Investigation of the hyaluronic acid-copper complex by N.M.R. spectroscopy

Analysis of the 13C and 1H relaxation data of the hyaluronic acid-copper complex indicates a binding site involving the carboxyl group and O-1 of the D-glucuronic acid moiety. The paramagnetic relaxation of Cu2+ is discussed within the framework of the Solomon-Bloembergen formalism and it is shown that various atoms experience, in addition to the dipolar paramagnetic relaxation, a strong scalar relaxation contribution. E.s.r. spectra have also been obtained in order to determine the binding constants, and measurements at 69 K gave the g-values of the complex.     

Transport of basic amino acids in Candida albicans

In Candida albicans, ATCC 46977, transport of basic amino acids is mediated by two systems (S1 and S2). Kinetic data and competitive inhibition studies of the different systems showed that transport of L-lysine, L-arginine and L-histidine have distinct specificities. System S1 of L-lysine and L-arginine was highly specific for the respective single basic amino acid. However, S2 of L-lysine and S1 of L-histidine were shown to be specific systems for most of basic amino acids. S2 of L-arginine was different from S2 of L-lysine and S1 of L-histidine. The effect of a thiol reagent, N-ethylmalemide, revealed that S2 of L-lysine and S1 of L-histidine were sensitive to this reagent, while all other systems were insensitive. The transport activity of different systems of L-lysine, L-arginine and L-histidine was followed during the growth of C. albicans. It was observed that different basic amino-acid systems have maximum activity during different stages of C. albicans growth.     

How amino acids control the binding of Cu(II) ions to DNA. Part III. A novel interaction of a histidine complex with DNA

L-Histidine Cu(II) complex bound to DNA showed broad EPR signals characteristic of the aggregated Cu(II) species, which could be observed even when the molar ratio of L-histidine to Cu(II) ion was smaller than unity. The signal for the DNA fibers changed with the orientation of the fibers in the static magnetic field. Based on these results, the signal was assigned to a mono-histidine Cu(II) complex stereospecifically aggregated in a groove or along a phosphodiester chain of the double helical DNA. In contrast to the L-histidine complex, the D-histidine complex bound to DNA did not show such broad signals and the observed spectra for the complex on B-form DNA fibers at -150 degrees C were simulated assuming that the g1 axis of the mono-D-histidine complex tilts by about 55 degrees from the DNA-fiber axis. Addition of some deoxy-nucleotides, but not deoxy-nucleosides, to a solution of a mono-histidine complex resulted in the formation of a dinuclear ternary complex with different structures for L- or D-histidine, suggesting the possibility that the stereospecific aggregation of the L-histidine complex on a double helical DNA was mediated by the phosphodiester backbones.     

Copper(II) complexation by pituitary adenylate cyclase activating polypeptide fragments.

Results are reported from potentiometric and spectroscopic (UV-Vis, CD, and ESR) studies of the protonation constants and Cu2+ complex stability constants of pituitary adenylate cyclase activating polypeptide fragments (HSDGI-NH2, TDSYS-NH2, RKQMAVKKYLAAVL-NH2). With HSDGI-NH2, the formation of a dimeric complex Cu2H-2L2 was found in the pH range 5-8, in which the coordination of copper(II) is glycylglycine-like, while the fourth coordination site is occupied by the imidazole N3 nitrogen atom, forming a bridge between two copper(II) ions. The formation of dimeric species does not prevent the deprotonation and coordination of the amide nitrogen, and in pH above 8 the CuH-2L complex is formed. Aspartic acid in the third position of peptide sequence stabilizes the CuH-2L species and prevents the coordination of a fourth nitrogen donor. Aspartic acid residue in the second position of TDSYS-NH2 stabilizes the CuL (2N) complex but does not prevent deprotonation and binding of the second and third peptide nitrogens to give 3N and 4N complexes at higher pH. The tetradecapeptide amide forms with copper(II) ions unusually stable 3N and 4N complexes compared to pentaalanine amide.     

Crystallization and preliminary X-ray diffraction studies of the metal-ion-mediated ternary complex of the HutP protein with L-histidine and its cognate RNA

HutP is an RNA-binding protein that regulates the expression of the Bacillus subtilis hut operon by binding to cis-acting regulatory sequences within hut mRNA, exclusively in the presence of L-histidine. We recently solved the crystal structure of a binary complex (HutP with an L-histidine analog) that revealed a novel RNA-binding fold, and identified the important residues that interact with the L-histidine analog. In addition, we have defined the minimal RNA binding segment that is required for HutP recognition. Interestingly, we showed that ternary complex formation depends on the availability of not only L-histidine but also divalent metal ions. Here we report the crystallization and preliminary X-ray diffraction analysis of the HutP ternary complex. The ternary complex was crystallized in the presence of Mg2+ along with L-histidine and hut mRNA, using the hanging drop vapor diffusion method. The crystal belongs to the R3 space group, with unit cell parameters a=b=75.30 A, c=133.8 A. A complete data set at 1.60 A was collected.     

Zinc stabilizes the SecB binding site of SecA

The molecular chaperone SecB targets preproteins to SecA at the translocation sites in the cytoplasmic membrane of Escherichia coli. SecA recognizes SecB via its carboxyl-terminal 22 aminoacyl residues, a highly conserved domain that contains 3 cysteines and 1 histidine residue that could potentially be involved in the coordination of a metal ion. Treatment of SecA with a zinc chelator resulted in a loss of the stimulatory effect of SecB on the SecA translocation ATPase activity, while the activity could be restored by the addition of ZnCl2. Interaction of SecB with the SecB binding domain of SecA is disrupted by chelators of divalent cations, and could be restored by the addition of Cu2+ or Zn2+. Atomic absorption and electrospray mass spectrometry revealed the presence of one zinc atom per monomeric carboxyl terminus of SecA. It is concluded that the SecB binding domain of SecA is stabilized by a zinc ion that promotes the functional binding of SecB to SecA.     

The effect of carboxylates and halides on L-lysine 6-aminotransferase-catalyzed reactions

L-Lysine:2-oxoglutarate 6-aminotransferase catalyzes very slow transamination between L-alanine and 2-oxoglutarate. A high concentration of anions such as formate, acetate and halides greatly accelerated this transamination without affecting the affinity of the enzyme for L-alanine. In contrast, the anions strongly inhibited the normal L-lysine 6-transamination in a competitive manner with L-lysine and in a non-competitive manner with 2-oxoglutarate. This result suggests that the enzyme has an anion binding site which normally binds the carboxyl group of L-lysine. The binding of halides or carboxylates to this site probably induces a conformational change of the enzyme, and results in the inhibition of L-lysine 6-transamination, and in the stimulation of L-alanine transamination. Treatment of the enzyme with an arginine-specific dicarbonyl reagent, phenylglyoxal, led to a loss of the enzyme activity for L-lysine. The activity for L-alanine was not affected, but the stimulating effect of anions on L-alanine transamination was impaired. Thus, it is suggested that an arginine residue(s) plays an important role in the anion binding site.     

Dicyclohexylcarbodiimide interaction with sarcoplasmic reticulum. Inhibition of Ca2+ efflux

Dicyclohexylcarbodiimide (DCCD), a hydrophobic carboxyl reagent, inhibited Ca2+ release from Ca2+-loaded sarcoplasmic reticulum vesicles, induced by elevated pH, tetraphenylboron, ATP + Pi, or membrane modification with acetic anhydride. Under the conditions used, the same concentrations of DCCD were required for inhibition of Ca2+ release, Ca2+-ATPase activity, and Ca2+ uptake. On the other hand, free Ca2+ or alkaline pH prevented the inhibition by DCCD of Ca2+-ATPase and coupled Ca2+ transport but not that of Ca2+ release. Moreover, several hydrophilic carboxyl reagents inhibited Ca2+-ATPase but not Ca2+ release. We suggest that a carboxyl residue(s), located in a hydrophobic region of a protein(s), is involved in the control of Ca2+ release, where DCCD interaction with this group blocks Ca2+ release. This group is distinct from the one involved in the inhibition of Ca2+-ATPase. DCCD also inhibited [3H]ryanodine binding to junctional sarcoplasmic reticulum membranes. The presence of Ca2+ or an alkaline pH only slightly affects the degree of inhibition of ryanodine binding by DCCD. Incubation of the membranes with [14C]DCCD resulted in labeling of 350-, 170-, 140-, 53-, and 30-kDa proteins in addition to the Ca2+-ATPase. The involvement of one or all of the DCCD-labeled proteins in Ca2+ release and ryanodine binding is discussed.     

Structural, motional, and kinetic features of the Cu(II)-(L-His)2 complex in aqueous solution

A careful analysis by 1H and 13C FT-NMR on the Cu(II) (L-histidine)2 complex was carried out which allows delineation of structure and dynamics in solution. A mixture of complexes was shown such that 24% of the Cu(II) (L-histidine)2 complex contains both histidines bound in the histaminelike way, while the remaining 76% contains one L-His molecule bound in the histaminelike way and the other L-His molecule bound in the glycinelike way. The motional correlation time and relevant features of the exchange process were also delineated.     

13C and 113Cd NMR studies of the chelation of metal ions by the calcium binding protein parvalbumin

13C NMR spectra are presented for the calcium binding protein parvalbumin (pI 4.25) from carp muscle in several different metal bound forms: with Ca2+ in both the CD and EF calcium binding sites, with Cd2+ in both sites, with 113Cd2+ in both sites, and with 113Cd2+ in the CD site and Lu3+ in the EF site. The different metals differentially shift the 13C NMR resonances of the protein ligands involved in chelation of the metal ion. In addition, direct 13C-113Cd spin-spin coupling is observed which allows the assignment of protein carbonyl and carboxyl 13C NMR resonances to ligands directly interacting with the metal ions in the CD and EF binding sites. The displacement of 113Cd2+ from the EF site by Lu3+ further allows these resonances to be assigned to the CD or EF site. The occupancy of the two sites in the two cadmium species and in the mixed Cd2+/Lu3+ species is verified by 113Cd NMR. The resolution in these 113Cd NMR spectra is sufficient to demonstrate direct interaction between the two metal binding sites.     

pH-dependence of 13C chemical shifts and 13C,H coupling constants in imidazole and L-histidine.

The pH-dependence of selected 13C chemical shifts reflects the state of ionization of the imidazole ring in both imidazole and L-histidine. Titration of the amino and carboxyl groups of histidine also perturbs the shifts. The coupling constants 1J (13C(2),H) and 1J (13C(5),H) for both compounds also vary with pH, but in L-histidine these constants are relatively insensitive to the titration of groups outside the imidazole ring.     

Copper inclusion in cellulose using sodium d-gluconate complexes

Copper containing cellulose material is of growing interest, e.g. offering alternative in the field of antimicrobials. Solutions of copper d-gluconate complexes (Cu(2+)-DGL) were used to introduce copper ions into a swollen cellulosic matrix. A ligand exchange mechanism forms the chemical basis of the sorption process. Copper sorption in cellulose was studied in the range between pH 6 and 13. An estimate for the complex stabilities of the Cu-cellulose system could be derived from the calculated species distribution of the different Cu(2+)-DGL complexes present. Spectrophotometry and cyclic voltammetry of Cu(2+)-DGL complex solution were used to confirm the presence of different species participating in the ligand exchange reaction. The pH dependent uptake of Cu(2+) ions in the cellulose matrix can be explained on the basis of the relative stabilities of Cu(2+)-DGL complex vs. Cu(2+)-cellulose complexes. In comparison to pH 10, higher copper content was observed at pH 6 and 13. Copper content was limited by carboxyl content of cellulosic materials, thus in analogy to the structure of Cu(2+)-DGL complexes participation of the carboxyl group as complex forming site is proposed. At high Cu(2+)-concentration and longer time of immersion in the copper complex solutions formation of solid deposits was observed on the surface of the treated fibres.     

Intramolecular stacking interactions in ternary copper(II) complexes formed by a heteroaromatic amine and 9-[2-(2-phosphonoethoxy)ethyl]adenine, a relative of the antiviral nucleotide analogue 9-[2-(phosphonomethoxy)ethyl]adenine

The stability constants of the mixed-ligand complexes formed between Cu(Arm)2+, where Arm=2,2'-bipyridine (Bpy) or 1,10-phenanthroline (Phen), and the dianions of 9-[2-(2-phosphonoethoxy)ethyl]adenine (PEEA2-) and (2-phosphonoethoxy)ethane (PEE2-), also known as [2-(2-ethoxy)ethyl]phosphonate, were determined by potentiometric pH titrations in aqueous solution (25 degrees C; I=0.1 M, NaNO3). The ternary Cu(Arm)(PEEA) complexes are considerably more stable than the corresponding Cu(Arm)(R-PO3) species, where R-PO3(2-) represents a phosph(on)ate ligand with a group R that is unable to participate in any kind of interaction within the complexes. The increased stability is attributed to intramolecular stack formation in the Cu(Arm)(PEEA) complexes and also, to a smaller extent, to the formation of 6-membered chelates involving the ether oxygen atom present in the -CH2-O-CH2-CH2-PO3(2-) residue of PEEA2-. This latter interaction is separately quantified by studying the ternary Cu(Arm)(PEE) complexes which can form the 6-membered chelates but where no intramolecular ligand-ligand stacking is possible. Application of these results allows a quantitative analysis of the intramolecular equilibria involving three structurally different Cu(Arm)(PEEA) species; e.g., of the Cu(Bpy)(PEEA) system about 11% exist with the metal ion solely coordinated to the phosphonate group, 4% as a 6-membered chelate involving the ether oxygen atom of the -CH2-O-CH2CH2-PO3(2-) residue, and 85% with an intramolecular stack between the adenine moiety of PEEA2- and the aromatic rings of Bpy. In addition, the Cu(Arm)(PEEA) complexes may be protonated, leading to Cu(Arm)(H;PEEA)+ species for which it is concluded that the proton is located at the phosphonate group and that the complexes are mainly formed (50 and 70%) by a stacking adduct between Cu(Arm)2+ and the adenine residue of H(PEEA)-. Finally, the stacking properties of adenosine 5'-monophosphate (AMP2-), of the dianion of 9-[2-(phophonomethoxy)ethyl]adenine (PMEA2-) and of several of its analogues (=PA2-) are compared in their ternary Cu(Arm)(AMP) and Cu(Arm)(PA) systems. Conclusions regarding the antiviral properties of several acyclic nucleoside phosphonates are shortly discussed.     

Potentiometric and spectroscopic studies of the Cu(II) complexes of Ala-Arg8-vasopressin and oxytocin: two vasopressin-like peptides.

The results are reported of a potentiometric and spectroscopic study of the H+ and Cu2+ complexes of Ala-Arg8-vasopressin (Ala-AVP) and oxytocin at 25 degrees C and an ionic strength of 0.10 mol dm-3 (KNO3). The coordination chemistry of oxytocin and Cu(II) has been shown to be virtually identical to that of Arg8-vasotocin, forming unusually stable complexes with four nitrogen coordination (4N complexes) below pH 7. Spectroscopic evidence suggests weak interaction between Cu(II) and the sulphur atom of the -Cys6- residue in the 2N species (pH congruent to 6) but this is absent in the 4N complex. Evidence is also presented for perturbation of electronic transitions within the aromatic ring of the Tyr residue by Cu(II). While the physiological potency of Ala-AVP is very high, its coordination chemistry differs significantly from that of Arg8-vasopressin. With Cu(II) it forms complexes of similar stability to those with tetraglycine, demonstrating that the addition of an alanyl residue to the amino-terminal of the peptide destroys the conformation which is particularly favorable for rapid 4N coordination.     

Spontaneous resolution of binary copper(II) complexes with racemic dipeptides: crystal structures of glycyl-L-alpha-amino-n-butyrato copper(II) monohydrate, glycyl-D-valinato copper(II) hemihydrate, and glycyl-L-valinato copper(II) hemihydrate

Copper(II) complexes with glycyl-DL-alpha-amino-n-butyric acid (H2gly-DL-but), glycyl-DL-valine (H2gly-DL-val), glycyl-DL-norleucine (H2gly-DL-norleu), glycyl-DL-threonine (H2gly-DL-thr), glycyl-DL-serine (H2gly-DL-ser), glycyl-DL-phenylalanine (H2gly-DL-phe), and glycyl-L-valine (H2gly-L-val), have been prepared and characterized by IR, powder diffuse reflection, CD and ORD spectra, and magnetic susceptibility measurements, and by single-crystal X-ray diffraction. The crystal structures of the copper complex with H2gly-DL-but, the copper complex with H2gly-DL-val, and [Cu(gly-L-val)]n.0.5nH2O have been determined by a single-crystal X-ray diffraction method. As for the structure of the copper complex with H2gly-DL-but, the configuration around the asymmetric carbon atom is similar to that of [Cu(gly-L-val)]n.0.5nH2O. Therefore it is concluded that the copper complex with H2gly-DL-but is [Cu(gly-L-but)]n.nH2O. On the contrary, as for the structure of the copper complex with H2gly-DL-val, the configuration around the asymmetric carbon atom is different from that of [Cu(gly-L-val)]n.0.5nH2O. Therefore it is concluded that the copper complex with H2gly-dl-val is [Cu(gly-D-val)]n.0.5nH2O. So during the crystallization of the copper(II) complexes with H2gly-DL-but and H2gly-DL-val, spontaneous resolution has been observed; the four complexes have separated as [Cu(gly-D-but)]n.nH2O, [Cu(gly-L-but)]n.nH2O, [Cu(gly-D-val)]n.0.5nH2O, and [Cu(gly-L-val)]n.0.5nH2O, respectively. [Cu(gly-L-but)]n.nH2O is orthorhombic with the space group P2(1)2(1)2(1). [Cu(gly-D-val)]n.0.5nH2O and [Cu(gly-L-val)]n.0.5nH2O are monoclinic with the space group C2. In these complexes, the copper atom is in a square-pyramidal geometry, ligated by a peptide nitrogen atom, an amino nitrogen atom, a carboxyl oxygen atom, and a carboxyl oxygen atom and a peptide oxygen atom from neighboring molecules. So these complexes consist of a two-dimensional polymer chain bridged by a carboxyl oxygen atom and a peptide oxygen atom from neighboring molecules. The axial oxygen atom is located above the basal plane and the side chain of an amino acid is located below it. These polymer chains consist of only one or the other type of optical isomers; no racemic dipeptides are found. Therefore, spontaneous resolution has been observed in the crystallization of copper(II) complexes with H2gly-DL-but and H2gly-DL-val. The crystal structure of [Cu(gly-D-val)]n.0.5nH2O agrees almost completely with that of [Cu(gly-L-val)]n.0.5nH2O, except for the configuration around the asymmetric carbon atom.     

Effects of modifying individual amino or carboxyl groups on the affinity of calmodulin for calcineurin

The effects of modifying individual lysyl, aspartyl, or glutamyl residues in calmodulin on its ability to bind to the neural phosphatase calcineurin have been investigated using a competitive binding method termed "label selection." Samples of calmodulin were radiochemically labeled at a low level (0.03-0.6 group/molecule) by acetylation of amino groups or coupling carboxyl groups with ethanolamine to produce preparations containing predominantly single-site modified and unmodified molecules. These preparations were incubated in a 5-10-fold molar excess with bovine calcineurin under conditions appropriate for complex formation. The bound population was isolated, and the level of modification of each reactive residue was compared with the level in the corresponding group in the intial unselected preparation to determine if molecules modified at specific sites had been selected for or against during the competition for complex formation. Significant selection was observed against molecules modified at Lys21, Asp64, Glu67, Lys75, Glu84, Glu114, Asp118, or Lys148, whereas modification of Glu83 increased binding. The modification of other groups, including components of the four Ca2+-binding sites, had no effect on the interaction. Glu67, a Ca2+-liganding residue in Ca2+-binding site II that may regulate the orientation of this site in relation to the central helix, had the strongest influence on complex formation. Most of the residues identified form a nearly linear array in the three-dimensional structure of calmodulin and indicate the location of an extended surface for interaction with calcineurin and other enzymes.     

Extent of metal ion-sulfur binding in complexes of thiouracil nucleosides and nucleotides in aqueous solution

Previously published stability constants of several metal ion (M2+) complexes formed with thiouridines and their 5'-monophosphates, together with recently obtained log K(M(U))(M) versus pK(U)(H) plots for M2+ complexes of uridinate derivatives (U-) allowed now a quantitative evaluation of the effect that the exchange of a (C)O by a (C)S group has on the stability of the corresponding complexes. For example, the stability of the Ni2+, Cu2+ and Cd2+ complexes of 2-thiouridinate is increased by about 1.6, 2.3, and 1.3 log units, respectively, by the indicated exchange of groups. Similar results were obtained for other thiouridinates, including 4-thiouridinate. The structure of these complexes and the types of chelates formed (involving (N3)- and (C)S) are discussed. A recently advanced method for the quantification of the chelate effect allows now also an evaluation of several complexes of thiouridinate 5'-monophosphates. In most instances the thiouracilate coordination dominates the systems, allowing only the formation of small amounts of phosphate-bound isomers. Among the complexes studied only the one formed by Cu2+ with 2-thiouridinate 5'-monophosphate leads to significant amounts of the macrochelated isomer, which means that in this case Cu2+ is able to force the nucleotide from the anti to the syn conformation, allowing thus metal ion binding to both potential sites and this results in the formation of about 58% of the macrochelated isomer. The remaining 42% are species in which Cu2+ is overwhelmingly coordinated to the thiouracilate residue; Cu2+ binding to the phosphate group occurs in this case only in trace amounts.     

Comparison of the Cu2+ binding to bovine, goat and human alpha-lactalbumin.

Cu2+ binds to bovine alpha-lactalbumin at two different sites, principally at a hystidyl residue and in second instance at a deprotonated amide group. In human alpha-lactalbumin, that is lacking His 68, only the second binding site was observed, so that evidence is given that His 68 in bovine alpha-lactalbumin is responsible for the major Cu2+ binding. In goat alpha-lactalbumin, the histidyl binding effectively occurs but only to a lesser degree as the accessibility of His 68 is reduced by the greater compactness of goat alpha-lactalbumin. In the three species the Cu2+ binding is independent on the occupation of the primary Ca(2+)- site.     

Molecular-level insights into furfural hydrogenation intermediates over single-atomic Cu catalysts on magnesia and silica nanoclusters

This paper addresses the geometrical, charge, topological, and thermochemical data for the adsorption of the relevant furan species during hydrogenation of furfural to furfuryl alcohol over Cu/SiO₂ and Cu/MgO catalysts. The calculations indicated that the binding of Cu to magnesia was stronger than that on silica. The results also indicated that the formation of an alkoxide intermediate is more favoured than that of a hydroxyalkyl species. The binding energetic data were in general agreement with the geometrical parameters, electron densities, and molecular orbital energy levels. The QTAIM data revealed the closed-shell interactions between Cu and O atoms on both of the catalysts. Finally, the analysis of the partial charges on the atoms revealed that the Cu atom acquires a positive charge upon interaction with the carbonyl group owing to a π-back-donation from Cu to the C?=?O bond.     

A nuclear magnetic resonance study of the metal binding sites in bacitracin.

Carbon-13 nuclear magnetic resonance spectroscopy has been used to identify sites in bacitracin which bind Cu2+ and Mn2+. Results are presented which implicate the free carboxyl groups of the aspartic and glutamic acid residues and the imidazole ring of the histidine residue as metal complexation sites between pH 6 and 8. Evidence is presented which also indicates that the thiazoline ring of bacitracin binds Mn2+. Bacitracin does not bind Cu2+ or Mn2+ at pH values of 2.5 or less.