Protein interactions with surface-immobilized metal ions: structure-dependent variations in affinity and binding capacity with temperature and urea concentration.

We have used equilibrium binding analyses to evaluate the influence of temperature and urea on the affinity of hen egg white lysozyme and bovine pancreatic ribonuclease A for surface-immobilized Cu(II) ions. Linear Scatchard plots suggested that these model proteins were interacting with immobilized metal ions via a single class of intermediate-affinity (Kd = 10-40 microM) binding sites. Alterations in temperature had little or no effect on the immobilized Cu(II) binding capacity of either protein. Temperature effects on the interaction affinity, however, were protein-dependent and varied considerably. The affinity of lysozyme for immobilized Cu(II) ions was significantly decreased with increased temperature (0 degree C-37 degrees C), yet the affinity of ribonuclease did not vary measurably over the same temperature range. The van 't Hoff plot (1n K vs 1/T) for lysozyme suggests a straight line relationship (single mechanism) with a delta H of approximately -5.5 kcal/mol. Urea effects also varied in a protein-dependent manner. A 10-fold reduction in the affinity of lysozyme for the immobilized Cu(II) was observed with the urea concentrations up to 3 M; yet urea had no effect on the affinity of ribonuclease for the immobilized metal ions. Although the interaction capacity of lysozyme with the immobilized Cu(II) ions was decreased by 50% in 3 M urea, ribonuclease interaction capacity was not diminished in urea. Thus, temperature- and urea-dependent alterations in protein-metal ion interactions were observed for lysozyme but not ribonuclease A. The complete, yet reversible, inhibition of lysozyme- and ribonuclease-metal ion interactions by carboxyethylation with low concentrations of diethylpyrocarbonate provided direct evidence of histidyl involvement. The differential response of these proteins to the effects of temperature and urea was, therefore, interpreted based on calculated solvent-accessibilities and surface distributions of His residues, individual His residue pKa values, and specific features of the protein surface structure in the immediate environment of the surface-exposed histidyl residues. Possible interaction mechanisms involved in protein recognition of macromolecular surface-immobilized metal ions are presented.     

Native and dry-heated lysozyme interactions with membrane lipid monolayers: Lipid packing modifications of a phospholipid mixture,model of the Escherichia coli cytoplasmic membrane

Antimicrobial resistance is currently an important public health issue. The need for innovative antimicrobials is therefore growing. The ideal antimicrobial compound should limit antimicrobial resistance. Antimicrobial peptides or proteins such as hen egg white lysozyme are promising molecules that act on bacterial membranes. Hen egg white lysozyme has recently been identified as active on Gram-negative bacteria due to disruption of the outer and cytoplasmic membrane integrity. Furthermore, dry-heating (7 days and 80 °C) improves the membrane activity of lysozyme, resulting in higher antimicrobial activity. These in vivo findings suggest interactions between lysozyme and membrane lipids. This is consistent with the findings of several other authors who have shown lysozyme interaction with bacterial phospholipids such as phosphatidylglycerol and cardiolipin. However, until now, the interaction between lysozyme and bacterial cytoplasmic phospholipids has been in need of clarification. This study proposes the use of monolayer models with a realistic bacterial phospholipid composition in physiological conditions. The lysozyme/phospholipid interactions have been studied by surface pressure measurements, ellipsometry and atomic force microscopy. Native lysozyme has proved able to absorb and insert into a bacterial phospholipid monolayer, resulting in lipid packing reorganization, which in turn has lead to lateral cohesion modifications between phospholipids. Dry-heating of lysozyme has increased insertion capacity and ability to induce lipid packing modifications. These in vitro findings are then consistent with the increased membrane disruption potential of dry heated lysozyme in vivo compared to native lysozyme. Moreover, an eggPC monolayer study suggested that lysozyme/phospholipid interactions are specific to bacterial cytoplasmic membranes.     

Stability studies in relation to IR data of some schiff base complexes of transition metals and their biological and pharmacological studies

Schiff bases derived from salicylaldehyde and 2-substituted anilines and their Cu(II), Ni(II) and Co(II) complexes have been synthesized and characterized by their elemental analysis, TGA, IR and electronic spectral studies, molar conductance and magnetic susceptibility measurements. The mode of bonding between Cu(II), Ni(II) and Co(Il) and Schiff bases has been studied by IR spectrophotometry. The shift in the band positions of the groups involved in coordination has been utilized to estimate the metal- nitrogen bond lengths. The results obtained are in good agreement with the values of metal-nitrogen modes and ligand-field splitting energy (10 Dq). The antimicrobial activities of the synthesized ligands and their metal complexes have been determined on Gram-positive (Staphylococcus aureus), Gram- negative (Escherichia coli) bacteria and on fungi like Aspergillus niger, Aspergillus nidulense and Candida albicans. The antimicrobial activity of the organic ligands increased several folds on chelation as compared to the ligand molecule alone. However, their anti-inflammatory activity showed a different pattern; the activity of some ligands was more than their respective metal chelates. It is interesting to note that only cobalt complexes exhibited anti-inflammatory activity.     

Interaction of Ribonucleotides with Metal Hexacyanocobaltate(III): A Possible Role in Chemical Evolution

It has been proposed that metal cyanide complexes would have acted as effective prebiotic catalysts. Insoluble metal cyanide complexes could have concentrated biomonomers from the dilute prebiotic soup, facilitating certain prebiotic reactions. In the light of the above hypothesis, interaction of four ribonucleotides, namely 5′-AMP, 5′-GMP, 5′-CMP, and 5′-UMP with copper(II)- and cadmium(II) hexacyanocobaltate(III) has been studied. The interaction was found to be maximum at neutral pH. 5′-GMP showed greater interaction with both the metal hexacyanocobaltate(III) while copper(II) hexacyanocobaltate(III) showed greater uptake than cadmium(II) hexacyanocobaltate(III) for all the four ribonucleotides studied. Infrared spectral studies of ribonucleotides, metal hexacyanocobaltate(III) and ribonucleotide – metal hexacyanocobaltate(III) adducts indicated that the nitrogen base and phosphate moiety of ribonucleotides interact with outer divalent metal ion present in the lattice of metal hexacyanocobaltate(III).     

Role of Lysozyme Inhibitors in the Virulence of Avian Pathogenic Escherichia coli

Lysozymes are key effectors of the animal innate immunity system that kill bacteria by hydrolyzing peptidoglycan, their major cell wall constituent. Recently, specific inhibitors of the three major lysozyme families occuring in the animal kingdom (c-, g- and i-type) have been discovered in Gram-negative bacteria, and it has been proposed that these may help bacteria to evade lysozyme mediated lysis during interaction with an animal host. Escherichia coli produces two inhibitors that are specific for c-type lysozyme (Ivy, Inhibitor of vertebrate lysozyme; MliC, membrane bound lysozyme inhibitor of c-type lysozyme), and one specific for g-type lysozyme (PliG, periplasmic lysozyme inhibitor of g-type lysozyme). Here, we investigated the role of these lysozyme inhibitors in virulence of Avian Pathogenic E. coli (APEC) using a serum resistance test and a subcutaneous chicken infection model. Knock-out of mliC caused a strong reduction in serum resistance and in in vivo virulence that could be fully restored by genetic complementation, whereas ivy and pliG could be knocked out without effect on serum resistance and virulence. This is the first in vivo evidence for the involvement of lysozyme inhibitors in bacterial virulence. Remarkably, the virulence of a ivy mliC double knock-out strain was restored to almost wild-type level, and this strain also had a substantial residual periplasmic lysozyme inhibitory activity that was higher than that of the single knock-out strains. This suggests the existence of an additional periplasmic lysozyme inhibitor in this strain, and indicates a regulatory interaction in the expression of the different inhibitors.     

Multiple Forms of Rice Seeds β-Amylases on Zymogram

Hen lysozyme modified with histamine (HML) and Japanese quail lysozyme (JQL) were treated with immobilized metal ion affinity chromatography to analyze the states of their imidazole groups. When Ni(II) was used as the metal ion immobilized, JQL was strongly retained in a Ni(II)-chelating Sepharose column, while hen lysozyme and HML were hardly retained in the same column. All of these lysozymes have a histidine imidazole group at the 15th position, while JQL has an additional histidine imidazole group at the 103rd position and HML has an additional imidazole group covalently attached to Asp101. Thus, I concluded that the imidazole group at the 103rd position of JQL is exposed to the solvent and recognized by the metal ion, but that the imidazole group attached to Asp101 in HML is localized to a hydrophobic region and not recognized by the metal ion.     

Magnetic susceptibility equation for dinuclear high-spin cobalt(II) complexes considering the exchange interaction between two axially distorted octahedral cobalt(II) ions

A new magnetic susceptibility equation has been obtained for dinuclear high-spin cobalt(II) complexes considering the exchange interaction between two axially distorted octahedral cobalt(II) ions. A computational program has been developed, and the temperature dependencies of the magnetic susceptibility have been demonstrated. A characteristic χ_A versus T curve due to the zero-field splitting was shown when the distortion parameter v was positive.     

Selectivity of Ni(II) and Zn(II) binding to Sporosarcina pasteurii UreE, a metallochaperone in the urease assembly: a calorimetric and crystallographic study

Urease is a nickel-dependent enzyme that plays a critical role in the biogeochemical nitrogen cycle by catalyzing the hydrolysis of urea to ammonia and carbamate. This enzyme, initially synthesized in the apo form, needs to be activated by incorporation of two nickel ions into the active site, a process driven by the dimeric metallochaperone UreE. Previous studies reported that this protein can bind different metal ions in vitro, beside the cognate Ni(II). This study explores the metal selectivity and affinity of UreE from Sporosarcina pasteurii (Sp, formerly known as Bacillus pasteurii) for cognate [Ni(II)] and noncognate [Zn(II)] metal ions. In particular, the thermodynamic parameters of SpUreE Ni(II) and Zn(II) binding have been determined using isothermal titration calorimetry. These experiments show that two Ni(II) ions bind to the protein dimer with positive cooperativity. The high-affinity site involves the conserved solvent-exposed His¹⁰⁰ and the C-terminal His¹⁴⁵, whereas the low-affinity site comprises also the C-terminal His¹⁴⁷. Zn(II) binding to the protein, occurring in the same protein regions and with similar affinity as compared to Ni(II), causes metal-driven dimerization of the protein dimer. The crystal structure of the protein obtained in the presence of equimolar amounts of both metal ions indicates that the high-affinity metal binding site binds Ni(II) preferentially over Zn(II). The ability of the protein to select Ni(II) over Zn(II) was confirmed by competition experiments in solution as well as by analysis of X-ray anomalous dispersion data. Overall, the thermodynamics and structural parameters that modulate the metal ion specificity of the different binding sites on the protein surface of SpUreE have been established.     

Characterizing metalloendonuclease mixed metal complexes by global kinetic analysis

To test the role of a secondary metal ion in a two metal ion metallonuclease mechanism, some groups have introduced a nonsupportive metal ion [usually Ca(II)] in cleavage reactions. Stimulation of Mg(II)- or Mn(II)-supported activity has been taken as evidence that the second metal ion is regulatory. However, this activity has yet to be dissected to determine what processes and species contribute to this observation. Here, we test global kinetic analysis as an approach to this problem. Taking advantage of the various binding and cleavage constants established for PvuII endonuclease, we apply cleavage data obtained under a range of Mg(II) and Ca(II) concentrations to a number of kinetic models which specify A and B sites for both metal ions and various active species. The data are best fit and simulated with models which feature Ca(II) being held more strongly in the B (or secondary) site. This mixed metal enzyme species is the only one which forms appreciably and exhibits a cleavage rate constant similar to that observed when there is only one Mg(II) per active site (approximately 0.01 s^?1). Thus, in the case of PvuII endonuclease, Ca(II) does not stimulate cleavage. However, a simulated increase in activity at moderate Ca(II) concentrations can be rationalized with a cleavage rate constant for the mixed species similar to that when two Mg(II) ions are present in the active site. This provides an important insight into the underlying basis for the Ca(II)-stimulated activity observed for some metallonucleases that is not accessible by any other means.     

Structural characterization of a metal-based perfusion tracer: Copper(II) pyruvaldehyde bis (N4-methylthiosemicarbazone)

Copper(II) pyruvaldehyde bis(N⁴-methylthiosemicarbazone), Cu(PTSM), has been obtained as a dark red crystalline solid from EtOH-DMSO solvent mixture and structurally characterized by x-ray crystallography. The molecule possesses the expected pseudo-square planar N₂S₂ metal coordination sphere; however, the copper center also interacts through its axial coordination site with the sulfur atom of an adjacent Cu(PTSM) molecule in the crystal lattice. The structure of this compound is compared with the structures of other metal complexes that have been proposed in the nuclear medicine literature as perfusion tracers.     

Chiral bimetallic complexes from chiral salen metal complexes and mercury (II) halides and acetates: the anionic groups interact with Cu(II) in apical position

A series of chiral bimetallic complexes have been prepared containing both Cu(II) and Hg(II) metal centers. The complexes possess chiral salen ligands which host Cu(II) in the center of the cis-N₂O₂ chromophore and Hg(II) via two oxygen atoms of the chromophore. Halogen and acetate groups from mercury salts interact with the Cu(II) center. The X-ray crystallographic data of 11 reveals a short distance of Cl?Cu (3.22-3.26 Å). EPR study also discloses a strong interaction, in particular, of acetate group with Cu.     

Hyperthermophilic archaeal prefoldin shows refolding activity at low temperature

Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin for correct folding. Previous studies of archaeal prefoldins have shown that prefoldin only possesses holdase activity and is unable to fold unfolded proteins by itself. In this study, we have demonstrated for the first time that a prefoldin from hyperthermophilic archaeon, Pyrococcus horikoshii OT3 (PhPFD), exhibits refolding activity for denatured lysozyme at temperatures relatively lower than physiologically active temperatures. The interaction between PhPFD and denatured lysozyme was investigated by use of a surface plasmon resonance sensor at various temperatures. Although PhPFD showed strong affinity for denatured lysozyme at high temperature, it exhibited relatively weak interactions at lower temperature. The protein-folding seems to occur through binding and release from PhPFD by virtue of the weak affinity. Our results also imply that prefoldin might be able to contribute to the folding of some cellular proteins whose affinity with prefoldin is weak.     

Cobalt(II) and μ-oxodiiron(III,IV) complexes of salen analog with aromatic thioether pendant group,N, N′-disalicylidene-2-methyl-4-(2-methylthiophenyl)- 1,2-butanediamine

A pentadentate salen analog containing a thioether group in the pendant tail, N,N′-disalicylidene-2- methyl-4-(2-methylthiophenyl)- 1,2-butanediamine, has been synthesized. The cobalt(II) complex of this ligand retains a planar configuration free from the coordination of the pendant group at room temperature but adopts a square-pyramidal configuration with the thioether at the apex near liquid nitrogen temperature. The iron complex obtained with this ligand is shown to be a μ-oxodiiron(III, IV) complex comprised of high-spin iron(III) and low-spin iron(IV), based on cryomagnetic data (80–300 K), ESR, and M:ossbauer spectra. An antiferromagnetic spin-exchange interaction (J = − 13.0 cm ⁻¹) operates between the metal ions.     

IR and Raman study on the interactions of the 5′-GMP and 5′-CMP phosphate groups with Mg(II), Ca(II), Sr(II), Ba(II), Cr(III), Co(II), Cu(II), Zn(II), Cd(II), Al(III) and Ga(III)

The chief motive behind this research is the interest provoked by the presence of metal ions as necessary stabilizers of the negative charges of phosphate groups in nucleic acids. The effect that the presence of different metal ions produces on the band principally assigned to the nu(s) PO(3)(2-) mode has been studied using FT-IR and FT-Raman spectroscopy. The results obtained reveal the diagnostic capacity of these techniques in determining the type of metal ion interaction with respect to the mononucleotides that form DNA and RNA, providing a tool for improving the knowledge of the stabilizing or destabilizing effects of these ions on such macromolecules. The metal complexes of the ribonucleotides 5'-CMP and 5'-GMP with Mg(II), Ca(II), Sr(II), Ba(II), Cr(III), Co(II), Cu(II), Zn(II), Cd(II), Al(III) and Ga(III) were obtained in this study. After studying and analyzing the IR and Raman spectra of all these complexes and comparing them with the spectra of the corresponding disodium salts, it was verified that, independently of the type of nucleotide involved, the presence of the metal in the vicinity of the phosphate group produces an alteration in the aforementioned nu(s) PO(3)(2-) band. This effect is related to the type of interaction that the phosphate group has with the metal. Three components are observed: (1) one near 983-975 cm(-1) (detectable in IR and Raman), associated with phosphate groups in an electrostatic type of interaction with the metal ion, separated by two or more water molecules; (2) another near 989-985 cm(-1) (only in IR), associated with phosphate groups in indirect interaction through the water molecules of the coordination sphere of the metal ions; and (3) the IR and Raman bands near 1014-1001 cm(-1), which represent phosphate groups directly bonded to the metal ion. These results are supported by the behavior of 5'-CMP in aqueous solution in the presence of Mg(II) ions.     

The effect of spermine concentration on the solution structure of complexes formed in copper(II)/adenosine 5′-triphosphate/phosphoserine system

Quaternary systems of copper(II) complexes with adenosine 5′-triphosphate, O-phospho l-serine and with equimolar or excessive amount of spermine have been investigated. The studies have been performed in aqueous solution. Types of complexes and the overall stability constants have been determined using the potentiometric method with computer analysis of the data. On the basis of the results of spectroscopic studies (nuclear magnetic resonance, visible, circular dichroism, Raman, infrared and electron paramagnetic resonance spectroscopies) as well as equilibrium studies, the mode of interactions has been proposed. The reaction centers in the systems studied are the phosphate, carboxyl and amine groups from phosphorylated serine, heterocyclic nitrogen atom from purine ring and phosphate groups from adenosine 5′-triphosphate as well as amine groups from polyamine. The influence of change in the concentration of the polyamine (spermine) on the mode of coordination is discussed. It has been shown that in the physiological conditions an increase in the polyamine concentration changes the mode of metal bonding in the CuH₃(ATP)(Ser-P)(Spm) complexes (isomer I — coordination {2 N,O_x}, isomer II — coordination {3 N,O_x} and significant differences in sites of interaction).     

Promiscuity comes at a price: Catalytic versatility vs efficiency in different metal ion derivatives of the potential bioremediator GpdQ

The glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) is a highly promiscuous dinuclear metallohydrolase with respect to both substrate specificity and metal ion composition. While this promiscuity may adversely affect the enzyme's catalytic efficiency its ability to hydrolyse some organophosphates (OPs) and by-products of OP degradation have turned GpdQ into a promising candidate for bioremedial applications. Here, we investigated both metal ion binding and the effect of the metal ion composition on catalysis. The prevalent in vivo metal ion composition for GpdQ is proposed to be of the type Fe(II)Zn(II), a reflection of natural abundance rather than catalytic optimisation. The Fe(II) appears to have lower binding affinity than other divalent metal ions, and the catalytic efficiency of this mixed metal center is considerably smaller than that of Mn(II), Co(II) or Cd(II)-containing derivatives of GpdQ. Interestingly, metal ion replacements do not only affect catalytic efficiency but also the optimal pH range for the reaction, suggesting that different metal ion combinations may employ different mechanistic strategies. These metal ion-triggered modulations are likely to be mediated via an extensive hydrogen bond network that links the two metal ion binding sites via residues in the substrate binding pocket. The observed functional diversity may be the cause for the modest catalytic efficiency of wild-type GpdQ but may also be essential to enable the enzyme to evolve rapidly to alter substrate specificity and enhance k_cat values, as has recently been demonstrated in a directed evolution experiment. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.     

Synthesis, structure and reactivity of the homoleptic iron(II) complex of the novel 4′-(4?-pyridyl-N-oxide)-2,2′:6′,2″-terpyridine ligand

The new ligand 4′-(4?-pyridyl-N-oxide)-2,2′:6′,2″-terpyridine (pyNoxterpy) and its homoleptic iron(II) complex have been synthesised, and structural and spectroscopic studies have been carried out. The obtained results have been compared with the reported data for the parent ligand 4′-(4?-pyridyl)-2,2′:6′,2″-terpyridine (pyterpy) and its homoleptic iron(II) complex. Significant differences between the spectral and electrochemical properties of the metal complexes have been found, derived from the changes in the electronic properties of the coordinated ligands.     

Interaction of Heavy Metals in Multimetal Biosorption by Galerina vittiformis from Soil

ABSTRACT Soil heavy metal contamination, a major threat due to industrialization, can be tackled by an efficient and economical process called bioremediation. Mushrooms are employed to accumulate heavy metals from soil due to their high metal accumulation potential and better adaptability. The bioaccumulation potential of Galerina vittiformis was already reported for individual metals. At natural conditions, since soil consists of more than one polluting metal, more focus has to be given to multimetal systems. In this study, multimetal accumulation potential was analyzed using central composite design, and the responses obtained were analyzed using response surface methodology. Heavy metals such as Cu(II), Cd(II), Cr(VI), Pb(II), and Zn(II) were subjected to biosorption at 10–250 mg/kg concentrations along with pH 5–8. The results showed that the preference of the organism for the five metals under study was in the order Pb(II) > Zn(II) > Cd(II) > Cu(II) > Cr(VI) at pH 6.5 under multimetal condition. The study also indicates that the metal interaction pattern in multimetal interaction is a property of their ionic radii. The response surface methodology clearly explains the effect of interaction of heavy metals on the accumulation potential of the organism using three-dimensional response plots. The present work suggests that the fungus Galerina vittiformis could be employed as a low-cost metal removal agent from heavy metal–polluted soil.     

Kinetic study of ferrous sulphate oxidation of Acidithiobacillus ferrooxidans in the presence of heavy metal ions

Acidophilic microorganisms such as Acidithiobacillus ferrooxidans have the capability to carry out processes of bioleaching, biosorption and bioprecipitation of heavy metal ions, which have important environmental applications. At. ferrooxidans derives the energy for their metabolism from ferrous iron oxidation, process, which can be affected by the presence of heavy metals in the medium. Moreover, organic matter produces an inhibitory effect over the ferrous iron oxidation of At. ferrooxidans. In this work, heterotrophic bacterium Acidiphilium sp. was added when the medium is supplemented with organic matter to reduce this negative effect. The purpose of this work is the kinetic study of ferrous sulphate oxidation by At. ferrooxidans in the presence of different concentrations of several heavy metal ions (Cr(III), Cu(II), Cd(II), Zn(II) and Ni(II)) and compare this kinetic behaviour with a mixed culture with Acidiphilium sp.The obtained results show a non-competitive inhibition of heavy metals over bacterial oxidation of ferrous sulphate. In accordance with this kind of inhibition, a kinetic equation has been proposed to predict the behaviour of At. ferrooxidans in the presence of heavy metals in the range of concentrations studied.