Determination of Cu Environments in the CyanobacteriumAnabaena flos-aquaeby X-Ray Absorption Spectroscopy

Abstract

Whole cells and peptidoglycan isolated from cell walls of the cyanobacterium Anabaena flos-aquae were lyophilized and used at pH 2 and pH 5 in Cu(II) binding studies. X-ray absorption spectra measured at the Cu K-edge were used to determine the oxidation states and chemical environments of Cu species in the whole-cell and peptidoglycan samples. In the whole-cell samples, most of the Cu retained at both pH values was coordinated by phosphate ligands. The whole-cell fractions contained significant concentrations of Cu(I) as well as Cu(II). An X-ray absorption near-edge spectrum analysis suggested that Cu(I) was coordinated by amine and thiol ligands. An analysis of the peptidoglycan fractions found that more Cu was adsorbed by the peptidoglycan fraction prepared at pH 5, due to increased chelation by amine and carboxyl ligands. The peptidoglycan fractions, also referred to as the cell wall fractions, contained little or no Cu(I). The Cu loading level was 30 times higher in the cell wall sample prepared at pH 5 than in the sample prepared at pH 2. Amine and bidentate carboxyl ligands had similar relative levels of importance in cell wall peptidoglycan samples prepared at both pH values, but phosphate coordination was insignificant.Heavy metals damage the environment since they are nonbiodegradable and have toxic effects on plants, animals, and humans. Precipitation, coagulation, membrane-based processes, and ion exchange are some of the current approaches to the remediation of contaminated sites, but these methods are expensive and lose effectiveness as metal concentrations fall (54). The use of microbes for the bioremediation of trace metals has shown potential for enabling a site to meet regulatory specifications. Experiments have demonstrated the ability of bacteria to immobilize and concentrate metal ions (6,14,28,37,46).Mechanisms by which cells accumulate, sequester, and detoxify metals have been investigated. One indirect approach has been the determination of uptake and retention as a function of pH or competition with some other cation (14,16,20,35). Another has been to chemically modify plant or cell matter (for example, by esterification) to render specific classes of prospective ligands unavailable and to evaluate the impact on metal binding capacity (7,21). In a study of binding sites in cell walls of gram-positive bacteria, teichoic acid, presenting phosphate sites, was extracted with aqueous alkali in order to quantify its contribution to metal binding (7).The chemical studies described are informative but involve many assumptions due to the complexity of biological systems. Also, because of that complexity, few analytical probes can illuminate the chemistry of metals associated with cells. X-ray absorption spectroscopy (XAS) is element specific, returning information for a single atomic number in an arbitrarily complicated mixture (52). XAS is nondestructive, and no sample reduction or digestion is required which would alter the chemistry of the element of interest. XAS can be used to determine the ...

Human serum γ-globulin binds copper cations

“…The concentrations of copper binding to γ-globulin did not surpass 20-24 µM and were below the concentrations toxic for bacterial and human cells [5,10,12] or destabilizing biomacromolecules by many folds [2]. Hence, γ-globulin, which bound copper under conditions of metal exchange, is nontoxic and will not promote cleavage of structures (cells and biopolymers) reacting with it (if we rule out the possibility that the protein releases all bound cations simultaneously into the local microenvironment).…”

“…We previously showed that γ-globulin fraction proteins interact with copper cations in solution. Obvious changes in the protein conformation manifesting (depending on copper concentration) by unfolding of the molecule into extramolecular space or by compactization of the globule indicate that the metal is bound by sites located on the surface of γ-globulin molecule or in its inter-domain space [8,9].Binding and subsequent retention of copper by the biopolymer molecule can cause changes determined by the chemical properties of cations (toxicity of copper in supraphysiological concentrations [5,10,12], redox activity of metal capable of inducing cleavage or destabilization of biological structures binding it [1,2]) and by physicochemical changes in immunoactive serum proteins under physiological conditions (manifestation of new effector properties in the proteins which bound cations and transition of this protein into a new conformation status) [3,9].We studied conformation changes in γ-globulin during copper cation binding and evaluated the parameters of this binding. …”

“…Binding and subsequent retention of copper by the biopolymer molecule can cause changes determined by the chemical properties of cations (toxicity of copper in supraphysiological concentrations [5,10,12], redox activity of metal capable of inducing cleavage or destabilization of biological structures binding it [1,2]) and by physicochemical changes in immunoactive serum proteins under physiological conditions (manifestation of new effector properties in the proteins which bound cations and transition of this protein into a new conformation status) [3,9].…”

Biphasic Interaction of γ-Globulin with Copper Cations

“…Copper-binding sites are widely presented on biomacromolecules [10]. In antibodies the complementary sites of variable regions form 11 sites for copper and zinc binding [15].…”

Biophysical Methods for the Elucidation of the S-Layer Proteins/Metal Interaction

“…This ability is of great interest because of its potential use in the removal of metals from contaminated water or soil, as well as for the recovery of precious metals (Au, Pt, Pd, Rh) from industrial wastes. Several genera of microorganisms have been used for such purposes [30][31][32][33][34][35][36].…”