Kamila Stokowa-Sołtys

Revised Coordination Model and Stability Constants of Cu(II) Complexes of Tris Buffer

2-Amino-2-hydroxymethyl-propane-1,3-diol, or tris(hydroxymethyl)aminomethane (Tris), is probably the most common biochemical buffer used alone or in combination with other buffers because it is stable, unreactive, and compatible with most proteins and other biomolecules. Being nontoxic, it has even found applications in medicine. Tris is known, however, to coordinate transition metal ions, Cu(II) among them. Although often ignored, this feature affects interactions of Cu(II) ions with biomolecules, as Tris is usually used in high molar excess. Therefore, it is important to have precise knowledge on the stoichiometry, stability, and reactivity of cupric Tris complexes. The literature data are incoherent in this respect. We reinvestigated the complex formation in the Tris-Cu(II) system by potentiometry, UV-vis, ESI-MS, and EPR at a broad range of concentrations and ratios. We found, contrary to several previous papers, that the maximum stoichiometry of Tris to Cu(II) is 2 and at neutral pH, dimeric complexes are formed. The apparent affinity of Tris buffer for Cu(II), determined by the competitivity index (CI) approach [Krężel, A.; Wójcik, J.; Maciejczyk, M.; Bal, W. Chem. Commun. 2003, 6, 704-705] at pH 7.4 varies between 2 × 10(6) and 4 × 10(4) M(-1), depending on the Tris and Cu(II) concentrations and molar ratio.

Selective control of Cu(II) complex stability in histidine peptides by β-alanine.

The cooperativity of formation of 5-membered and 6-membered chelate rings is the driving force for specificity and selectivity in Cu(II) peptidic complexes. α-Amino acids enable the formation of 5-membered rings, while a 6-membered ring is provided by the coordination of the His side chain imidazole. Introduction of β-alanine is another way of creating a 6-membered ring in the Cu(II) complex. The potentiometric and spectroscopic (UV-vis and CD) study of Cu(II) complexation by a series of four peptides, AAH-am, ABH-am, BAH-am, and BBH-am (where B stands for β-alanine, and -am for C-terminal amide) revealed a very strong effect of the sizes of individual rings, with the order of complex stability AAH-am (5,5,6)>BAH-am (6,5,6)>ABH-am (5,6,6)≫BBH-am (6,6,6). The stabilities of ABH-am and BAH-am complexes are intermediate between those of strong His-3 peptides but these complexes are still able to saturate the coordination sphere of the Cu(II) ion at neutral pH. This fact opens up new possibilities in engineering specific peptide-based chelates.

High affinity of copper(II) towards amoxicillin, apramycin and ristomycin. Effect of these complexes on the catalytic activity of HDV ribozyme.

Three representatives of the distinct antibiotics groups: amoxicillin, apramycin and ristomycin A were studied regarding their impact on hepatitis D virus (HDV) ribozyme both in the metal-free form and complexed with copper(II) ions. Hence the Cu(II)-ristomycin A complex has been characterized by means of NMR, EPR, CD and UV-visible spectroscopic techniques and its binding pattern has been compared with the coordination modes estimated previously for Cu(II)-amoxicillin and Cu(II)-apramycin complexes. It has thus been found that all three antibiotics bind the Cu(II) ion in a very similar manner, engaging two nitrogen and two oxygen donors into coordination with the square planar symmetry in physiological conditions. All three tested antibiotics were able to inhibit the HDV ribozyme catalysis. However, in the presence of the complexes, the catalytic reactions were almost completely inhibited. It was important therefore to check whether the complexes used in lower concentrations could inhibit the HDV ribozyme catalytic activity, thus creating opportunities for their practical application. It turned out that the complexes used in the concentrations of 50μM influenced the catalysis much less effectively comparing to the 200 micromolar concentration. The kobs values were lower than those observed in the control reaction, in the absence of potential inhibitors: 2-fold for amoxicillin, ristomycin A and 3.3-fold for apramycin, respectively.

Capreomycin and hygromycin B modulate the catalytic activity of the delta ribozyme in a manner that depends on the protonation and complexation with Cu2+ ions of these antibiotics

Catalytic RNA molecules (ribozymes) have often been used for the testing of interactions of antibiotics with ribonucleic acids. We showed that the impact of capreomycin and hygromycin B on delta ribozyme catalysis might change dramatically, from stimulation to inhibition, depending on conditions. In order to evaluate possible mechanisms of modulation of the ribozyme catalytic activity we used our earlier data on species distribution for protonated forms of capreomycin and hygromycin B and their complexes with Cu(2+) ions at different pH values. We proposed that, upon inhibition, the protonated amino group of capreomycin was located in the ribozyme catalytic cleft interfering with binding catalytic Mg(2+). Such a mechanism was also supported by the results of ribozyme inhibition with capreomycin complexed with Cu(2+). The effects of stimulation of the delta ribozyme activity by capreomycin and hygromycin B were less pronounced than inhibition. Possibly, the amino functions of these antibiotics might be involved in a general acid-base catalysis performed by the ribozyme, acting as proton acceptors/donors.

Impact of Cu2 + ions on the structure of colistin and cell-free system nucleic acid degradation

Colistin and transition metal ions are commonly used as feed additives for livestock animals. This work presents the results of an analysis of combined potentiometric and spectroscopic (UV-vis, EPR, CD, NMR) data which lead to conclude that colistin is able to effectively chelate copper(II) ions. In cell-free system the oxidative activity of the complex manifests itself in the plasmid DNA destruction with simultaneous generation of reactive OH species, when accompanied by hydrogen peroxide or ascorbic acid. The degradation of RNA occurs most likely via a hydrolytic mechanism not only for complexed compound but also colistin alone. Therefore, huge amounts of the used antibiotic for nontherapeutic purposes might have a potential influence on livestock health.

A rice fungicide blasticidin S efficiently binds Cu(II) ions and prevents DNA from metal-induced damage

Blasticidin S is a representative of the aminoacylnucleoside class of antibiotics and it possesses fungicidal properties against the virulent fungus which causes a serious rice blast disease in Asia. It is widely used to control rice blast by foliar application as a crop protection agent. Serious copper contamination is observed in some areas of China. Moreover, some paddy soils present a potential risk of copper accumulation in the human body through the food chain, leading to several disorders. This work presents the results of combined potentiometric and spectroscopic (UV-visible, EPR, CD, NMR) data which lead to the conclusion that the antibiotic is capable of binding copper, and the resulting complexes are likely to form in the soil. The process of complex formation has a potential influence on the population feeding on a rice-based diet. Moreover, the results of electrophoretic experiments revealed that complexes do not cleave DNA. On the contrary, the presence of blasticidin S may prevent DNA from a Cu(II)-induced damage.

Mapping the interactions of selected antibiotics and their Cu2+ complexes with the antigenomic delta ribozyme

The interactions of selected antibiotics with the trans‐acting antigenomic delta ribozyme were mapped. Ribozyme with two oligonucleotide substrates was used, one uncleavable with deoxycytidine at the cleavage site, mimicking the initial state of ribozyme, and the other with an all‐RNA substrate mimicking, after cleavage, the product state. Mapping was performed with a set of RNA structural probing methods: Pb2+ ‐induced cleavage, nuclease digestion, and the selective 2′‐hydroxyl acylation analyzed by primer extension (SHAPE) approach. The experimental results combined with molecular modeling revealed different binding sites for neomycin B, amikacin and actinomycin D inside the ribozyme structure. Neomycin B, an aminoglycoside antibiotic, which strongly inhibited the catalytic properties of delta ribozyme, was bound to the pocket formed by the P1 stem, the P1.1 pseudoknot, and the J4/2 junction. Amikacin showed less effective binding to the ribozyme catalytic core, resulting in weak inhibition. Complexes of these aminoglycosides with Cu2+ ions were bound to the same ribozyme regions, but more effectively, showing lower Kd values. On the other hand, the Cu2+ complex of the cyclopeptide antibiotic actinonomycin D was preferentially intercalated into the P2 and the P4 double‐stranded region, and was three times more potent in ribozyme inhibition than the free antibiotic. In addition, some differences in SHAPE reactivities between the ribozyme forms containing all‐RNA and deoxycytidine‐modified substrates in the J4/2 region were detected, pointing to different ribozyme conformations before and after the cleavage event.

Peptides having antimicrobial activity and their complexes with transition metal ions

Peptide antibiotics are produced by bacterial, mammalian, insect or plant organisms in defense against invasive microbial pathogens. Therefore, they are gaining importance as anti-infective agents. There are a number of antibiotics that require metal ions to function properly. Metal ions play a key role in their action and are involved in specific interactions with proteins, nucleic acids and other biomolecules. On the other hand, it is well known that some antimicrobial agents possess functional groups that enable them interacting with metal ions present in physiological fluids. Some findings support a hypothesis that they may alter the serum metal ions concentration in humans. Complexes usually have a higher positive charge than uncomplexed compounds. This means that they might interact more tightly with polyanionic DNA and RNA molecules. It has been shown that several metal ion complexes with antibiotics promote degradation of DNA. Some of them, such as bleomycin, form stable complexes with redox metal ions and split the nucleic acids chain via the free radicals mechanism. However, this is not a rule. For example blasticidin does not cause DNA damage. This indicates that some peptide antibiotics can be considered as ligands that effectively lower the oxidative activity of transition metal ions.

Characterization of Highly Efficient RNA-Cleaving DNAzymes that Function at Acidic pH with No Divalent Metal-Ion Cofactors

Abstract Here, we describe the characterization of new RNA‐cleaving DNAzymes that showed the highest catalytic efficiency at pH 4.0 to 4.5, and were completely inactive at pH values higher than 5.0. Importantly, these DNAzymes did not require any divalent metal ion cofactors for catalysis. This clearly suggests that protonated nucleic bases are involved in the folding of the DNAzymes into catalytically active structures and/or in the cleavage mechanism. The trans‐acting DNAzyme variants were also catalytically active. Mutational analysis revealed a conservative character of the DNAzyme catalytic core that underpins the high structural requirements of the cleavage mechanism. A significant advantage of the described DNAzymes is that they are inactive at pH values close to physiological pH and under a wide range of conditions in the presence of monovalent and divalent metal ions. These pH‐dependent DNAzymes could be used as molecular cassettes in biotechnology or nanotechnology, in molecular processes that consist of several steps. The results expand the repertoire of DNAzymes that are active under nonphysiological conditions and shed new light on the possible mechanisms of catalysis.

Copper(II) complexes with Fusobacterium nucleatum adhesin FadA: Coordination pattern, physicochemical properties and reactivity

Fusobacterium nucleatum is an anaerobic, Gram-negative bacteria linked to colon cancer. It is interesting to determine how metal ions interact with bacterial adhesin proteins. To this end, the coordination of ATDAAS-NH2 and MKKFL-NH2 fragments of Fusobacterium adhesin A (FadA) to copper(II) ions was studied by potentiometry, spectroscopic techniques (UV-Vis, CD, EPR and NMR) and the density functional theory (DFT) methods. At pH 6.8 (colon physiological pH), the metal ion in the first peptide (ATDAAS-NH2) is coordinated by one oxygen and three nitrogen donors while in the second one (MKKFL-NH2) - by sulfur and three nitrogen atoms. Both complexes form two five- and one six-membered stable chelate rings. Moreover, reactivity studies confirmed the production of reactive oxygen species such as hydroxyl radical, superoxide anion radical and singlet oxygen. Generation of reactive oxygen species (ROS) was observed during gel electrophoresis and spectroscopic assays with reporting molecules like NDMA (N,N-dimethyl-p-nitrosoaniline) and NBT (Nitrotetrazolium Blue Chloride). All reactions were conducted in the presence of hydrogen peroxide as endogenous oxidant.