A theoretical study of the principles regulating the specificity for Al(III) against Mg(II) in protein cavities

Several toxic effects arise from Al's presence in living systems, one of them being the alteration of the natural role of enzymes and non-enzyme proteins. Al(III) is capable of entering protein active sites that in normal conditions should be occupied by other metals. Even if Mg(II) is an ubiquitous metal in biological systems, the interference of aluminium in magnesium metabolism is not clear yet. In this work, a systematic study of the affinity of Al(III) for different protein binding sites is presented, with special attention on structural parameters, the role of the charge and the presence of different ligands in the protein cavity. The specificity of the binding site for Al(III) against Mg(II) has been studied, and also the thermodynamical propensity of a Mg(II)/Al(III) exchange. Quantum mechanical methods that proved to be reliable in previous works have been used, namely, the density functional theory (DFT) and polarizable continuum model (PCM).     

Nonequivalence of the metal binding sites of conalbumin. Calorimetric and spectrophotometric studies of aluminum binding.

Differential scanning calorimetric experiments show that addition of Al(III) to conalbumin increases its denaturation temperature by 5 degrees, from 60 to 68 degrees. Only one Al(III) bound per conalbumin molecule produces this change in heat stability; additional bound Al(III) does not affect the heat stability. Since Al(III) displaces both Cu(II) bound at the metal binding sites of conalbumin, binding of aluminum takes place at the same metal binding sites. The binding constant for the second Al(III) is at least 100-fold less than that for the binding of the first Al(III), and both are displaced by added iron. The order of increasing heat stability of the metal ion complexes of conalbumin, Cu(II), Al(III), Fe(III), is the order of increasing binding constant for these metal ions.     

Synthesis and characterization of metal complexes of Calcein Blue: Formation of monomeric, ion pair and coordination polymeric structures

A series of metal complexes containing the 4-methylumbelliferone-8-methyleneiminodiacetic acid (H₃muia, also named as Calcein Blue) has been synthesized and characterized. Complexes of Cu(II), Ni(II), Mn(II), Zn(II) and Mg(II) have been structurally characterized while Ca(II) and Al(III) complexes by elemental analysis and thermogravimetry. The Cu(II) and Ni(II) complexes are neutral and mononuclear in the solid state. Interestingly, the Mn(II), Zn(II) and Mg(II) muia complexes exist as ion-pairs containing hydrated or solvated metal cation and dimeric metal(II) muia anions. Owing to the presence of hydrogen-bond donors and acceptors in the ligand, hydrogen bonding interactions are dominant along with π-π stacking in their solid-state structures. The solid-state fluorescence studies indicate that the family of muia complexes exhibit comparable emission properties as in solution state, in which only main group and post-transition complexes show bright blue fluorescence while transition metal complexes do not fluoresce.     

Metalloporphyrins inhibit beta-hematin (hemozoin) formation

Metal-substituted protoporphyrin IXs (Cr(III)PPIX (1), Co(III)PPIX (2), Mn(III)PPIX (3), Cu(II)PPIX (4), Mg(II)PPIX (5), Zn(II)PPIX (6), and Sn(IV)PPIX (7)) act as inhibitors to beta-hematin (hemozoin) formation, a critical detoxification biopolymer of malarial parasites. The central metal ion plays a significant role in the efficacy of the metalloprotoporphyrins to inhibit beta-hematin formation. The efficacy of these compounds correlates well with the water exchange rate for the octahedral aqua complexes of the porphyrin's central metal ion. Under these in vitro reaction conditions, metalloporphyrins 5, 6 and 7 are as much as six times more efficacious than the free ligand protoporphyrin IX in preventing beta-hematin formation and four times as efficacious as chloroquine, while metalloporphyrins 3 and 4 are three to four times more effective at preventing beta-hematin formation than the free protoporphyrin IX base. In contrast, the relatively exchange inert metalloporphyrins 1 and 2 are only as efficacious as the free ligand and only two-thirds as effective as chloroquine. Aggregation studies of the heme:MPPIX using UV-Vis and fluorescence spectroscopies are indicative of the formation of pi-pi hetero-metalloporphyrin assemblies. Thus, hemozoin inhibition is likely prevented by the formation of heme:MPPIX complexes through pi-stacking interactions. The ramifications of such hetero-metalloporphyrin assemblies, in the context of the emerging structural picture of hemozoin, are discussed.     

Fe(III) and Cu(II) conalbumin visible circular dichroism spectra.

The single polypeptide chain of conalbumin strongly binds two Fe(III) or two Cu(II) ions to yield intense absorption in the visible region similar to that shown by the related protein transferrin. Comparison of the metal-ion-binding sites in the two proteins is made by exploiting the sensitivity to ligand geometry of circular dichroism (CD). For the Fe(III) proteins strong similarities of the CD spectra outweigh marginal differences. For Cu(II) conalbumin an additional negative extremum near 506 nm appears between two positive ones at 634 and 410 nm suggesting greater subtraction of oppositely signed CD components leading to lesser magnitudes for the two positive peaks than are found in Cu(II)-transferrin. The two Fe(III)-binding sites within conalbumin are compared by noting the strong similarities of the CD and MCD of proteins with Fe(III) in one site and Ga(III) in the other site, and vice versa, with the protein containing Fe(III) in both sites. Due to features of the amino acid sequences of the single protein chains, the four strong metal ion binding sites in conalbumin and transferrin cannot be identical in all particulars, yet CD spectra of their metal ion complexes are closely similar. From a study of model phenolate complexes and the wavelength maxima of visible absorption in the Fe(III), Cu(II), and Co(III) proteins near 465, 440, and 405 nm, respectively, these strong absorption bands are identified as ligand to metal ion electron-transfer transitions. It is suggested that tyrosyl residues are the donors in the electron transfer transitions and that they lock in the metal ions after being keyed into position by binding of bicarbonate or other anions.     

Binding of the Activating Ion Co(II) to myo-Inositol Monophosphatase Monitored by Fluorescence and Phosphorescence Spectroscopy

Two extrinsic probes, pyrene-maleimide and eosin-maleimide, were used to label specific SH groups of the enzyme myo-inositol monophosphatase. The fluorescence of pyrene-monophosphatase is enhanced upon addition of the activating metal ions Co(II) and Mg(II). Co(II) ions bind with a dissociation constant of 4 μM, whereas the apparent activation constant K _a is 0.4 mM. Energy transfer measurements demonstrated that the pyrene chromophore, covalently linked to Cys-218, is within 9 Å of the metal ion Tb(III) coordinated to the metal-binding site. The phosphorescence emitted by eosin covalently linked to the protein is quenched by the addition of the activating cations Co(II) and Mg(II). Phosphorescence titrations conducted under anaerobic conditions were used to determine a dissociation constant of approximately 3 μM for the binding of Co(II) ions. The results are consistent with the hypothesis that two activating ions per monomeric subunit participate in the catalytic mechanism. The affinity of the tightly bound ion is at least 100-fold greater than the affinity of the weakly bound ion.     

Antimicrobial, spectral, magnetic and thermal studies of Cu(II), Ni(II), Co(II), UO(2)(VI) and Fe(III) complexes of the Schiff base derived from oxalylhydrazide

The Schiff base ligand, oxalic bis[(2-hydroxybenzylidene)hydrazide], H(2)L, and its Cu(II), Ni(II), Co(II), UO(2)(VI) and Fe(III) complexes were prepared and tested as antibacterial agents. The Schiff base acts as a dibasic tetra- or hexadentate ligand with metal cations in molar ratio 1:1 or 2:1 (M:L) to yield either mono- or binuclear complexes, respectively. The ligand and its metal complexes were characterized by elemental analyses, IR, (1)H NMR, Mass, and UV-Visible spectra and the magnetic moments and electrical conductance of the complexes were also determined. For binuclear complexes, the magnetic moments are quite low compared to the calculated value for two metal ions complexes and this shows antiferromagnetic interactions between the two adjacent metal ions. The ligand and its metal complexes were tested against a Gram + ve bacteria (Staphylococcus aureus), a Gram -ve bacteria (Escherichia coli), and a fungi (Candida albicans). The tested compounds exhibited high antibacterial activities.     

A Comparative study of Fe(II) and Fe(III) interactions with DNA duplex: major and minor grooves bindings

The involvement of the Fe cations in autoxidation in cells and tissues is well documented. DNA is a major target in such reaction, and can chelate Fe cation in many ways. The present study was designed to examine the interaction of calf-thymus DNA with Fe(II) and Fe(III), in aqueous solution at pH 6.5 with cation/DNA (P) (P = phosphate) molar ratios (r) of 1:160 to 1:2. Capillary electrophoresis and Fourier transform infrared (FTIR) difference spectroscopic methods were used to determine the cation binding site, the binding constant, helix stability and DNA conformation in Fe-DNA complexes. Structural analysis showed that at low cation concentration (r = 1/80 and 1/40), Fe(II) binds DNA through guanine N-7 and the backbone PO(2) group with specific binding constants of K(G) = 5.40 x 10(4) M(1) and K(P) = 2.40 x 10(4) M(1). At higher cation content, Fe(II) bindings to adenine N-7 and thymine O-2 are included. The Fe(III) cation shows stronger interaction with DNA bases and the backbone phosphate group. At low cation concentration (r = 1:80), Fe(III) binds mainly to the backbone phosphate group, while at higher metal ion content, cation binding to both guanine N-7 atom and the backbone phosphate group is prevailing with specific binding constants of K(G) = 1.36 x 10(5) M(-1) and K(P) = 5.50 x 10(4) M(-1). At r = 1:10, Fe(II) binding causes a minor helix destabilization, whereas Fe(III) induces DNA condensation. No major DNA conformational changes occurred upon iron complexation and DNA remains in the B-family structure.     

Negative cooperativity of chicken ovotransferrin on Al(III)-binding

Chicken ovotransferrin, an iron binding protein, has two metal binding sites (amino (N) and carboxy (C) terminal sites). It binds Cu(II), Al(III), Co(II), and other metals, as well as Fe(III). In this study, the selectivity and cooperativity of the N and C sites on Al(III), Co(II), and Tb(III) binding were investigated. Metals were classified into two groups according to their site preference. Co(II) and Al(III) bound to the N site more preferably than to the C site, whereas Tb(III) bound to the C site more preferably. On Fe(III) binding, the binding constant of Fe(III) becomes larger when the other site is already occupied. Thus, positive cooperativity is seen. In the present study, the binding cooperativities of Co(II), Tb(III), and Al(III) as to the N and C sites were investigated. On Co(II) and Tb(III) binding, no cooperativity was observed, as in the case of Cu(II) [Yamamura, T. et al. (1985) in Proteins of Iron Storage and Transport (Spik, G., Montreuil, J., Crichton, R.R., & Mazurier, J., eds.) pp. 53-56, Elsevier Science Publ. B.V., Amsterdam]. In contrast, negative cooperativity was observed on Al(III) binding. Based on a model proposed by Yamamura et al. [Yamamura, T. et al. (1985) ibid.], the ratio of the binding constants, KC/KN, and the stacking coefficient, Kst, were estimated. KC/KN is 2.2 +/- 0.4 for the Tb(III) ion, 0.5 +/- 0.1 for the Co(II) ion, and 0.12 +/- 0.02 for the Al(III) ion. Kst (= 1 in a non-cooperative case) is 0.98 +/- 0.02 for the Tb(III) ion, 1.03 +/- 0.02 for the Co(II) ion, and 0.55 +/- 0.22 for the Al(III) ion.     

Electronic property and reactivity of (hydroperoxo)metal compounds

DFT calculations were done for the (hydroperoxo)metal complexes with eta1-coordination mode, where metal ions are Fe(III), Al(III), Cu(II) and Zn(II). Results shows that 1) the electron density at the two oxygen atoms of the hydroperoxide ion is highly dependent on the angle O-O-H in M-OOH species and the difference in electron density between the two oxygen atoms reaches a maximum at the angle O-O-H = 180 degrees, 2) total electron density at the two oxygen atoms of the peroxide ion increases by approach of methane to the (hydroperoxo)metal species in the cases of Fe(III) and Cu(II); on the other hand, significant decrease of the electron density on peroxide oxygen atoms was observed for the cases of Al(III) and Zn(II) compounds. These findings suggest that the (hydroperoxo)metal species acts as an electrophile in the former cases (M = Fe(III), Cu(II)) and as a nucleophile for the latter two compounds (M = Zn(II), Al(III)). The electrophilicity observed for the Fe(III) and Cu(II) complexes is attributed to the presence of unoccupied- or half-filled d-orbitals interacting with the hydroperoxide ion. 3) Two oxygen atoms of the (hydroperoxo)-compounds of Fe(III) and Cu(II) complexes exhibit quite different reactivity toward the substrate, such as methane. When methane approaches the oxygen atom which is coordinated to a metal ion, a strong decrease of electron density at the methane carbon atom occurs with concomitant increase of electron density at the peroxide oxygen atoms inducing its heterolytic O-O cleavage. When methane approaches the terminal oxygen atom, an oxidative coupling reaction occurs between peroxide ion and methane; at first a nucleophilic attack by the terminal electron-rich oxygen atom occurs at the carbon atom to induce C-O bond formation, and a subsequent oxidative electron transfer proceeds from substrate to the metal-peroxide species yielding CH3-OOH, CH3OH, or other oxidized products. These results clearly demonstrate that the (hydroperoxo)-metal compound itself is a rather stable compound, and activation of the peroxide ion is induced by interaction with the substrate, and the products obtained by the oxygenation reaction are dependent on the chemical property of the substrate, redox property of a metal ion, and stability of the compounds formed in the intermediate process.     

Antimicrobial,spectral, magnetic and thermal studies of Cu(II), Ni(II), Co(II), UO2(VI) and Fe(III) complexes of the Schiff base derived from oxalylhydrazide

The Schiff base ligand, oxalic bis[(2-hydroxybenzylidene)hydrazide], H₂L, and its Cu(II), Ni(II), Co(II), UO₂(VI) and Fe(III) complexes were prepared and tested as antibacterial agents. The Schiff base acts as a dibasic tetra- or hexadentate ligand with metal cations in molar ratio 1:1 or 2:1 (M:L) to yield either mono- or binuclear complexes, respectively. The ligand and its metal complexes were characterized by elemental analyses, IR, ¹H NMR, Mass, and UV-Visible spectra and the magnetic moments and electrical conductance of the complexes were also determined. For binuclear complexes, the magnetic moments are quite low compared to the calculated value for two metal ions complexes and this shows antiferromagnetic interactions between the two adjacent metal ions. The ligand and its metal complexes were tested against a Gram + ve bacteria (Staphylococcus aureus), a Gram -ve bacteria (Escherichia coli), and a fungi (Candida albicans). The tested compounds exhibited high antibacterial activities.     

NMR study of a lymphocyte differentiating thymic factor. An investigation of the Zn(II)-nonapeptide complexes (thymulin)

Detailed investigations of a serum peptide (less than Glu1-Ala2-Lys3-Ser4-Gln5-Gly6-Gly7-Ser8-++ +Asn9) were carried out by 1H and 13C NMR spectroscopy to elucidate the structure of the complex formed with Zn(II), thymulin, which has been found to be active in vivo. These experiments were performed in dimethyl sulfoxide-d6 solution at different metal:peptide ratios. The results suggest the following conclusions. (i) The Zn(II) complexation corresponds to a fast exchange on the NMR time scale. (ii) The evolution of 1H and 13C NMR chemical shifts indicates the existence of two types of complexes: a 1:2 species associating two peptide molecules and one Zn(II) ion and a complex with 1:1 stoichiometry. The former is predominant for metal:peptide ratios below unity. (iii) In the 1:2 complex, Zn(II) is coordinated by the Ser4-O gamma H and Asn9-CO2- sites, while in the 1:1 complex, Ser8-O gamma H is the third ligand to the Zn(II) ion. The results are compared with those for the [Ala4] and [Ala8] analogues, and those for the complexes of thymulin with other metal ions (Cu2+ and Al3+) in terms of its biological activity. These comparative studies suggested that the 1:1 complex is the only conformation recognized by the antibodies.     

Analysis of aluminum and divalent cation binding to wheat root plasma membrane proteins using terbium phosphorescence

A phosphorescent trivalent cation, terbium [Tb(III)], has been used to study the binding of different polyvalent cations to the proteins of wheat (Triticum aestivum L.) root plasma membranes. The phosphorescence emission intensity of Tb(III) was enhanced after Tb(III) binding to wheat root plasma membranes as a result of nonradiative resonance energy transfer from the membrane protein tyrosine and phenylalanine residues. Complex, saturable Tb(III) binding was observed, suggesting multiple binding sites. Bound Tb(III) could be displaced by divalent cations in the general order: Mn(II) > Ca(II) > Mg(II). Al(III) was very effective in reducing the protein-enhanced Tb(III) phosphorescence at pH values below 5. Al(III) also altered the Tb(III) phosphorescence lifetime, suggesting Al(III)-induced changes in membrane protein conformation. The more Al(III)-sensitive wheat cultivar (Anza) bound Al(III) with higher affinity than the more tolerant cultivar (BH 1146). At pH 5.5 where Al(III) did not displace bound Tb(III), low levels of Al(III) reduced the ability of Mn(II) to decrease Tb(III) phosphorescence. The significance of these results is discussed with respect to the mechanisms of Al(III) tolerance in wheat and the potential beneficial effects of Al(III) in reducing Mn(II) phytotoxicity.     

Al(III)-binding ability of an octapeptide and its phosphorylated derivative

A synthetic octapeptide, H-GlyGluGlyGluGlySerGlyGly-OH, and its phosphorylated Ser derivative were synthetized and their solution speciation and binding modes in their complexes with Al(III) were measured. One goal of the work was find a lead compound for the design of a selective peptide-based Al(III) chelator. pH-potentiometry was used to characterize the stoichiometry and the stability of the species formed in the interactions of the metal ion and the peptides, while multinuclear NMR was applied to characterize the binding sites of the metal ion in the complexes. CD spectroscopy revealed a difference in the conformational behaviour of the phosphorylated peptide as compared with its non-phosphorylated parent derivative. The Al(III) is presumed to enhance aggregation through the -PO3H(-)-Al(3+)-PO3(2-)-Al(3+)- intermolecular bindings between the peptide chains. The results of molecular dynamics calculations supported the experimentally obtained secondary structures and the binding position of Al(III).     

The reaction mechanism of the Ga(III)Zn(II) derivative of uteroferrin and corresponding biomimetics

Purple acid phosphatase from pig uterine fluid (uteroferrin), a representative of the diverse family of binuclear metallohydrolases, requires a heterovalent Fe(III)Fe(II) center for catalytic activity. The active-site structure and reaction mechanism of this enzyme were probed with a combination of methods including metal ion replacement and biomimetic studies. Specifically, the asymmetric ligand 2-bis{[(2-pyridylmethyl)-aminomethyl]-6-[(2-hydroxybenzyl)(2-pyridylmethyl)]aminomethyl}-4-methylphenol and two symmetric analogues that contain the softer and harder sites of the asymmetric unit were employed to assess the site selectivity of the trivalent and divalent metal ions using (71)Ga NMR, mass spectrometry and X-ray crystallography. An exclusive preference of the harder site of the asymmetric ligand for the trivalent metal ion was observed. Comparison of the reactivities of the biomimetics with Ga(III)Zn(II) and Fe(III)Zn(II) centers indicates a higher turnover for the former, suggesting that the M(III)-bound hydroxide acts as the reaction-initiating nucleophile. Catalytically active Ga(III)Zn(II) and Fe(III)Zn(II) derivatives were also generated in the active site of uteroferrin. As in the case of the biomimetics, the Ga(III) derivative has increased reactivity, and a comparison of the pH dependence of the catalytic parameters of native uteroferrin and its metal ion derivatives supports a flexible mechanistic strategy whereby both the mu-(hydr)oxide and the terminal M(III)-bound hydroxide can act as nucleophiles, depending on the metal ion composition, the geometry of the second coordination sphere and the substrate.     

Stopped-flow kinetic studies of Ca(II) and Mg(II) dissociation in cod parvalbumin and bovine alpha-lactalbumin

The dissociation kinetics of complexes of bovine alpha-lactalbumin and cod parvalbumin with Ca(II) and Mg(II) ions induced by mixing of a Ca(II)- or MG(II)-loaded protein with a chelator of divalent cations (EDTA or EGTA) have been studied by means of the stopped-flow method with intrinsic protein fluorescence registration. Within the temperature interval from 10 to approx. 37 degrees C kinetic curves for Ca(II) removal from alpha-lactalbumin are monoexponential with a rate constant ranging from 0.006 to 1 s. Taking into account the rather low rate of fluorescence changes, one can assume that the limiting stage in this case is the dissociation of the single bound Ca(II) ion from the protein and not a conformational transition which occurs after Ca(II) dissociation. At temperatures above 37 degrees C the kinetic curves require at least two exponential terms for a satisfactory fit. The second exponential seems to be due to denaturation of the apo form of alpha-lactalbumin which takes place at these temperatures. The values of the dissociation rate constants for Mg(II) bound to alpha-lactalbumin practically coincide with those for Ca(II). Within the temperature interval 10-30 degrees C the kinetic curves for Ca(II) and Mg(II) removal from parvalbumin are best fitted by a sum of two exponential terms identified as arising from the dissociation of cations from the two binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metalloporphyrins obtained in aqueous solution based on the 5,10,15,20-tetra-p-(N,N-dimethyl)anilinporphyrin

The modified method of preparation of water soluble metalloporphyrins is presented. As a ligand 5,10,15,20-tetra-p(N-ethyl-N,N-dimethyl)anilinporphyrinium disulphate was used. The structure of the obtained metalloporphyrins for the following metal cations: Mg(II), Zn(II), Cd(II), Ag(II), Ru(Il), Rh(II), Ni(II), Fe(III), Mn(III), Co(III) and Sn(IV), was confirmed by electron, IR spectra and elemental analyses.     

Binding of the Activating Ion Co(II) to myo-Inositol Monophosphatase Monitored by Fluorescence and Phosphorescence Spectroscopy

Two extrinsic probes, pyrene-maleimide and eosin-maleimide, were used to label specific SH groups of the enzyme myo-inositol monophosphatase. The fluorescence of pyrene-monophosphatase is enhanced upon addition of the activating metal ions Co(II) and Mg(II). Co(II) ions bind with a dissociation constant of 4 M, whereas the apparent activation constant K _a is 0.4 mM. Energy transfer measurements demonstrated that the pyrene chromophore, covalently linked to Cys-218, is within 9 Å of the metal ion Tb(III) coordinated to the metal-binding site. The phosphorescence emitted by eosin covalently linked to the protein is quenched by the addition of the activating cations Co(II) and Mg(II). Phosphorescence titrations conducted under anaerobic conditions were used to determine a dissociation constant of approximately 3 M for the binding of Co(II) ions. The results are consistent with the hypothesis that two activating ions per monomeric subunit participate in the catalytic mechanism. The affinity of the tightly bound ion is at least 100-fold greater than the affinity of the weakly bound ion.     

Iron reduction: a mechanism for dynamic cycling of occluded cations in tropical forest soils?

Nutrient cations can limit plant productivity in highly weathered soils, but have received much less attention than phosphorus and nitrogen. The reduction of iron (Fe) in anaerobic microsites of surface soils can solubilize organic matter and P sorbed or occluded with short-range-ordered (SRO) Fe phases. This mechanism might also release occluded cations. In the Luquillo Experimental Forest, Puerto Rico, we measured cation release during anaerobic laboratory incubations, and assessed patterns of cation availability in surface soils spanning ridge-slope-valley catenas. During anaerobic incubations, potassium (K), calcium (Ca) and magnesium (Mg) significantly increased with reduced Fe (Fe(II)) in both water and 0.5 M HCl extractions, but did not change during aerobic incubations. In the field, 0.5 M HCl-extractable Fe(II) and Fe(III) were the strongest predictors of K, Mg, and Ca on ridges (R² 0.57–0.75). Here, both Ca and Mg decreased with Fe(III), while K, Ca, and Mg increased with Fe(II), consistent with release of Fe-occluded cations following Fe reduction. Manganese in ridge soils was extremely low, consistent with leaching following reductive dissolution of Mn(IV). On slopes, soil C was the strongest cation predictor, consistent with the importance of organic matter for cation exchange in these highly weathered Oxisols. In riparian valleys, cation concentrations were up to 16-fold greater than in other topographic positions but were weakly or unrelated to measured predictors, potentially reflecting cation-rich groundwater. Predictors of cation availability varied with topography, but were consistent with the potential importance of microsite Fe reduction in liberating occluded cations, particularly in the highly productive ridges. This mechanism may explain discrepancies among indices of “available” soil cations and plant cation uptake observed in other tropical forests.