Coordination abilities of piperyd-1-yl-methane-1,1-diphosphonic acids towards zinc(II), magnesium(II) and calcium(II): potentiometric and NMR studies

The combination of the pH-metric and NMR studies is used to examine the stabilities and coordination modes as well as related structural aspects of zinc(II), magnesium(II) and calcium(II) complexation to piperyd-1-yl-methane-1,1-diphosphonic acid (1) and its derivatives containing a topologically modified piperidine ring (2-7). The studied compounds coordinate metal ions exclusively via the phosphonate functions with a nitrogen atom remaining protonated over the whole range of studied pH. Compounds 1-6 readily form soluble multinuclear complexes of type [M(3)(HL)(2)] and [M(3)(HL)(3)](3-) with Zn(2+) or [M(2)(H(2)L)(2)] with Ca(2+) and Mg(2+). These species are formed based on dimers consisting of two head-to-head arranged molecules linked by strong symmetrical hydrogen bonds. The placement of the two methyl groups at 2- and 6-positions on the piperidine ring precludes the molecular recognition via similar hydrogen bonds and accounts for different complexation properties of 7 compared to 1-6. The role that the metal coordination plays on conformation dynamics in 1-7 is also discussed.     

Effect of calcium (II) and magnesium (II) ions on the 18-23 gamma-carboxyglutamic acid containing cyclic peptide loop of bovine prothrombin. An AMBER molecular mechanics study

The effect of calcium (II) and magnesium (II) ions on the conformation of the 18-23 cyclic peptide loop of bovine prothrombin are investigated by the molecular mechanics program AMBER (Assisted Model Building with Energy Refinement). The work is an extension of an earlier paper (Eastman et al., Int. J. Peptide Protein Res. 27, 1986, 530-553) that employed the program ECEPP (Empirical Conformational Energy Program for Peptides). In the absence of either metal ion, or in the presence of either one Ca(II) or one Mg(II) ion, the lowest-energy forms found by AMBER have the Gla21-Pro22 peptide bond in a trans conformation. In the presence of two Ca(II) or Mg(II) ions, the loop form of lowest energy is decidedly cis. The coordination about the Ca(II) and Mg(II) ions is different in both the single and double metal cases. In addition, the peptide chains that emerge from the loop are oriented parallel to each other in the lowest-energy complex with two Ca(II) ions, but are not parallel in the lowest-energy complex with two Mg(II) ions.     

A study of the coordination shell of aluminum(III) and magnesium(II) in model protein environments: thermodynamics of the complex formation and metal exchange reactions

Al(III) toxicity in living organisms is based on competition with other metal cations. Mg(II) is one of the most affected cations, since the size similarity dominates over the charge identity. The slow ligand exchange rates for Al(III) render the ion useless as a metal ion at the active sites of enzymes and provide a mechanism by which Al(III) inhibits Mg(II) dependent biochemical processes. Al(III) cation interactions with relevant bioligands have been studied in a protein-model environment in gas and aqueous phases using density functional theory methods. The protein model consists of the metal cation bound to two chosen bioligands (functional groups of the amino acid side chains, one of them being always an acetate) and water molecules interacting with the cation to complete its first coordination shell. Analogous Mg(II) complexes are calculated and compared with the Al(III) ones. Formation energies of the complexes are calculated in both phases and magnesium/aluminum exchange energies evaluated. The effect of different dielectric media is also analyzed. The presence of an acetate ligand in the binding site is found to promote both, complex formation and metal exchange reactions. In addition, buried binding sites (with low dielectric constant) of the protein favor metal exchange, whereas fully solvated environments of high dielectric constant require the presence of two anionic ligands for metal exchange to occur.     

Interrelationships among biological activity, disulfide bonds, secondary structure, and metal ion binding for a chemically synthesized 34-amino-acid peptide derived from alpha-fetoprotein.

A 34-amino-acid peptide has been chemically synthesized based on a sequence from human alpha-fetoprotein. The purified peptide is active in anti-growth assays when freshly prepared in pH 7.4 buffer at 0.20 g/l, but this peptide slowly becomes inactive. This functional change is proven by mass spectrometry to be triggered by the formation of an intrapeptide disulfide bond between the two cysteine residues on the peptide. Interpeptide cross-linking does not occur. The active and inactive forms of the peptide have almost identical secondary structures as shown by circular dichroism (CD). Zinc ions bind to the active peptide and completely prevents formation of the inactive form. Cobalt(II) ions also bind to the peptide, and the UV-Vis absorption spectrum of the cobalt-peptide complex shows that: (1) a near-UV sulfur-to-metal-ion charge-transfer band had a molar extinction coefficient consistent with two thiolate bonds to Co(II); (2) the lowest-energy visible d-d transition maximum at 659 nm, also, demonstrated that the two cysteine residues are ligands for the metal ion; (3) the d-d molar extinction coefficient showed that the metal ion-ligand complex was in a distorted tetrahedral symmetry. The peptide has two cysteines, and it is speculated that the other two metal ion ligands might be the two histidines. The Zn(II)- and Co(II)-peptide complexes had similar peptide conformations as indicated by their ultraviolet CD spectra, which differed very slightly from that of the free peptide. Surprisingly, the cobalt ions acted in the reverse of the zinc ions in that, instead of stabilizing anti-growth form of the peptide, they catalyzed its loss. Metal ion control of peptide function is a saliently interesting concept. Calcium ions, in the conditions studied, apparently do not bind to the peptide. Trifluoroethanol and temperature (60 degrees C) affected the secondary structure of the peptide, and the peptide was found capable of assuming various conformations in solution. This conformational flexibility may possibly be related to the biological activity of the peptide.     

Assignment of oxidation states in metal complexes. Cerium(III) or cerium(IV) and other questions

The use of the bond valence sum to assign correctly the oxidation state of a metal ion in a complex is discussed. Cerium complexes are used as examples since the oxidation state of Ce has been incorrectly assigned in a surprising number of publications. The recommended R₀ values for Ce(III)-O of 2.118 Å, Ce(IV)-O of 2.070 Å, Ce(III)-N of 2.251 Å, and for Ce(IV)-N of 2.202 Å were derived from analyses of homoleptic Ce-O, Ce-N, and heteroleptic Ce-O and -N complexes. These R₀ values can be used to assign correctly the oxidation state of Ce in complexes containing any combination of Ce-O or Ce-N bonds. An incorrect oxidation state assignment usually arises when the oxidation state of Ce or Pr in the product is assumed to be same as that of the starting Ce or Pr compound, but an oxidation or reduction has occurred. Problems with two related Sn complexes may have arisen because of a mix up in the starting materials.     

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).     

1H NMR spectroscopic studies of calcium-binding proteins. 3. Solution conformations of rat apo-alpha-parvalbumin and metal-bound rat alpha-parvalbumin

Lacking the extraordinary thermal stability of its metal-bound forms, apo-alpha-parvalbumin from rat muscle assumes two distinct conformations in aqueous solution. At 25 degrees C, its highly structured form predominates (Keq = 5.7; delta G degree = -4.3 kJ X mol-1); as deduced from both 1H NMR and circular dichroism (CD) spectroscopy, this conformation is exceedingly similar to those of its Mg(II)-, Ca(II)-, and Lu(III)-bound forms. The temperature dependences of several well-resolved aromatic and upfield-shifted methyl 1H NMR resonances and several CD bands indicate that the native, highly helical structure of rat apo-alpha-parvalbumin is unfolded by a concerted mechanism, showing no indication of partially structured intermediates. The melting temperature, TM, of rat apo-alpha-parvalbumin is 35 +/- 0.5 degrees C as calculated by both spectroscopic techniques. By 45 degrees C, rat apo-alpha-parvalbumin unfolds entirely, losing the tertiary structure that characterizes its folded form: not only are the ring-current-shifted aromatic and methyl 1H NMR resonances leveled, but the 262- and 269-nm CD bands are also severely reduced. As judged by the decrease in the negative ellipticity of the 222-nm CD band, this less-structured form of rat apo-alpha-parvalbumin shows an approximate 50% loss in apparent alpha-helical content compared to its folded state. Several changes in the 1H NMR spectrum of rat apo-alpha-parvalbumin were exceptionally informative probes of the specific conformational changes that accompany metal ion binding and metal ion exchange. In particular, the line intensities of the ortho proton resonance of Phe-47, the unassigned downfield-shifted alpha-CH resonances from the beta-sheet contacts between the metal-binding loops, the C2H resonance of His-48, and the epsilon-CH3 resonance of an unassigned Met residue were monitored as a function of added metal to determine the stability constants of several metal ion-parvalbumin complexes. We conclude that Mg(II) binds to the CD and EF sites independently, its affinity for the EF site being almost twice that for the CD site. Mg(II)----Ca(II) exchange showed that the CD-site Mg(II) is displaced first, in contrast to Lu(III)'s preferential displacement of the EF-site Ca(II) as determined from the Ca(II)----Lu(III) exchange experiments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insight into the catalytic mechanism of DNA polymerase beta: structures of intermediate complexes

The catalytic reaction mediated by DNA polymerases is known to require two Mg(II) ions, one associated with dNTP binding and the other involved in metal ion catalysis of the chemical step. Here we report a functional intermediate structure of a DNA polymerase with only one metal ion bound, the DNA polymerase beta-DNA template-primer-chromium(III).2'-deoxythymidine 5'-beta,gamma-methylenetriphosphate [Cr(III).dTMPPCP] complex, at 2.6 A resolution. The complex is distinct from the structures of other polymerase-DNA-ddNTP complexes in that the 3'-terminus of the primer has a free hydroxyl group. Hence, this structure represents a fully functional intermediate state. Support for this contention is provided by the observation of turnover in biochemical assays of crystallized protein as well as from the determination that soaking Pol beta crystals with Mn(II) ions leads to formation of the product complex, Pol beta-DNA-Cr(III).PCP, whose structure is also reported. An important feature of both structures is that the fingers subdomain is closed, similar to structures of other ternary complexes in which both metal ion sites are occupied. These results suggest that closing of the fingers subdomain is induced specifically by binding of the metal-dNTP complex prior to binding of the catalytic Mg(2+) ion. This has led us to reevaluate our previous evidence regarding the existence of a rate-limiting conformational change in Pol beta's reaction pathway. The results of stopped-flow studies suggest that there is no detectable rate-limiting conformational change step.     

Studies on Ca(II) binding to gamma-carboxyglutamic acid. Use of thermal decarboxylation to probe metal ion/gamma-carboxyglutamic acid interactions

The thermal decarboxylation of N-benzyloxycarbonyl-L-gamma-carboxyglutamic acid alpha-methyl ester [Z)-L-Gla-OMe) has been studied. In the presence of increasing amounts of calcium or magnesium ions, lyophilized powders of (Z)-L-Gla-OMe exhibit a corresponding increase in thermal stability. Both magnesium and calcium form relatively tight, thermally stable complexes with (Z)-L-Gla-OMe at high metal ion concentrations. Differences between Ca(II) and Mg(II) binding are noted at low metal ion concentrations, where (Z)-L-Gla-OMe is in excess. Under these conditions, complex formation with Mg(II) apparently favors a 2:1 Gla-magnesium ion complex in which both Gla residues are unstable to thermal decarboxylation. Calcium ion complexes, however, are found to favor a 3:1 Gla-calcium ion complex in which 1 of the 3 Gla residues is thermally stable.     

Carbonic anhydrase inhibitors. metal complexes of 5-(2-chlorophenyl)-1,3,4-thiadiazole-2-sulfonamide with topical intraocular pressure lowering properties: the influence of metal ions upon the pharmacological activity

Metal complexes of a sulfonamide possessing strong carbonic anhydrase (CA) inhibitory properties, 5-(2-chlorophenyl)-1,3,4-thiadiazole-2-sulfonamide (chlorazolamide) have been obtained from the sodium salt of the sulfonamide and the following metal ions: Mg(II), Zn(II), Mn(II), Cu(II), Co(II), Ni(II), Be(II), Cd(II), Pb(II), Al(III), Fe(III) and La(III). The original sulfonamide and its complexes were assayed for the in vitro inhibition of three CA isozymes, CA I, II, and IV, some of which play a critical role in ocular fluid secretion. All these compounds (the sulfonamide and its metal complexes) behaved as powerful inhibitors against the three investigated isozymes. The parent sulfonamide possessed an extremely weak topical pressure lowering effect when administered as a 1-2% suspension into the rabbit eye, but some of its metal complexes, such as the Mg(II), Zn(II), Mn(II) and Cu(II) derivatives, lower intraocular pressure (IOP) in experimental animals very well. Ex vivo data showed a 99.5-99.9% CA II inhibition in ocular fluids and tissues of rabbits treated with these agents, proving that the observed IOP lowering is due to CA inhibition. The influence of the different metal ions upon the efficiency of the obtained complexes as pressure lowering drugs are discussed, leading to the possibility of designing more selective/potent pharmacological agents from this class.     

Carbonic Anhydrase Inhibitors. Metal Complexes of 5-(2-Chlorophenyl)-1, 3, 4-Thiadiazole-2-Sulfonamide with Topical Intraocular Pressure Lowering Properties: The Influence of Metal Ions Upon the Pharmacological Activity

Metal complexes of a sulfonamide possessing strong carbonic anhydrase (CA) inhibitory properties, 5-(2-chlorophenyl)-1, 3, 4-thiadiazole-2-sulfonamide (chlorazolamide) have been obtained from the sodium salt of the sulfonamide and the following metal ions: Mg(II), Zn(II), Mn(II), Cu(II), Co(II), Ni(II), Be(II), Cd(II), Pb(II), AI(III), Fe(III) and La(III). The original sulfonamide and its complexes were assayed for the in vitro inhibition of three CA isozymes, CA I, II, and IV, some of which play a critical role in ocular fluid secretion. All these compounds (the sulfonamide and its metal complexes) behaved as powerful inhibitors against the three investigated isozymes. The parent sulfonamide possessed an extremely weak topical pressure lowering effect when administered as a 1-2% suspension into the rabbit eye, but some of its metal complexes, such as the Mg(II), Zn(II), Mn(II) and Cu(II) derivatives, lower intraocular pressure (IOP) in experimental animals very well. Ex vivo data showed a 99.5-99.9% CA II inhibition in ocular fluids and tissues of rabbits treated with these agents, proving that the observed IOP lowering is due to CA inhibition. The influence of the different metal ions upon the efficiency of the obtained complexes as pressure lowering drugs are discussed, leading to the possibility of designing more selective; potent pharmacological agents from this class     

Cytotoxicity and DNA binding properties of a chloro glycylhistidinate gold(III) complex (GHAu)

The chloro glycylhistidinate gold(III) complex (GHAu) is shown to be fairly cytotoxic towards the established A2780 ovarian carcinoma human cell line either sensitive or resistant to cisplatin. Remarkably, GHAu is far more cytotoxic than the corresponding zinc(II), palladium(II), platinum(II) and cobalt(II) complexes implying that cytotoxicity is essentially to be ascribed to the presence of a gold(III) center. Circular dichroism (CD) spectra, atomic absorption measurements and DNA melting profiles suggest that GHAu in vitro is able to bind DNA, the presumed target for several antitumor metal complexes, and to modify its conformation, even if the observed changes are generally small. Implications of these findings for the mechanism of action of cytotoxic gold(III) complexes are discussed.     

Complexation of the fungal metabolite tenuazonic acid with copper (II), iron (III), nickel (II), and magnesium (II) ions

Tenuazonic acid (TA) is a phytotoxin produced by a fungal pathogen of rice, Pyricularia oryzae. We have synthesized and characterized the metal complexes of TA with copper (II), iron (III), nickel (II), and magnesium (II). The stoichiometry of the complexes determined by microanalysis and mass spectroscopy (D/CI) are Cu(II)TA2, Fe(III)TA3, Ni(II)TA2, and Mg(TA)2. Voltammograms of Fe(III)TA3, and Cu(II)TA2 in methanolic solutions confirmed this stoichiometry. Ni(II)TA2 paramagnetism and visible absorption data suggest an octahedral geometry. Fe(III)TA3 showed a characteristic visible absorption at 450 nm. Addition of Fe(III)Cl3 and Mg(II)Cl2 did not reverse the toxicity of NaTA to rice and bacterial cells, showing that this toxicity is not due to the privation of the cells of these metals essential for cell growth.     

Herbicides interacting with lanthanide(III) and calcium(II) ions: structural and biological similarities of Gd and Ca polymers

In this paper we report the synthesis and characterization of Ca(II), Gd(III) and Ce(III) complexes with chlorophenoxyalkanoic acids, which are commonly used as herbicides. The Gd(III) and Ca(II) complexes were characterized by the typical formulas [Gd(III)(L)(3)(H(2)O)(2).2dmf](n) and [Ca(L)(2)(MeOH)(2)](n) [L=[2,4-D=2,4-dichlorophenoxyacetic acid, 2,4,5-T=2,4,5-trichlorophenoxyacetic acid, MCPA=2-methyl-4-chlorophenoxy acetic acid and 2,4-DP=2-(2,4-dichlorophenoxy)propanoic acid]]. The crystal structure of the Gd(III) complex with 2,4-D shows that the compound is a one-dimensional polymer with a [Gd(III)(2)(2,4-D)(6)(H(2)O)(4)] dimeric repeat unit and the gadolinium atoms are in a nine-coordination environment, while the crystal structure of the Ca analog shows that it also has a polymeric structure with a [Ca(2)(2,4-D)(4)(CH(3)OH)(4)] dimeric repeat unit and the calcium atoms are in an eight-coordination environment. The gadolinium compound displays three different coordination modes for the carboxylato moiety, bidentate chelate, bidentate double bound and bidentate triple bound, while the calcium compound displays only one, bidentate triple bound. Coordination spheres are completed with oxygens of H(2)O or MeOH molecules, respectively. The complexes were tested against a few common bacteria by minimum inhibitory concentration (MIC) experiments and did not exhibit any antimicrobial action at concentrations up to 1600 microg/ml.     

Kinetics and mechanisms of reduction of Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha

The reduction of low-molecular-weight Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha was characterized using both kinetic analysis and 1H-NMR experiments. Whereas Fe(III) (CN)6(3-) was reduced through an outer sphere transfer over the exposed heme edge, all other Cu(II) and Fe(III) complexes investigated were reduced via a site-specific binding of the metal to the protein. Reduction of all metal complexes was enhanced by decreasing pH while only Fe(III)NTA reduction kinetics were altered by changes in ionic strength. Rates of reduction for both Cu(II) and Fe(III) were also affected inversely by the effective binding constant of the metal chelate used. NMR data confirmed that both Cu(II)NTA and Fe(III)NTA were bound to specific sites on the protein. Cu(II) bound preferentially to distal His-61 and Fe(III) exerted its greatest effect on two surface lysine residues with epsilon proton resonances at 3.04 and 3.12 ppm. The Fe(III)NTA complex also had a mild but noticeable line broadening effect on the distal His-61 singlet resonance near 5.3 ppm. Like hemoglobin and myoglobin, leghemoglobin might function not only as an oxygen carrier, but also as a biological reductant for low-molecular-weight Cu(II) and Fe(III) complexes.     

Ion-exchange and adsorption of Fe(III) by Sepia melanin

Sepia eumelanin is associated with many metal ions, yet little is known about its metal binding capacity and the chemical nature of the binding site(s). Herein, the natural concentrations of metal ions are presented and the ability to remove metals by exposure of the melanin granules to EDTA is quantified. The results reveal that the binding constants of melanin at pH 5.8 for Mg(II), Ca(II), Sr(II) and Cu(II) are, respectively, 5, 4, 14 and 34 times greater than the corresponding binding constants of these ions with EDTA. By exposing Sepia eumelanin to aqueous solutions of FeCl(3), the content of bound Fe(III) can be increased from a natural concentration of approximately 180 ppm to a saturation limit of approximately 80 000 ppm or 1.43 mmol/g of melanin. Similar saturation limits are found for Mg(II) and Ca(II). Exposure of Sepia melanin granules to aqueous solutions containing Ca(II) results in the stoichiometric replacement of the initially bound Mg(II), arguing that these two ions occupy the same binding site(s) in the pigment. The pH-dependent binding of Mg(II) and Ca(II) suggests coordination of these ions to carboxylic acid groups in the pigment. Mg(II) and Ca(II) can be added to a Fe(III)-saturated melanin sample without affecting the amount of Fe(III) pre-adsorbed, clearly establishing Fe(III) and Mg(II)/Ca(II) occupy different binding sites. Taking recent Raman spectroscopic data into account, the binding of Fe(III) is concluded to involve coordination to o-dihydroxyl groups. The effects of metal ion content on the surface morphology were analyzed. No significant changes were found over the full range of Fe(III) concentration studied, which is supported by the Brunauer-Emmett-Teller surface area analysis. These observations imply the existence of channels within the melanin granules that can serve to transport metal ions.     

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.     

Iron(III) reduction by D-galacturonic acid. Part II. Influence of uranyl(VI), lead(II), nickel(II), and cadmium(II) complexes formation

As a part of our study on iron reduction-mobilization in biological systems, in particular at root-soil interface, the effect of the addition of different metal ions to the iron(III)-D-galacturonic acid system has been investigated. The ions which are found to form particularly stable complexes with the galacturonate ligand strongly increase the yield of the reduction of iron(III) to iron(II). These findings are in agreement with the capability of some metal ions to form stable complexes through interaction both with the carboxylate group and with the ring oxygen atom of the sugar molecule, inducing opening of the ring and formation of a free aldehydic group. The importance of these processes in availability of iron to plant roots is emphasized.