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Nonylphenol (NP), an environmentally persistent and toxic endocrine-disrupting chemical with estrogenic properties, has severe implications on humans and wildlife. Accumulating evidence demonstrates the toxic response of NP on the developmental process, nervous system, and reproductive parameters. Although NP exposure has been implicated in chronic liver injury, the underlying events associated with hepatic pathophysiology remain less investigated. Using male zebrafish (Danio rerio) as the model, the present study investigates the impact of environmentally relevant concentrations of NP (50 and 100 μg/L, 21 days) on hepatic redox homeostasis vis-a-vis cellular energy sensors, inflammatory response, and cell death involving a mechanistic insight into estrogen receptor (ER) modulation. Our results demonstrate that congruent with significant alteration in transcript abundance of antioxidant enzymes (SOD1, SOD2, Catalase, GPx1a, GSTα1), chronic exposure to NP promotes ROS synthesis, more specifically superoxide anions and H2O2 levels, and lipid peroxidation potentially through elevated NOX4 expression. Importantly, NP perturbation of markers associated with fatty acid biosynthesis (srebf1/fasn) and cellular energy-sensing network (sirt1/ampkα/pgc1α) indicates dysregulated energy homeostasis, metabolic disruption, and macrovesicular steatosis, albeit with differential sensitivity at the dose level tested. Besides, elevated p38-MAPK phosphorylation (activation) together with loss of ER homeostasis at both mRNA (esr1, esr2a, esr2b) and protein (ERα, ERβ) levels suggest that NP modulation of ER abundance may have a significant influence on hepatic events. Elevated expression of inflammatory markers (TLR4, p-NF-κB, TNF-α, IL-6, IL-1β, and NOS2) and pro-apoptotic and necrotic regulators, e.g., Bax, caspase- 8, -9 and cleaved PARP1 (50 kDa), indicate chronic inflammation and hepatotoxicity in NP-exposed males. Collectively, elevated oxidative stress, metabolic dysregulation and immune modulation may lead to chronic liver injury in organisms exposed to metabolic disrupting chemicals.

The HisJ protein from Escherichia coli and related Gram negative bacteria is the periplasmic component of a bacterial ATP-cassette (ABC) transporter system. Together these proteins form a transmembrane complex that can take up L-histidine from the environment and translocate it into the cytosol. We have studied the specificity of HisJ for binding L-His and many related naturally occurring compounds. Our data confirm that L-His is the preferred ligand, but that 1-methyl-L-His and 3-methyl-L-His can also bind, while the dipeptide carnosine binds weakly and D-histidine and the histidine degradation products, histamine, urocanic acid and imidazole do not bind. L-Arg, homo-L-Arg, and post-translationally modified methylated Arg-analogs also bind with reasonable avidity, with the exception of symmetric dimethylated-L-Arg. In contrast, L-Lys and L-Orn have considerably weaker interactions with HisJ and methylated and acetylated Lys variants show relatively poor binding. It was also observed that the carboxylate group of these amino acids and their variants was very important for proper recognition of the ligand. Taken together our results are a key step towards designing HisJ as a specific protein-based reagentless biosensor.

Brucella is a zoonotic pathogen requiring iron for its survival and acquires this metal through the expression of several high-affinity uptake systems. Of these, the newly discovered ferrous iron transporter, FtrABCD, is proposed to take part in ferrous iron uptake. Sequence homology shows that, FtrA, the proposed periplasmic ferrous-binding component, is a P19-type protein (a periplasmic protein from C. jejuni which shows Cu2+ dependent iron affinity). Previous structural and biochemical studies on other P19 systems have established a Cu2+ dependent Mn2+ affinity as well as formation of homodimers for these systems. The Cu2+ coordinating amino acids from these proteins are conserved in Brucella FtrA, hinting towards similar properties. However, there has been no experimental evidence, till date, establishing metal affinities and the possibility of dimer formation by Brucella FtrA. Using wild-type FtrA and Cu2+-binding mutants (H65A, E67A, H118A, and H151A) we investigated the metal affinities, folding stabilities, dimer forming abilities, and the molecular basis of the Cu2+ dependence for this P19-type protein employing homology modeling, analytical gel filtration, calorimetric, and spectroscopic methods. The data reported here confirm a Cu2+-dependent, low-μM Mn2+ (Fe2+ mimic) affinity for the wild-type FtrA. In addition, our data clearly show the loss of Mn2+ affinity, and the formation of less stable protein conformations as a result of mutating these conserved Cu2+-binding residues, indicating the important roles these residues play in producing a native and functional fold of Brucella FtrA.

Bordetella pertussis is the causative agent of whooping cough. This pathogenic bacterium can obtain the essential nutrient iron using its native alcaligin siderophore and by utilizing xeno-siderophores such as desferrioxamine B, ferrichrome, and enterobactin. Previous genome-wide expression profiling identified an iron repressible B. pertussis gene encoding a periplasmic protein (FbpABp). A previously reported crystal structure shows significant similarity between FbpABp and previously characterized bacterial iron binding proteins, and established its iron-binding ability. Bordetella growth studies determined that FbpABp was required for utilization of not only unchelated iron, but also utilization of iron bound to both native and xeno-siderophores. In this in vitro solution study, we quantified the binding of unchelated ferric iron to FbpABp in the presence of various anions and importantly, we demonstrated that FbpABp binds all the ferric siderophores tested (native and xeno) with μM affinity. In silico modeling augmented solution data. FbpABp was incapable of iron removal from ferric xeno-siderophores in vitro. However, when FbpABp was reacted with native ferric-alcaligin, it elicited a pronounced change in the iron coordination environment, which may signify an early step in FbpABp-mediated iron removal from the native siderophore. To our knowledge, this is the first time the periplasmic component of an iron uptake system has been shown to bind iron directly as Fe(3+) and indirectly as a ferric siderophore complex.

Haptoglobin (Hp) is an abundant and conserved plasma glycoprotein, which binds acellular adult hemoglobin (Hb) dimers with high affinity and facilitates their rapid clearance from circulation after hemolysis. Humans possess three main phenotypes of Hp, designated Hp 1-1, Hp 2-1, and Hp 2-2. These variants exhibit diverse structural configurations and have been reported to be functionally nonequivalent. We have investigated the functional and redox properties of Hb-Hp complexes prepared using commercially fractionated Hp and found that all forms exhibit similar behavior. The rate of Hb dimer binding to Hp occurs with bimolecular rate constants of ~0.9 μM(-1) s(-1), irrespective of the type of Hp assayed. Although Hp binding does accelerate the observed rate of HbO2 autoxidation by dissociating Hb tetramers into dimers, the rate observed for these bound dimers is three- to fourfold slower than that of Hb dimers free in solution. Co-incubation of ferric Hb with any form of Hp inhibits heme loss to below detectable levels. Intrinsic redox potentials (E1/2) of the ferric/ferrous pair of each Hb-Hp complex are similar, varying from +54 to +59 mV (vs NHE), and are essentially the same as reported by us previously for Hb-Hp complexes prepared from unfractionated Hp. All Hb-Hp complexes generate similar high amounts of ferryl Hb after exposure to hydrogen peroxide. Electron paramagnetic resonance data indicate that the yields of protein-based radicals during this process are approximately 4 to 5% and are unaffected by the variant of Hp assayed. These data indicate that the Hp fractions examined are equivalent to one another with respect to Hb binding and associated stability and redox properties and that this result should be taken into account in the design of phenotype-specific Hp therapeutics aimed at countering Hb-mediated vascular disease.

We compared oxygenation and anaerobic oxidation reactions of a purified complex of human hemoglobin (Hb) and haptoglobin (Hb-Hp) to those of uncomplexed Hb. Under equilibrium conditions, Hb-Hp exhibited active-site heterogeneity and noncooperative, high-affinity O(2) binding (n(1/2)=0.88, P(1/2)=0.33 mm Hg in inorganic phosphate buffer at pH 7 and 25 °C). Rapid-reaction kinetics also exhibited active-site heterogeneity, with a slower process of O(2) dissociation and a faster process of CO binding relative to uncomplexed Hb. Deoxygenated Hb-Hp had significantly reduced absorption at the λ(max) of 430 nm relative to uncomplexed Hb, as occurs for isolated Hb subunits that lack T-state stabilization. Under comparable experimental conditions, the redox potential (E(1/2)) of Hb-Hp was found to be +54 mV, showing that it is much more easily oxidized than uncomplexed Hb (E(1/2)=+125 mV). The Nernst plots for Hb-Hp oxidation showed no cooperativity and slopes less than unity indicated active-site heterogeneity. The redox potential of Hb-Hp was unchanged by pH over the range of 6.4-8.3. Exposure of Hb-Hp to excess hydrogen peroxide (H(2)O(2)) produced ferryl heme, which was found to be more kinetically inert in the Hb-Hp complex than in uncomplexed Hb. The negative shift in the redox potential of Hb-Hp and its stabilized ferryl state may be central elements in the protection against Hb-induced oxidative damage afforded by formation of the Hb-Hp complex.

Described here is a mass spectrometry-based protocol to study the thermodynamic stability of proteins and protein-ligand complexes using the chemical denaturant dependence of the slow H/D exchange reaction of the imidazole C(2) proton in histidine side chains. The protocol is developed using several model protein systems including: ribonuclease (Rnase) A, myoglobin, bovine carbonic anhydrase (BCA) II, hemoglobin (Hb), and the hemoglobin-haptoglobin (Hb-Hp) protein complex. Folding free energies consistent with those previously determined by other more conventional techniques were obtained for the two-state folding proteins, Rnase A and myoglobin. The protocol successfully detected a previously observed partially unfolded intermediate stabilized in the BCA II folding/unfolding reaction, and it could be used to generate a K(d) value of 0.24 nM for the Hb-Hp complex. The compatibility of the protocol with conventional mass spectrometry-based proteomic sample preparation and analysis methods was also demonstrated in an experiment in which the protocol was used to detect the binding of zinc to superoxide dismutase in the yeast cell lysate sample. The yeast cell sample analyses also helped define the scope of the technique, which requires the presence of globally protected histidine residues in a protein's three-dimensional structure for successful application.

From a mononuclear Cu(II)-hydrazone complex [Cu(PBH)2] (1), one μ1,1-azido bridged dinuclear Cu(II) complex having the formula [{Cu(PBH)(μ1,1-NNN)}2] (2) (where HPBH = 2-pyridinecarboxaldehyde benzoyl hydrazone) has been synthesised. Both the complexes are characterised by elemental analyses, IR and UV–Vis spectroscopic studies. The tridentate hydrazone pro-ligand (HPBH) is obtained by the condensation of benzhydrazide and pyridine-2-carboxaldehyde. The structures of the complexes have conclusively been established by the X-ray single crystal diffraction method. Complex 1 and 2 both display DNA binding ability, which is ascertained by UV–Vis titration and cyclic voltammetric studies using calf thymus DNA (CT-DNA). The apparent binding constants (Kapp) are of moderate values and are 2.048 × 104 M−1 (±0.006) and 1.644 × 104 M−1 (±0.005), respectively. The modes of binding of the complexes with CT-DNA has been investigated using circular dichroism, ethidium bromide displacement assay and viscosity measurements. The cleavage properties of these complexes as well as the free pro-ligand with super coiled (SC) pUC19 are studied using the gel electrophoresis method, where both the complexes displayed chemical nuclease activity in the presence of H2O2 via an oxidative mechanism. The antimicrobial study using the free pro-ligand, 1 and 2 against both Gram positive and Gram negative bacteria are performed, 2 showed antimicrobial activity against both Gram negative and Gram positive bacteria whereas the free ligand and 1 show no antibacterial activity.

International audience; Metal complexes of Schiff bases derived from an aromatic hydrazide are well characterised and have been reported several times. Single crystal structures of metal hydrazone complexes derived from an aliphatic hydrazide are very rare and still remain unexplored. In this paper we report two new end-on azido bridged dinuclear copper(II) and cobalt(III) hydrazone complexes derived from an aliphatic hydrazide. The Schiff base [LH = CH3C(O)NHNCHC5H4N] has resulted from the condensation of acetic hydrazide and pyridine-2-carboxaldehyde. The ligand and the complexes [Cu2(L)2(μ1,1-N3)2] (1) and [Co2(L)2(μ1,1-N3)2(N3)2] (2) are characterised by elemental analysis, IR and UV–Vis spectral studies and in addition mass spectroscopy has been also carried out for LH. The structures of 1 and 2 have been established by X-ray crystallography. Variable temperature magnetic susceptibility measurement studies of 1 indicate the χMT values remain constant in the whole range of temperature 10 to 300 K at a value close of 0.75 cm3 K mol−1, which is in good agreement with the one expected for two uncoupled copper(II) centers.

A nickel(II) and a cobalt(III) complex of two different potentially tetradentate Schiff bases with different binding modes have been synthesised. The nickel(II) complex [NiL1] · CH3OH (1) was formed, on reacting the metal salt with a perfectly symmetrical N2O2 tetradentate Schiff base ligand H 2 L 1 , which is the 1:2 condensation product of 1,3-diamino propane and 2-hydroxyacetophenone. The cobalt(III) complex [Co(HL2)3] · (ClO4)3 · H2O (2) was synthesised using an asymmetric N2O2 tetradentate Schiff base ligand HL 2 on condensing N,N-dimethyl-1,3-diamino propane with o-vanillin in 1:1 mmol ratio. Although both Schiff bases are N2O2 functionalised, they showed variation in their coordinative property with nickel(II) and cobalt(III) ions. Both the complexes were characterised by IR spectroscopy and cyclic voltammetry and their single crystal structures clearly indicate that 1 is a mononuclear species whereas 2 is a hydrogen-bonded dimer.

Three new dinuclear Zn(II) complexes [Zn(L)(μ1,1-N3)Zn(L)(N3)] · 1.5H2O (1), [Zn(L)(μ1,1-NCO)Zn(L)(NCO)] · 1.5H2O (2) and [Zn(L)(μ1,1-NCS)Zn(L)(NCS)(OH2)] (3) have been synthesized from a potentially tetradentate N2O2-donor Schiff base ligand LH, [LH = (OCH3)(OH)C6H3CHN(CH2)2N(CH3)2], which is the condensation product of o-vanillin and 2-dimethylaminoethylamine. All the three complexes 1, 2 and 3 have been characterized by elemental analysis, IR and 1H NMR spectroscopy, TGA and fluorescence studies. Finally, their structures have been established by the single crystal X-ray diffraction method. Structural studies reveal that in complexes 1, 2 and 3 the two Zn(II) centers are held together by a μ2-phenolato oxygen atom and also by an end-on pseudohalide nitrogen (azide for 1; cyanate for 2; thiocyanate for 3) atom. Among the two deprotonated Schiff base ligands present in each complex, one acts as a tetradentate ligand (N2O2 donor set) while the other acts as a tridentate ligand (N2O donor set), having a non-coordinated methoxy group. All the synthesized complexes display intraligand 1(π–π∗) fluorescence and can potentially serve as photoactive materials.

The Escherichia coli Fec system, consisting of an outer membrane receptor (FecA), a periplasmic substrate binding protein (FecB) and an inner membrane permease-ATPase type transporter (FecC/D), plays an important role in the uptake and transport of Fe(3+)-citrate. Although several FecB sequences from various organisms have been reported, there are no biophysical or structural data available for this protein to date. In this work, using isothermal titration calorimetry (ITC), we report for the first time the ability of FecB to bind different species of Fe(3+)-citrate as well as other citrate complexes with trivalent (Ga(3+), Al(3+), Sc(3+) and In(3+)) and a representative divalent metal ion (Mg(2+)) with low μM affinity. Interestingly, ITC experiments with various iron-free di- and tricarboxylic acids show that FecB can bind tricarboxylates with μM affinity but not biologically relevant dicarboxylates. The ability of FecB to bind with metal-free citrate is also observed in (1)H,(15)N HSQC-NMR titration experiments reported here at two different pH values. Further, differential scanning calorimetry (DSC) experiments indicate that the ligand-bound form of FecB has greater thermal stability than ligand-free FecB under all pH and ligand conditions tested, which is consistent with the idea of domain closure subsequent to ligand binding for this type of periplasmic binding proteins.

Three new complexes, [{Cu(L)I}2]n (1), [Ag(L)]n·nClO4 (2) and [{Ag(L)(CF3COO)}2]n (3), have been synthesised and characterized by different physico-chemical methods with the aim of exploring the ligating properties of pyridine-4-amidoxime (L). Single crystal X-ray structure analysis reveals that 1 is a 2D coordination polymer, while 2 and 3 are wavy 1D chains in spite of the same coordination mode adopted by the ligand. The crystal structures also show that the coordination geometry of the metals is different in each complex, being tetrahedral in 1, but distorted trigonal planar and distorted linear in 2 and 3, respectively. The asymmetric units of 1 and 3 consist of two independent metal centres and each is associated with a counter anion of the metal salt used, while the asymmetric unit of 2 is cationic in nature, the perchlorate anion being non-coordinated. Structural diversities are also evident as the 2D structure of 1 is stabilized by O–H⋯I interactions, while several classical and non-classical hydrogen bonds lead to 3D supra-molecular structures for complexes 2 and 3. The solid state fluorescence of L and of its complexes were studied revealing that both 1 and 2 show a broad emission band with a maxima at 525 and 522 nm, respectively, strongly red shifted when compared to the free ligand band (463 nm), while 3 is found surprisingly to be non-emissive.

α-Hemoglobin stabilizing protein (AHSP) is a molecular chaperone that binds monomeric α-subunits of human hemoglobin A (HbA) and modulates heme iron oxidation and subunit folding states. Although AHSP·αHb complexes autoxidize more rapidly than HbA, the redox mechanisms appear to be similar. Both metHbA and isolated met-β-subunits undergo further oxidation in the presence of hydrogen peroxide (H(2)O(2)) to form ferryl heme species. Surprisingly, much lower levels of H(2)O(2)-induced ferryl heme are produced by free met-α-subunits as compared with met-β-subunits, and no ferryl heme is detected in H(2)O(2)-treated AHSP·met-α-complex at pH values from 5.0 to 9.0 at 23 °C. Ferryl heme species were similarly not detected in AHSP·met-α Pro-30 mutants known to exhibit different rates of autoxidation and hemin loss. EPR data suggest that protein-based radicals associated with the ferryl oxidation state exist within HbA α- and β-subunits. In contrast, treatment of free α-subunits with H(2)O(2) yields much smaller radical signals, and no radicals are detected when H(2)O(2) is added to AHSP·α-complexes. AHSP binding also dramatically reduces the redox potential of α-subunits, from +40 to -78 mV in 1 m glycine buffer, pH 6.0, at 8 °C, demonstrating independently that AHSP has a much higher affinity for Fe(III) versus Fe(II) α-subunits. Hexacoordination in the AHSP·met-α complex markedly decreases the rate of the initial H(2)O(2) reaction with iron and thus provides α-subunits protection against damaging oxidative reactions.

In this paper we report the temperature and pH dependent syntheses and systematic characterization of four new Co(II)/Co(III) and Ni(II) complexes with a pentadentate Schiff base ligand H(3)L obtained by condensing 1,3,-diaminopropan-2-ol with 2-hydroxyacetophenone in 1:2 molar ratio. The room temperature syntheses involving Co(II) and Ni(II) nitrates and the ligand H(3)L lead to the isolation of the dinuclear species [Co(2)L(2)(H(2)O)] (1), and the mononuclear complex [Ni(LH)] (3), respectively, whereas refluxing at basic pH leads to the tetranuclear complexes, [Co(II)(2)Co(III)(2)L(2)(μ(3)-OMe)(2)(NO(3))(H(2)O)(2)]NO(3)·2(H(2)O) (2), and [Ni(4)L(2)(μ(3)-OMe)(2)(H(2)O)(2)]·2H(2)O (4). 1 is found to be a simple mono alkoxo-bridged Co(III) dinuclear species, whereas 2 and 4 are both rhomb-like tetrameric complexes with double oxo bridges and μ(3)-methoxo bridges, derived from the methanol solvent, in an open dicubane arrangement. Moreover 2 shows six coordinate ordered Co(II) and Co(III) ions and 4 has both six- and five-coordinate Ni(II) centers. Compound 3 is assigned a tentative mononuclear structure based on IR, UV-Vis spectroscopic, (1)H-NMR and ESI mass study results and is supposed to have one Ni(II) center coordinated with a ligand fragment in square planar geometry. The variable temperature magnetic susceptibility study for 2 and 4 is performed which indicate for both 2 and 4 the presence of intracluster dominant ferromagnetic interactions.

Syntheses and crystal structures of four new hydrazone-based Cu(ii) complexes, [{Cu(L(1))(H(2)O)}(2)(mu-pyraz)](ClO(4))(2) (), [{Cu(L(1))(OClO(3))}(2)(mu-4,4'-bipy)] (), [{Cu(L(2)H)}(mu-pyraz){Cu(L(2)H)(OClO(3))}].(ClO(4)) () and [{Cu(L(2))}(2)(mu-bpe)] () [L(1)H = condensation product of benzhydrazide and pyridine-2-carbaldehyde and L(2)H(2) = condensation product of benzoyl acetone and benzhydrazide], bridged by various organic spacers [pyrazine (pyraz), 4,4'-bipyridine (4,4'-bipy) and 1,2-di(4-pyridyl)ethane (bpe)] are reported in this paper. The single-crystal X-ray crystallographic studies reveal that all are dinuclear units where and form strong intermolecular H-bonding to form sheets of interconnected ions, whereas forms sheets of dinuclear chains through pi-pi interactions; in , molecules are linked only through van der Waals interactions. The variable-temperature magnetic moment studies reveal that and show antiferromagnetic coupling between the Cu(ii) centers at lower temperatures. The binding ability of with calf thymus DNA [CT-DNA] is reported using various spectroscopic studies (UV-Vis titration, circular dichroism and fluorescence). The binding constants of with CT-DNA, as calculated by different methodologies, are of the order of 10(5) M(-1). The mode of interaction between and CT-DNA has been predicted using circular dichroic (CD) spectroscopy, where it has been shown that most probably interacts with DNA via intercalation between the base pairs leading to a change in B-DNA conformation. is also able to cleave supercoiled (SC) plasmid DNA pUC19 in a time and dose dependent manner as demonstrated by agarose gel electrophoresis, and also demonstrates its potential to cleave the SC plasmid DNA via both oxidative and hydrolytic mechanisms. Approximately 50% of leukemic cells are found to be dead when two representative leukemic cell lines are exposed to ( approximately 80 muM) even for 24 h as determined by different cell cytotoxicity assays. Preliminary results also showed that, at 20 muM, could selectively induce apoptosis in leukemic cells without affecting normal lymphocytes.

International audience; Two new pseudohalide-bridged copper(II) complexes [{Cu(PBH)(μ1,1-CNO)}2] (1) and {Cu(PBH)(μ1,5-NCNCN)}n (2) (where HPBH = 2-pyridinecarboxaldehyde benzoyl hydrazone) have been synthesised and characterised by elemental analysis, CV, IR and UV–Vis spectral studies. The tridentate hydrazone pro-ligand (HPBH) was obtained by the condensation of benzhydrazide and pyridine-2-carboxaldehyde. Structures of both complexes have been established by X-ray crystallography which shows that 1 is a μ1,1-CNO−-bridged dimer whereas 2 is a μ1,5-dca-bridged (dca = dicyanamide) linear polynuclear structure. Variable temperature magnetic susceptibility studies indicate weak antiferromagnetic interactions with J values −0.50 cm−1 and −0.10 cm−1 for 1 and 2, respectively.

The aerial oxidation of cobalt(II) salt, in a methanolic solution, containing a hydrazone ligand, (E)-N'-(4-oxo-4-phenylbutan-2-ylidene)benzohydrazide (condensation product of benzoyl acetone and benzhydrazide, LH2) leads to the coupling of two such ligand units through the formation of a rather long C-C bond [1.601(6) A] giving rise to a dinuclear Co (III) hydrazone complex, [Co2(L)2(L')(].0.25H2O (L' = C-C coupled hydrazone ligand). The structure of the complex has been determined by X-ray crystallography and IR, UV-Vis spectroscopy and elemental analysis have characterized the complex.