosa SES-938-1

Among the numerous virulance factors produced byPseudomonas aeruginosa, elastase is the one most often associated with pathogenesis. In this study, effects of various metal ions on elastase from a new isolate ofP. aeruginosa (Strain SES-938-1) was investigated. Crude elastase was prepared from culture supernatant via salting out by ammonium sulfate, and then desalting and concentrating the sample using a centricon microconcentrator. Activities were measured at 450 nm usingN-succinyl-l-(ala)₃-p-nitroanilide as the substrate. The metal chelating agents EDTA and EGTA inhibited thePseudomonas elastase, which shows that the enzyme is a typical metalloproteinase. At a 10-mM concentration, Mn²⁺, Ni²⁺, and Zn²⁺ strongly inhibited the elastase, whereas Mg²⁺ effect was negligable. There was a gradual decrease in the enzyme activity in accordance with an increase in the concentration of metal ions.     

Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli

ZnuA is the periplasmic Zn²⁺-binding protein associated with the high-affinity ATP-binding cassette ZnuABC transporter from Escherichia coli. Although several structures of ZnuA and its homologs have been determined, details regarding metal ion stoichiometry, affinity, and specificity as well as the mechanism of metal uptake and transfer remain unclear. The crystal structures of E. coli ZnuA (Eco-ZnuA) in the apo, Zn²⁺-bound, and Co²⁺-bound forms have been determined. ZnZnuA binds at least two metal ions. The first, observed previously in other structures, is coordinated tetrahedrally by Glu59, His60, His143, and His207. Replacement of Zn²⁺ with Co²⁺ results in almost identical coordination geometry at this site. The second metal binding site involves His224 and several yet to be identified residues from the His-rich loop that is unique to Zn²⁺ periplasmic metal binding receptors. Electron paramagnetic resonance and X-ray absorption spectroscopic data on CoZnuA provide additional insight into possible residues involved in this second site. The second site is also detected by metal analysis and circular dichroism (CD) titrations. Eco-ZnuA binds Zn²⁺ (estimated K _d < 20 nM), Co²⁺, Ni²⁺, Cu²⁺, Cu⁺, and Cd²⁺, but not Mn²⁺. Finally, conformational changes upon metal binding observed in the crystal structures together with fluorescence and CD data indicate that only Zn²⁺ substantially stabilizes ZnuA and might facilitate recognition of ZnuB and subsequent metal transfer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Some metals inhibit the glutathione S‐transferase from Van Lake fish gills

Glutathione S‐transferases (GSTs) are the superfamily of multifunctional detoxification isoenzymes and play important role cellular signaling. The present article focuses on the role of Cd²⁺, Cu²⁺, Zn²⁺, and Ag⁺ in vitro inhibition of GST. For this purpose, GST was purified from Van Lake fish (Chalcalburnus tarichii Pallas) gills with 110.664 EU mg^?1 specific activity and 79.6% yield using GSH‐agarose affinity chromatographic method. The metal ions were tested at various concentrations on in vitro GST activity. IC₅₀ values were found for Cd⁺², Cu⁺², Zn⁺², Ag⁺ as 450.32, 320.25, 1510.13, and 16.43 μM, respectively. K _i constants were calculated as 197.05 ± 105.23, 333.10 ± 152.76, 1670.21 ± 665.43, and 0.433 ± 0.251 μM, respectively. Ag⁺ showed better inhibitory effect compared with the other metal ions. The inhibition mechanisms of Cd²⁺ and Cu²⁺ were non‐competitive, whereas Zn²⁺ and Ag⁺ were competitive. Co²⁺, Cr²⁺, Pb²⁺, and Fe³⁺ had no inhibitory activity on GST.