Reducing centers on the surface of Escherichia coli bacteria and their role in copper-induced plasma membrane permeability

The reducing properties of Escherichia coli and their role in the induction of nonselective cationic permeability of plasma membrane by the action of Cu2+ ions were studied. The ability of cells to reduce exogenous dithiopyridine was shown to be maximal in freshly collected culture and to decrease upon starvation or exhaustion of bacteria by dinitrophenol, in the presence of other oxidants of cell thiols in the medium, and after the disturbance of the barrier properties of membrane by tetrachloracetic acid or butanol. The alkylation of cell thiols accessible for N-ethyl maleimide completely disrupted the reducing activity of bacteria. These data are consistent with the conception that the reduction of dithiopyridine and Cu2+ ions by bacteria occurs on the thiol-containing centers of the cell surface, which are continuously reduced by the transfer of cell reducing equivalents from the inner to the outer surface of plasma membrane. The analysis of data on the effect of external oxidizing and reducing agents on the copper-induced plasmolysis of bacteria showed that the induction of membrane permeability by the action of copper can occur upon interaction with critical targets on the surface of Cu+ ions formed in the periplasmic space in the reaction of Cu2+ ions with reducing centers.     

Structural modifications of the surface of Escherichia coli bacteria and copper-induced permeability of plasma membrane

The effect of Cu2+ on the structural organization of the cell surface of Escherichia coli bacteria during the induction of conductivity of a plasma membrane was studied. A fluorescent study did not reveal any substantial changes in the microviscosity of lipids by the action of copper ions. At the same time, a substantial reorganization of membrane proteins during plasmolysis was observed. A model of the copper-induced structural reorganization of membrane lipids was constructed, according to which the reorganization leads to the opening in the membrane of channels of nonspecific conductivity for cations. The opening of conductivity channels results from the break of disulfide bonds in critical membrane proteins during the interaction with Cu+, which form either due to the reduction of Cu2+ on specific sites of cell surface or by means of external reducing agents.     

Mutation spectrum of copper-induced DNA damage.

The ability of metal ions to damage DNA and cause mutagenesis has been analyzed with reversion and forward mutation assays using single-stranded DNA templates. We previously reported that incubation of phi X174 am3 DNA with Fe2+ in vitro results in mutagenesis when the treated DNA is transfected into Escherichia coli spheroplasts (Loeb, L. A., James, E. A., Waltersdorph, A. M., and Klebanoff, S. J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3918-3922, 1988). We now extend these studies to other metal ions. Of the metal ions tested, copper ions were the most mutagenic; the frequency of mutants produced was equal to or greater than that produced by Fe2+. Mutagenesis by Cu+ was diminished by catalase, mannitol, and superoxide dismutase suggesting the involvement of H2O2, hydroxyl ions, and superoxide, respectively. However, the findings that Cu+ and Cu2+ are nearly equally mutagenic and that the mutagenic activities are not completely inhibited by oxygen free radical scavengers make it unlikely that the mechanism for mutagenesis is simply the production of hydroxyl free radicals. The spectra of mutations produced by either copper ion using the lacZ gene as a target are very similar and differ from those reported with other agents. The predominant mutagenic sequence changes are single-base substitutions, the most frequent being replacement of a template C by a T. This transition presumably results from mispairing of an altered C with deoxyadenosine. Copper-induced mutations are not randomly distributed. Instead, they are found predominantly in clusters suggesting direct interaction of copper ions with specific nucleotide sequences in DNA. Evidence is considered that the high frequency of C----T transitions may be a common manifestation of DNA damage by oxygen radicals.     

Analysis of the binding site of copper with intact and Escherichia coli bacteria modified by N-ethylmaleimide by ESR]

Characteristics of copper binding sites in the bacteria E. coli were studied using ESR spectroscopy. It was found that these cations had high local density on the strong binding range represented by the two type sites. The former include nitrogen and oxygen ligands and the second ones--sulfur of the thiol biomolecules. The weak coupling Cu(II) sites of E. coli represent more polar nitrogen-oxygen environment. Blocking SH-groups by N-ethylmaleimide makes them inaccessible for copper ligation, sharply increases the percentage of ESR-detectable copper of the strong-binding sites and prevents the membrane breakdown by these cations. The results suggest that the Cu(2+)-induced membrane damage is the effect of Cu2+ binding with the SH-containing sites of the bacterial membrane.     

Electron paramagnetic resonance study of water distribution in starch granules

An electron paramagnetic resonance (EPR) study was performed for potato and wheat starch containing Cu2+ ions as a paramagnetic probe. Distribution of water in the starch granules as well as the interactions between the copper and starch matrix of different crystalline structures were determined. EPR spectra of the native starches consisted of two different centers of Cu2+. One of them, giving at 293 and 77 K an EPR signal of axial symmetry with a well-resolved hyperfine structure (HFS), was assigned to the Cu2+ -starch complex in which Cu2+ ions strongly interacted with oxygen atoms of the starch matrix. Another Cu2+ species, exhibiting an isotropic signal at 293 K and an axial signal with resolved HFS at 77 K, was attributed to a [Cu(H2O)6]2+ complex freely rotating at room temperature and immobilized at low temperatures. Interaction of Cu2+ with the starch matrix and the relative number of the particular copper species depended on the crystallographic type of starch. Dehydration at 393 K resulted in elimination of the rotating complex signal and decrease of the total intensity of the EPR spectrum caused by clustering of the Cu2+ ions. Freezing at 77 K and thawing led to restoring of the spectrum intensity and reappearing of the signal of the [Cu(H2O)6]2+ complex. This effect, related to liberation of water molecules from the granule semicrystalline growth rings on freezing/thawing, was especially visible for wheat starch, indicating differences in the water retention ability of starch granules of different crystallographic structure.     

Stimulation of oxygen evolution in photosystem II by copper(II) ions

We have found that copper(II) ions at about equimolar Cu2+/photosystem II (PS II) reaction center proportions stimulate oxygen evolution nearly twofold. This high affinity Cu-binding site is different from the binding sites of Mn and Ca ions. The analysis of the Cu2+ content in PS II preparations isolated from wild-type tobacco and a tobacco mutant deficient in light-harvesting complex suggests that Cu2+ may be a native component of PS II and may take part in the oxygen evolution process. At higher concentrations, Cu2+ ions inhibit oxygen evolution and quench fluorescence.     

Modification of radiosensitivity of chlorpromazine with biological metal ions in Thiobacillus ferrooxidans

Effect of chlorpromazine with biological metal ions, viz. calcium, magnesium, zink and copper was studied on T. ferrooxidans cell system. Chlorpromazine, calcium and magnesium alone could produce radioprotection. Maximum radioprotection was exhibited by chlorpromazine at lower concentration while copper and zink offered radiosensitization. However, combination of chlorpromazine with all biological metal ions exhibited radiosensitization. Dose modifying factor by chlorpromazine at lower concentration (0.025 mM) was 0.754 while in combination with Ca2+, Mg2+, Cu2+ and Zn2+ was 1.08, 1.25, 1.37 and 1.389 respectively. The possible interaction between chlorpromazine and biological metal ions is discussed at cellular membrane level.     

DNA structural transitions induced by divalent metal ions in aqueous solutions

Using methods of IR spectroscopy, light scattering, gel-electrophoresis DNA structural transitions are studied under the action of Cu2+, Zn2+, Mn2+, Ca2+ and Mg2+ ions in aqueous solution. Cu2+, Zn2+, Mn2+ and Ca2+ ions bind both to DNA phosphate groups and bases while Mg2+ ions-only to phosphate groups of DNA. Upon interaction with divalent metal ions studied (except for Mg2+ ions) DNA undergoes structural transition into a compact form. DNA compaction is characterized by a drastic decrease in the volume occupied by DNA molecules with reversible formation of DNA dense particles of well-defined finite size and ordered morphology. The DNA secondary structure in condensed particles corresponds to the B-form family. The mechanism of DNA compaction under Mt2+ ion action is not dominated by electrostatics. The effectiveness of the divalent metal ions studied to induce DNA compaction correlates with the affinity of these ions for DNA nucleic bases: Cu2+>Zn2+>Mn2+>Ca2+>Mg2+. Mt2+ ion interaction with DNA bases (or Mt2+ chelation with a base and an oxygen of a phosphate group) may be responsible for DNA compaction. Mt2+ ion interaction with DNA bases can destabilize DNA causing bends and reducing its persistent length that will facilitate DNA compaction.     

Assessment of the anti-biofouling potentials of a copper iodide-doped nylon mesh

We propose a copper iodide (CuI)-doped nylon mesh prepared using polyiodide ions as a precursor toward anti-biofouling polymer textile. The CuI-doped nylon mesh was subjected to the prevention of biofouling in marine environments. The attachment of the marine organisms was markedly inhibited on the CuI-doped nylon mesh surface until 249 days. Scanning electron microscopy-energy dispersive X-ray analysis indicated that copper compounds were maintained in the nylon mesh after the field experiment, although copper content in the nylon mesh was reduced. Therefore, the copper ions slowly dissolved from nylon mesh will contribute to the long-term prevention of biofouling. Furthermore, electron spin resonance analysis revealed the generation of reactive oxygen species (ROS) from CuI-doped nylon mesh after the field experiment. One of the possibilities for toxic action of copper ions will be the direct effect of Cu+ -induced ROS on biofilm forming on nylon mesh surface. The proposed polymer textile can be applied to fishing and aquafarming nets, mooring rope for ship, or silt fence to restrict polluted water in marine environments.     

Identification and characterization of a copper-binding site in αA-crystallin

Previous studies have shown that both αA- and αB-crystallins bind Cu2+, suppress the formation of Cu2+-mediated active oxygen species, and protect ascorbic acid from oxidation by Cu2+. αA- and αB-crystallins are small heat shock proteins with molecular chaperone activity. In this study we show that the mini-αA-crystallin, a peptide consisting of residues 71-88 of αA-crystallin, prevents copper-induced oxidation of ascorbic acid. Evaluation of binding of copper to mini-αA-crystallin showed that each molecule of mini-αA-crystallin binds one copper molecule. Isothermal titration calorimetry and nanospray mass spectrometry revealed dissociation constants of 10.72 and 9.9 μM, respectively. 1,1'-Bis(4-anilino)naphthalene-5,5'-disulfonic acid interaction with mini-αA-crystallin was reduced after binding of Cu2+, suggesting that the same amino acids interact with these two ligands. Circular dichroism spectrometry showed that copper binding to mini-αA-crystallin peptide affects its secondary structure. Substitution of the His residue in mini-αA-crystallin with Ala abolished the redox-suppression activity of the peptide. During the Cu2+-induced ascorbic acid oxidation assay, a deletion mutant, αAΔ70-77, showed about 75% loss of ascorbic acid protection compared to the wild-type αA-crystallin. This difference indicates that the 70-77 region is the primary Cu2+-binding site(s) in human native full-size αA-crystallin. The role of the chaperone site in Cu2+ binding in native αA-crystallin was confirmed by the significant loss of chaperone activity by the peptide after Cu2+ binding.     

Mechanism of catalysis by o-diphenol oxidase]

The reaction of terminal oxidation of the substrate (catechol) by molecular oxygen catalyzed by o-diphenoloxidase (o-diphenol: oxygen oxydoreductase; EC 1.10.3.1) is found to occur via a free radical mechanism. The copper of the active center changes its valency during the reaction. The spectra of substrate radicals and of the Cu2+ ions were registered by means of a high sensitivity ESR-spectrometer and their concentrations were determined.     

Oxygen-independent induction of enzyme activities related to oxygen metabolism in yeast by copper

Aerobic growth of Saccharomyces cerevisiae in the presence of CuSO4 (between 0.1 and 1 mM) caused a generalized induction of major enzyme activities involved in 'housekeeping' routes of oxygen metabolism (cytochrome oxidase, glutathione peroxidases and catalase) which were comparable to or higher than that observed with Cu,Zn-superoxide dismutase. Fumarase and glutathione transferase, tested as controls for oxygen-unrelated activities, were found to decrease under the same conditions. In the absence of oxygen, copper addition to yeast resulted in significant increases of Cu,Zn-superoxide dismutase and glutathione peroxidases and a slight increase of cytochrome oxidase, with catalase remaining undetectable irrespective of whether or not copper was present. Other metal ions tested (Mn2+, Co2+) were unable to produce such effects. It is concluded that copper has a general inducing effect on enzymes related to metabolism of oxygen and oxygen derivatives, which is mediated neither by formation of O2-. and H2O2 nor by interaction with copper-specific apoproteins. These results point to a general role of copper as regulator of the expression of major enzyme activities involved in biological oxygen activation.     

Redox silencing of copper in metal-linked neurodegenerative disorders: reaction of Zn7metallothionein-3 with Cu2+ ions

Dysregulation of copper and zinc homeostasis in the brain plays a critical role in Alzheimer disease (AD). Copper binding to amyloid-beta peptide (Abeta) is linked with the neurotoxicity of Abeta and free radical damage. Metallothionein-3 (MT-3) is a small cysteine- and metal-rich protein expressed in the brain and found down-regulated in AD. This protein occurs intra- and extracellularly, and it plays an important role in the metabolism of zinc and copper. In cell cultures Zn7MT-3, by an unknown mechanism, protects neurons from the toxicity of Abeta. We have, therefore, used a range of complementary spectroscopic and biochemical methods to characterize the interaction of Zn7MT-3 with free Cu2+ ions. We show that Zn7MT-3 scavenges free Cu2+ ions through their reduction to Cu+ and binding to the protein. In this reaction thiolate ligands are oxidized to disulfides concomitant with Zn2+ release. The binding of the first four Cu2+ is cooperative forming a Cu(I)4-thiolate cluster in the N-terminal domain of Cu4,Zn4MT-3 together with two disulfides bonds. The Cu4-thiolate cluster exhibits an unusual stability toward air oxygen. The results of UV-visible, CD, and Cu(I) phosphorescence at 77 K suggest the existence of metal-metal interactions in this cluster. We have demonstrated that Zn7MT-3 in the presence of ascorbate completely quenches the copper-catalyzed hydroxyl radical (OH.) production. Thus, zinc-thiolate clusters in Zn7MT-3 can efficiently silence the redox-active free Cu2+ ions. The biological implication of our studies as to the protective role of Zn7MT-3 from the Cu2+ toxicity in AD and other neurodegenerative disorders is discussed.     

Fre1p Cu2+ reduction and Fet3p Cu1+ oxidation modulate copper toxicity in Saccharomyces cerevisiae

Fre1p is a metalloreductase in the yeast plasma membrane that is essential to uptake of environmental Cu2+ and Fe3+. Fet3p is a multicopper oxidase in this membrane essential for high affinity iron uptake. In the uptake of Fe3+, Fre1p produces Fe2+ that is a substrate for Fet3p; the Fe3+ produced by Fet3p is a ligand for the iron permease, Ftr1p. Deletion of FET3 leads to iron deficiency; this deletion also causes a copper sensitivity not seen in wild type. Deletion of FTR1 leads to copper sensitivity also. Production in the ftr1delta strain of an iron-uptake negative Ftr1p mutant, Ftr1p(RAGLA), suppressed this copper sensitivity. This Ftr1p mutant supported the plasma membrane targeting of active Fet3p that is blocked in the parental ftr1delta strain. A ferroxidase-negative Fet3p did not suppress the copper sensitivity in a fet3delta strain, although it supported the plasma membrane localization of the Fet3p.Ftr1p complex. Thus, loss of membrane-associated Fet3p oxidase activity correlated with copper sensitivity. Furthermore, in vitro Cu1+ was shown to be an excellent substrate for Fet3p. Last, the copper sensitivity of the fet3delta strain was suppressed by co-deletion of FRE1, suggesting that the cytotoxic species was Cu1+. In contrast, deletion of CTR1 or of FET4 did not suppress the copper sensitivity in the fet3delta strain; these genes encode the two major copper transporters in laboratory yeast strains. This result indicated that the apparent cuprous ion toxicity was not due to excess intracellular copper. These biochemical and physiologic results indicate that at least with respect to cuprous and ferrous ions, Fet3p can be considered a metallo-oxidase and appears to play an essential role in both iron and copper homeostasis in yeast. Its functional homologs, e.g. ceruloplasmin and hephaestin, could play a similar role in mammals.     

The metal centers of particulate methane monooxygenase from Methylosinus trichosporium OB3b

Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains approximately 2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended X-ray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 A. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.     

The octapeptide repeat region of prion protein binds Cu(II) in the redox-inactive state

The octapeptide repeat region of human prion protein is known to bind four Cu(II) ions per molecule. A peptide, Octa(4), representing this region was tested for inhibitory effects on copper-catalyzed oxidation of l-ascorbate or glutathione and on generation of OH(*) during the former reaction. The result indicated that the catalytic activity of the first Cu(II) ion bound to an Octa(4) molecule was completely suppressed. The valence state of the copper under reducing conditions was Cu(II), as determined by a newly developed method using bathocuproinedisulfonate under acidic conditions. Furthermore, it was shown that Escherichia coli cells expressing the octapeptide repeat region were significantly resistant to copper treatment compared with control cells. The results taken together indicate that prion protein can function to sequester copper ions in the redox-inactive state, rendering copper-induced generation of reactive oxygen species impossible.     

The role of oxygen in the inhibition of Escherichia coli respiration by copper ions

Effect of Cu2+ on E. coli respiration and the role of oxygen in toxic action of copper has been studied. Stimulation of respiration is observed at initial time after introduction of Cu2+. It is based on a nonspecific cell response to membrane damage. After finishing of transitory processes, Cu2+ influenced respiration by noncompetitive inhibition, i.e. copper-sensitive enzyme can be oxygenated or nonoxygenated, and only the latter form of the enzyme is inhibited.     

Biochemical oxygen demand sensor using Serratia marcescens LSY 4

A microbial biochemical oxygen demand (BOD) sensor consisting of Serratia marcescens LSY 4 and an oxygen electrode was prepared for estimation of the biochemical oxygen demand. The response of the BOD sensor was insensitive to pH in the range of pH 6.0-8.0, and the baseline drift of the signal was nearly absent even in unbuffered aqueous solution. Because heavy metal ions were precipitated from the phosphate buffer solution, unbuffered solution was used to investigate the effect of the concentration of heavy metal ions on the sensor response. Contrary to previous studies, not only Cu2+ and Ag+ but also Cd2+ and Zn2+ significantly decreased the response of the BOD sensor in unbuffered solution. Graft polymerization of sodium styrene sulfonate on the surface of the porous teflon membrane was carried out to absorb the heavy metal ions permeating through the membrane. Tolerance against Zn2+ was induced for S. marcescens LSY 4 to make the cells less sensitive to the presence of heavy metal ions. The membrane modification and the Zn2+ tolerance induction showed some positive effects in such a way that they reduced the inhibitory effects of Zn2+ and Cd2+ on the sensitivity of the BOD sensor. However, they had no effect on the protection of the cells against the interference of Cu2+ and Ag+ on the performance of the sensor.     

Copper ion redox state is critical for its effects on ion transport pathways and methaemoglobin formation in trout erythrocytes.

We have studied the mechanism of copper uptake by the cells, its oxidative action and effects on ion transport systems using rainbow trout erythrocytes. Cupric ions enter trout erythrocytes as negatively charged complexes with chloride and hydroxyl anions via the band 3-mediated Cl-/HCO3- exchanger. Replacement of Cl- by gluconate, and complexation of cupric ions with histidine abolish rapid Cu2+ uptake. Within the cell cupric ions interact with haemoglobin, causing methaemoglobin formation by direct electron transfer from heme Fe2+ to Cu2+, and consecutive proton release. Ascorbate-mediated reduction of cupric ions to cuprous decreases copper-induced metHb formation and proton release. Moreover, cuprous ions stimulate Na+H+ exchange and residual Na+ transport causing net Na+ accumulation in the cells. The effect requires copper binding to an externally facing thiol group. Copper-induced Na+ accumulation is accompanied by K+ loss occurring mainly via K+-Cl- cotransporter. Taurine efflux is also stimulated by copper exposure. However, net loss of osmolytes is not as pronounced as Na+ uptake and modest swelling of the cells occurs after 5 min of copper exposure. Taken together the results indicate that copper toxicity, including copper transport into the cells and its interactions with ion transport processes, depend on the valency and complex formation of copper ions.