Damage to the cytoplasmic membrane of Escherichia Coli by catechin-copper (II) complexes

In the presence of a nonlethal concentration of Cu(II), washed Escherichia coli ATCC11775 cells were killed by (-)-epigallocatechin (EGC) and (-)-epicatechin (EC). Cell killing was accompanied by a depletion in both the ATP and potassium pools of the cells, but the DNA double strand was not broken, indicating that the bactericidal activity of catechins in the presence of Cu(II) results from damage to the cytoplasmic membrane. Induction of endogenous catalase in E. coli cells increased their resistance to being killed by the combination of catechins and Cu(II). In all cases studied, EGC and EC with Cu(II) were found to generate hydrogen peroxide, but its concentration was too low to account for the bactericidal activity. The bactericidal activity of EGC in the presence of Cu(II) was completely suppressed by ethylenediaminetetraacetate, bathocuproine, catalase, superoxide disumutase (SOD), heated catalase, and heated SOD, but not by dimethyl sulfoxide. When catalase, either heated or unheated, was added to the cells incubated with EGC in the presence of Cu(II), it completely inhibited further killing of the cells. These findings suggest that recycling redox reactions between Cu(II) and Cu(I), involving catechins and hydrogen peroxide on the cell surface, must be important in the mechanism of the killing.     

On the role of bicarbonate in peroxidations catalyzed by Cu,Zn superoxide dismutase

The interaction of Cu,ZnSOD with H2O2 generates an oxidant at the active site that can then cause either the inactivation of this enzyme or the oxidation of a variety of exogenous substrates. We show that the rate of inactivation, imposed by 10-mM H2O2 at 25 degrees C and pH 7.2, is not influenced by 10-mM HCO3-; whereas the oxidation of 2,2'-azino-bis-[3-ethylbenzothiazoline sulfonate] (ABTS=) is virtually completely dependent upon HCO3-. The reduction of the active site Cu(II) by H2O2, which precedes inactivation of the enzyme, occurred at the same rate in phosphate buffer with or without bicarbonate added. These results indicate that HCO3- does not play a role in facilitating the interaction of H2O2 with the active site copper, but they can be accommodated by the proposal that HCO3- is oxidized to HCO3*, which then diffuses from that site and causes the oxidation of substrates, such as ABTS=, that are too large to traverse the solvent access channel to the Cu(II).     

Manganese and copper promote the binding of dopamine to “serotonin binding proteins” in bovine frontal cortex

The authors previously reported that Fe²⁺ is capable of increasing the binding of dopamine and of serotonin to “serotonin binding proteins” which are present in soluble extracts from calf brain. In this study, it is shown that Mn²⁺ and Cu²⁺ are also capable of increasing the binding, but for dopamine only. As for Fe²⁺, Mn²⁺ and Cu²⁺ are likely to promote the binding by virtue of their ability to enhance the oxidation of dopamine into dopamine-O-quinone, a derivative which is known to undergo covalent association with sulfhydryl groups of proteins. Data such as the irreversible nature of the majority of the binding, the inhibitory action of reducing agents (sodium ascorbate) and of reagents which contain, or modify sulfhydryl groups (reduced glutathione) are compatible with such a mechanism. The three metal ions are also capable of inactivating part of the binding sites on SBP directly; this effect is more pronounced for Cu²⁺ than for Fe²⁺ and it is only weak for Mn²⁺. The Fe²⁺-mediated binding of dopamine is inhibited by the superoxide dismutase enzyme, and it was therefore suggested that Fe²⁺ enhances the oxidation of dopamine by virtue of its ability to produce superoxide radicals out of dissolved molecular oxygen. Such a mechanism does not appear to take place in the case of Mn²⁺ and Cu²⁺. Instead, it is likely that Cu²⁺ and dopamine form a complex which is highly susceptible towards oxidation by dissolved molecular oxygen. Mn²⁺, on the other hand, can easily be oxidized into Mn³⁺, which is capable to oxidize dopamine by itself. Chronic manganese intoxication (from exposure to manganese) and Wilson's disease (related to inadequate elimination of copper) go along with neurological symptoms which are very similar to those encountered in Parkinson's disease. Our data indicate that manganese and copper ions accelerate the oxidation of catecholamines to produce toxic quinones. These quinones could, at least in part, account for the degeneration of dopamininergic neurons in such pathologies.