JBIC Journal of Biological Inorganic Chemistry (v.21, #3)

Zinc oxide and titanium dioxide nanoparticles induce oxidative stress, inhibit growth, and attenuate biofilm formation activity of Streptococcus mitis by Shams Tabrez Khan; Javed Ahmad; Maqusood Ahamed; Javed Musarrat; Abdulaziz A. Al-Khedhairy (295-303).
Streptococcus mitis from the oral cavity causes endocarditis and other systemic infections. Rising resistance against traditional antibiotics amongst oral bacteria further aggravates the problem. Therefore, antimicrobial and antibiofilm activities of zinc oxide and titanium dioxide nanoparticles (NPs) synthesized and characterized during this study against S. mitis ATCC 6249 and Ora-20 were evaluated in search of alternative antimicrobial agents. ZnO and TiO2-NPs exhibited an average size of 35 and 13 nm, respectively. The IC50 values of ZnO and TiO2-NPs against S. mitis ATCC 6249 were 37 and 77 µg ml−1, respectively, while the IC50 values against S. mitis Ora-20 isolate were 31 and 53 µg ml−1, respectively. Live and dead staining, biofilm formation on the surface of polystyrene plates, and extracellular polysaccharide production show the same pattern. Exposure to these nanoparticles also shows an increase (26–83 %) in super oxide dismutase (SOD) activity. Three genes, namely bapA1, sodA, and gtfB like genes from these bacteria were identified and sequenced for quantitative real-time PCR analysis. An increase in sodA gene (1.4- to 2.4-folds) levels and a decrease in gtfB gene (0.5- to 0.9-folds) levels in both bacteria following exposure to ZnO and TiO2-NPs were observed. Results presented in this study verify that ZnO-NPs and TiO2-NPs can control the growth and biofilm formation activities of these strains at very low concentration and hence can be used as alternative antimicrobial agents for oral hygiene.
Keywords: ZnO; TiO2 ; Nanoparticles; Oral hygiene; Alternative antimicrobials; S. mitis

Smokers have an elevated risk of cardiovascular disease, but the origin(s) of this increased risk are incompletely defined. Evidence supports an accumulation of the oxidant-generating enzyme myeloperoxidase (MPO) in the inflamed artery wall, and smokers have high levels of SCN, a preferred MPO substrate, with this resulting in HOSCN formation. We hypothesised that HOSCN, a thiol-specific oxidant may target the iron-sulphur cluster of aconitase (both isolated, and within primary human coronary artery endothelial cells; HCAEC) resulting in enzyme dysfunction, release of iron, and conversion of the cytosolic isoform to iron response protein-1, which regulates intracellular iron levels. We show that exposure of isolated aconitase to increasing concentrations of HOSCN releases iron from the aconitase [Fe-S]4 cluster, and decreases enzyme activity. This is associated with protein thiol loss and modification of specific Cys residues in, and around, the [Fe-S]4 cluster. Exposure of HCAEC to HOSCN resulted in increased intracellular levels of chelatable iron, loss of aconitase activity and increased iron response protein-1 (IRP-1) activity. These data indicate HOSCN, an oxidant associated with oxidative stress in smokers, can induce aconitase dysfunction in human endothelial cells via Cys oxidation, damage to the [Fe-S]4 cluster, iron release and generation of IRP-1 activity, which modulates ferritin protein levels and results in dysregulation of iron metabolism. These data may rationalise, in part, the presence of increased levels of iron in human atherosclerotic lesions and contribute to increased oxidative damage and endothelial cell dysfunction in smokers. Similar reactions may occur at other sites of inflammation.
Keywords: Myeloperoxidase; Aconitase; Iron response protein-1; Iron release; Thiol oxidation; Hypothiocyanous acid; Inflammation; Cardiovascular disease

Studying metal ion binding properties of a three-way junction RNA by heteronuclear NMR by Simona Bartova; Maria Pechlaner; Daniela Donghi; Roland K. O. Sigel (319-328).
Self-splicing group II introns are highly structured RNA molecules, containing a characteristic secondary and catalytically active tertiary structure, which is formed only in the presence of Mg(II). Mg(II) initiates the first folding step governed by the κζ element within domain 1 (D1κζ). We recently solved the NMR structure of D1κζ derived from the mitochondrial group II intron ribozyme Sc.ai5γ and demonstrated that Mg(II) is essential for its stabilization. Here, we performed a detailed multinuclear NMR study of metal ion interactions with D1κζ, using Cd(II) and cobalt(III)hexammine to probe inner- and outer-sphere coordination of Mg(II) and thus to better characterize its binding sites. Accordingly, we mapped 1H, 15N, 13C, and 31P spectral changes upon addition of different amounts of the metal ions. Our NMR data reveal a Cd(II)-assisted macrochelate formation at the 5′-end triphosphate, a preferential Cd(II) binding to guanines in a helical context, an electrostatic interaction in the ζ tetraloop receptor and various metal ion interactions in the GAAA tetraloop and κ element. These results together with our recently published data on Mg(II) interaction provide a much better understanding of Mg(II) binding to D1κζ, and reveal how intricate and complex metal ion interactions can be.
Keywords: RNA; Metal ion binding; NMR

Copper(II) binding properties of hepcidin by Kanokwan Kulprachakarn; Yu-Lin Chen; Xiaole Kong; Maria C. Arno; Robert C. Hider; Somdet Srichairatanakool; Sukhvinder S. Bansal (329-338).
Hepcidin is a peptide hormone that regulates the homeostasis of iron metabolism. The N-terminal domain of hepcidin is conserved amongst a range of species and is capable of binding CuII and NiII through the amino terminal copper–nickel binding motif (ATCUN). It has been suggested that the binding of copper to hepcidin may have biological relevance. In this study we have investigated the binding of CuII with model peptides containing the ATCUN motif, fluorescently labelled hepcidin and hepcidin using MALDI-TOF mass spectrometry. As with albumin, it was found that tetrapeptide models of hepcidin possessed a higher affinity for CuII than that of native hepcidin. The log K 1 value of hepcidin for CuII was determined as 7.7. CuII binds to albumin more tightly than hepcidin (log K 1 = 12) and in view of the serum concentration difference of albumin and hepcidin, the bulk of kinetically labile CuII present in blood will be bound to albumin. It is estimated that the concentration of CuII-hepcidin will be less than one femtomolar in normal serum and thus the binding of copper to hepcidin is unlikely to play a role in iron homeostasis. As with albumin, small tri and tetra peptides are poor models for the metal binding properties of hepcidin.
Keywords: Peptide; Mass spectrometry; Homeostasis; Binding affinity; Biomedicine

Crystal retention on tubular cell surface inside renal tubules is considered as the earliest and crucial step for kidney stone formation. Therapeutics targeting this step would cease the development of kidney stone. This study thus aimed to investigate the potential role of epigallocatechin-3-gallate (EGCG), a major antioxidant found in green tea leaves, in the reduction of calcium oxalate monohydrate (COM) crystal binding onto renal tubular cells. Pretreatment of the cells with EGCG for up to 6 h significantly diminished crystal-binding capability in a dose-dependent manner. Indirect immunofluorescence assay without and with cell permeabilization followed by laser-scanning confocal microscopy revealed that EGCG significantly reduced surface expression of alpha-enolase, whereas its intracellular level was increased. Western blot analysis confirmed such contradictory changes in membrane and cytosolic fractions of EGCG-treated cells, whereas the total level in whole cell lysate remained unchanged. Moreover, overexpression of surface alpha-enolase and enhancement of cell–crystal adhesion induced by 10 mM sodium oxalate were completely abolished by EGCG. Taken together, these data indicate that EGCG decreases binding of COM crystals onto renal tubular cells by decreasing the surface expression of alpha-enolase via re-localization or inhibition of alpha-enolase shuttling from the cytoplasm to the plasma membrane. These findings may also explain the effects of EGCG in reducing COM crystal deposition in previous animal models of kidney stone disease. Thus, EGCG may be useful for the prevention of new or recurrent stone formation.
Keywords: Calcium oxalate; Crystal binding; EGCG; Enolase; Kidney stone; Renal tubular cells

Biological properties of novel ruthenium- and osmium-nitrosyl complexes with azole heterocycles by Maria S. Novak; Gabriel E. Büchel; Bernhard K. Keppler; Michael A. Jakupec (347-356).
Since the discovery that nitric oxide (NO) is a physiologically relevant molecule, there has been great interest in the use of metal nitrosyl compounds as antitumor pharmaceuticals. Particularly interesting are those complexes which can deliver NO to biological targets. Ruthenium- and osmium-based compounds offer lower toxicity compared to other metals and show different mechanisms of action as well as different spectra of activity compared to platinum-based drugs. Novel ruthenium- and osmium-nitrosyl complexes with azole heterocycles were studied to elucidate their cytotoxicity and possible interactions with DNA. Apoptosis induction, changes of mitochondrial transmembrane potential and possible formation of reactive oxygen species were investigated as indicators of NO-mediated damage by flow cytometry. Results suggest that ruthenium- and osmium-nitrosyl complexes with the general formula (indazolium)[cis/trans-MCl4(NO)(1H-indazole)] have pronounced cytotoxic potency in cancer cell lines. Especially the more potent ruthenium complexes strongly induce apoptosis associated with depolarization of mitochondrial membranes, and elevated reactive oxygen species levels. Furthermore, a slight yet not unequivocal trend to accumulation of intracellular cyclic guanosine monophosphate attributable to NO-mediated effects was observed.
Keywords: Ruthenium; Nitrosyl complexes; Cancer; Apoptosis; cGMP level

Anion–π interactions in complexes of proteins and halogen-containing amino acids by Sunčica Z. Borozan; Mario V. Zlatović; Srđan Đ. Stojanović (357-368).
We analyzed the potential influence of anion–π interactions on the stability of complexes of proteins and halogen-containing non-natural amino acids. Anion–π interactions are distance and orientation dependent and our ab initio calculations showed that their energy can be lower than −8 kcal mol−1, while most of their interaction energies lie in the range from −1 to −4 kcal mol−1. About 20 % of these interactions were found to be repulsive. We have observed that Tyr has the highest occurrence among the aromatic residues involved in anion–π interactions, while His made the least contribution. Furthermore, our study showed that 67 % of total interactions in the dataset are multiple anion–π interactions. Most of the amino acid residues involved in anion–π interactions tend to be buried in the solvent-excluded environment. The majority of the anion–π interacting residues are located in regions with helical secondary structure. Analysis of stabilization centers for these complexes showed that all of the six residues capable of anion–π interactions are important in locating one or more of such centers. We found that anion–π interacting residues are sometimes involved in simultaneous interactions with halogens as well. With all that in mind, we can conclude that the anion–π interactions can show significant influence on molecular organization and on the structural stability of the complexes of proteins and halogen-containing non-natural amino acids. Their influence should not be neglected in supramolecular chemistry and crystal engineering fields as well.
Keywords: Anion–π interactions; Halogen-containing amino acids; Proteins; Stabilization centers; Interaction energy

Spectroscopic and biological activity studies of the chromium-binding peptide EEEEGDD by Hirohumi Arakawa; Machender R. Kandadi; Evgeniy Panzhinskiy; Kenneth Belmore; Ge Deng; Ebony Love; Preshus M. Robertson; Juliette J. Commodore; Carolyn J. Cassady; Sreejayan Nair; John B. Vincent (369-381).
While trivalent chromium has been shown at high doses to have pharmacological effects improving insulin resistance in rodent models of insulin resistance, the mechanism of action of chromium at a molecular level is not known. The chromium-binding and transport agent low-molecular-weight chromium-binding substance (LMWCr) has been proposed to be the biologically active form of chromium. LMWCr has recently been shown to be comprised of a heptapeptide of the sequence EEEEDGG. The binding of Cr3+ to this heptapeptide has been examined. Mass spectrometric and a variety of spectroscopic studies have shown that multiple chromic ions bind to the peptide in an octahedral fashion through carboxylate groups and potentially small anionic ligands such as oxide and hydroxide. A complex of Cr and the peptide when administered intravenously to mice is able to decrease area under the curve in intravenous glucose tolerance tests. It can also restore insulin-stimulated glucose uptake in myotubes rendered insulin resistant by treating them with a high-glucose media.
Keywords: Chromium; Peptide; Spectroscopy; Glucose tolerance test; Myotubes

The [NiFe] hydrogenases catalyse the reversible conversion of H2 to protons and electrons. The active site consists of a Fe ion with one carbon monoxide, two cyanide, and two cysteine (Cys) ligands. The latter two bridge to a Ni ion, which has two additional terminal Cys ligands. It has been suggested that one of the Cys residues is protonated during the reaction mechanism. We have used combined quantum mechanical and molecular mechanics (QM/MM) geometry optimisations, large QM calculations with 817 atoms, and QM/MM free energy simulations, using the TPSS and B3LYP methods with basis sets extrapolated to the quadruple zeta level to determine which of the four Cys residues is more favourable to protonate for four putative states in the reaction mechanism, Ni-SIa, Ni-R, Ni-C, and Ni-L. The calculations show that for all states, the terminal Cys-546 residue is most easily protonated by 14–51 kJ/mol, owing to a more favourable hydrogen-bond pattern around this residue in the protein.
Keywords: [NiFe] hydrogenase; Protonation; Reaction mechanism; QM/MM; Big-QM calculations

The DNA sequence specificity of the cancer chemotherapeutic agent, bleomycin, was determined with high precision in purified plasmid DNA using an improved technique. This improved technique involved the labelling of the 5′- and 3′-ends of DNA with different fluorescent tags, followed by simultaneous cleavage by bleomycin and capillary electrophoresis with laser-induced fluorescence. This permitted the determination of bleomycin cleavage specificity with high accuracy since end-label bias was greatly reduced. Bleomycin produces single- and double-strand breaks, abasic sites and other base damage in DNA. This high-precision method was utilised to elucidate, for the first time, the DNA sequence specificity of bleomycin-induced DNA damage at abasic sites. This was accomplished using endonuclease IV that cleaves DNA at abasic sites after bleomycin damage. It was found that bleomycin-induced abasic sites formed at 5′-GC and 5′-GT sites while bleomycin-induced phosphodiester strand breaks formed mainly at 5′-GT dinucleotides. Since bleomycin-induced abasic sites are produced in the absence of molecular oxygen, this difference in DNA sequence specificity could be important in hypoxic tumour cells.
Keywords: Abasic DNA damage, bleomycin; Cleavage sites; DNA sequence specificity; End-label bias; Endonuclease IV

The combination of cytotoxic copper–thiosemicarbazone complexes with phenoxazines results in an up to 50-fold enhancement in the cytotoxic potential of the thiosemicarbazone against the MCF-7 human breast adenocarcinoma cell line over the effect attributable to drug additivity—allowing minimization of the more toxic copper–thiosemicarbazone component of the therapy. The combination of a benzophenoxazine with all classes of copper complex examined in this study proved more effective than combinations of the copper complexes with related isoelectronic azines. The combination approach results in rapid elevation of intracellular reactive oxygen levels followed by apoptotic cell death. Normal fibroblasts representative of non-cancerous cells (MRC-5) did not display a similar elevation of reactive oxygen levels when exposed to similar drug levels. The minimization of the copper–thiosemicarbazone component of the therapy results in an enhanced safety profile against normal fibroblasts.
Keywords: Copper; Cytotoxicity; Reactive oxygen species (ROS); Thiosemicarbazone; Benzophenoxazine

The nitrite reductase activity of horse heart carboxymethylated-cytochrome c is modulated by cardiolipin by Paolo Ascenzi; Diego Sbardella; Federica Sinibaldi; Roberto Santucci; Massimo Coletta (421-432).
Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO2 -mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k on = (7.3 ± 0.7) × 10−2 M−1 s−1; at pH 7.4], whereas the value of k on for NO2 reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M−1 s−1 (at pH 7.4). CL facilitates the NO2 -mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k on for the NO2 -mediated conversion of CL–CM-cytc-Fe(II) to CL–CM-cytc-Fe(II)-NO (5.6 ± 0.6 M−1 s−1; at pH 7.4) being slightly higher than that for the NO2 -mediated conversion of CL–cytc-Fe(II) to CL–cytc-Fe(II)-NO (2.6 ± 0.3 M−1 s−1; at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10−6 M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k on versus pH are −1.05 ± 0.07 and −1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH. These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL–CM-cytc.
Keywords: Horse heart carboxymethylated-cytochrome c ; Cardiolipin liposomes; Nitrite reductase activity; Kinetics