Vibrational circular dichroism and IR absorption of DNA complexes with Cu2+ ions

Vibrational circular dichroism (VCD) spectroscopy and simultaneous IR absorption measurements are applied to study the interaction of natural calf thymus DNA with Cu2+ ions at room temperature in a Cu2+ concentration range of 0-0.4M (a Cu2+/phosphate molar ratio [Cu]/[P] of 0-0.7). In some important instances, VCD provides more detailed insights than previous IR investigations whereas in several others it leads to the same interpretations. The Cu2+ ions bind to phosphate groups at a low metal concentration. Upon increasing the ion concentration, chelates are formed in which Cu2+ binds to the N7 of guanine (G) and a phosphate group. Detectable only by VCD, significant distortion of most guanine-cytosine (GC) base pairs occurs at a [Cu]/[P] ratio of 0.5 with only a minor affect on adenine-thymine (AT) base pairs, which favors a "sandwich" complex in which a Cu2+ ion is inserted between two adjacent guanines in a GpG sequence. The AT base pairs become significantly distorted when the metal concentration is increased to 0.7 [Cu]/[P]. A number of GC base pairs, which are possibly involved in sandwich complexes, remain stacked and paired even at 0.7 [Cu]/[P], preventing complete strand separation. The DNA secondary structure changes considerably from the standard B-form geometry at a [Cu]/[P] ratio of 0.4 and higher. A further transition to some intermediate conformation that is inconsistent with either the A- or Z-form or a completely denatured state is suggested in agreement with other works. In general, VCD proves to be a reliable indicator of the 3-dimensional structure of the DNA-metal ion complexes, which reveals structural details that cannot be deduced from the IR absorption spectra alone.     

Inhibition of Mn2+-induced error-prone DNA synthesis with Cd2+ and Zn2+

Bivalent metal cations are key components in the reaction of DNA synthesis. They are necessary for all DNA polymerases, being involved as cofactors in catalytic mechanisms of nucleotide polymerization. It is also known that in the presence of Mn²⁺ the accuracy of DNA synthesis is considerably decreased. The findings of this work show that Cd²⁺ and Zn²⁺ selectively inhibit the Mn²⁺-induced error-prone DNA polymerase activity in extracts of cells from human and mouse tissues. Moreover, these cations in low concentrations also can efficiently inhibit the activity of homogeneous preparations of DNA polymerase iota (Pol ?), which is mainly responsible for the Mn²⁺-induced error-prone DNA polymerase activity in cell extracts. Using a primary culture of granular cells from postnatal rat cerebellum, we show that low concentrations of Cd²⁺ significantly increase cell survival in the presence of toxic Mn²⁺ doses. Thus, we have shown that in some cases low concentrations of Cd²⁺ can display a positive influence on cells, whereas it is widely acknowledged that this metal is not a necessary microelement and is toxic for organisms.     

Effect of Mn2+ and Mg2+ ions on DNA conformation

The DNA conformation was studied at different relation between Na+ and Me2+ (Mn2+ or Mg2+) ions in solution at the fixed total ionic strength mu. At low mu the intrinsic viscosity of DNA [eta] decreased to the limited fixed value with the increasing of Mn2+ or Mg2+ concentration (CMe2+). At higher mu greater than or equal to 0.1 M [eta] doesn't depend on CMe2+. The presence of Mn2+ in solution caused a decrease of the optical anisotropy of DNA and the value of epsilon 260 (p) independent on ionic strengths. In contrary, these parameters of DNA didn't change in solution with Mg2+-concentration. The observed differences in the effects of Mn2+ and Mg2+ on the optical properties of the macromolecule suggest that there are different modes of binding of these ions to DNA. It has been concluded, that Mn2+ interacts with bases and phosphate groups of DNA, but Mg2+--only with phosphates. The persistence length of DNA doesn't depend on Me2+ concentration under the conditions of the experiment (mu greater than or equal to 0.005 M).     

Spectroscopic and theoretical investigations of Schiff base metal complexes with intraligand charge-transfer behavior

Nickel and zinc complexes of hydroxy or mercapto substituted glyoxaldianiles exhibit unusual long-wavelength absorptions. The electronic structure and spectroscopic properties of the compounds are investigated by means of electron spectroscopic studies and qualitative MO considerations. The deep color of the complexes is caused by intraligand charge-transfer transitions from the oxygen or sulphur-containing donor part to the diimine substructure of the ligand. The transition energies depend sensitively on the nature of the donor part of the ligand, and on the solvent polarity. The close structural relationship between these complexes and the d⁸ mixed-ligand complexes with S/O-donor and N-acceptor ligands is discussed.     

Stability and structure of binary and ternary complexes of alpha-lipoate and lipoate derivatives with Mn2+, Cu2+, and Zn2+ in solution.

The stabilities of the 1:1 complexes of Mn²⁺, Cu²⁺, and Zn²⁺ with lipoate and its chainshortened catabolites, viz., bisnorlipoate and tetranorlipoate, were studied by potentiometric titrations in water containing 50% dioxane (I = 0.1, NaClO₄; 25 °C). A comparison of the stabilities of these complexes with those of simple carboxylates reveals that the catabolite complexes formed with Cu²⁺ and Zn²⁺ are more stable than expected from only the basicity of the carboxylate groups. This is evidence that chelates involving the disulfide group are formed. The stability of all Mn²⁺ complexes is determined by the basicity of the carboxylate groups. The same pattern of stability holds for the mixed-ligand complexes formed by Cu²⁺ or Zn²⁺, 2,2′-bipyridyl, and lipoate or one of its derivatives. It is evident that the disulfide group of the 1,2-dithiolane moiety can participate in the formation of binary and ternary complexes. The somewhat less-pronounced coordinating properties of the 1,2-dithiolane moiety compared with the tetrahydrothiophene moiety are discussed. It is apparent that the electron density at S(1) and S(2) in the dithiolane moiety of lipoate is not equivalent: S(1) is favored over S(2) in electrophilic reactions; possible biological implications are indicated.     

Mn2+, Co2+, Cu2+ and Zn2+ complexes with two macrocyclic ligands bearing L-lactate-like functions: potentiometric studies and evaluation of superoxide-scavenging properties of the Mn2+ complex

Some aerobic organisms devoid of SOD use Mn2+ chelates to scavenge the O2- radical. Since the Mn2+-bis(lactato)diaquo complex is known as having a high SOD-like activity, we prepared manganese(II) complexes with triazamacrocyclic ligands bearing L-lactate-like functions in order to obtain model compounds able to disproportionate the superoxide radical. Thus, two macrocyclic ligands, N,N',N"-tris[2(S)-hydroxybutyric acid]-1,4,7-triazacyclononane, L1, and N,N',N"-tris[2(S)-hydroxybutyric acid]-1,5,9-triazacyclododecane, L2, were prepared and their capacity to retain the Mn2+ ion in aqueous solution was determined from potentiometric experiments. The chelating properties in aqueous solution of each ligand towards Co2+, Cu2+ and Zn2+ ions were also determined. L1 forms complexes with Mn2+, Co2+, Cu2+ and Zn2+ ions with stability constants of 8.33(5), 15.78(5), 17.65(3) and 14.32(1), respectively. L2 forms complexes with Cu2+ and Zn2+ ions with stability constants of 10.67(1) and 6.98(3), respectively. But the constants related to the Mn2+ and Co2+ complexes were too low to be determined by the method used. The stability constants values calculated for L2 complexes are significantly lower than those for the corresponding complexes of L1. Additional spectroscopic measurements were carried out on the Mn2+-L1 system. The electronic spectrum of this system showed a pH-dependence that may be consistent with the formation of hydroxo-species as the ESR spectra recorded at 120 K did not show oxidation of the Mn2+ ion in the pH range studied. The superoxide-scavenging activity of the manganese(II)-L1 complex was investigated using the cytochrome c assay. The Mn2+-L1 system showed an IC50 value of 1.7 microM which indicates that it appears as a potent SOD mimic.     

The effects of Cu2+ and Pb2+ on the solution structure of calf thymus DNA: DNA condensation and denaturation studied by fourier transform IR difference spectroscopy

The interaction of calf thymus DNA with Cu²⁺and Pb²⁺ was studied in aqueous solution at pH 6.5 with metal/DNA (P) (P = phosphate) molar ratios (r) 1/80, 1/40, 1/20, 1/10, 1/4, 1/2, and 1, using Fourier Transform ir (FTIR) spectroscopy. Correlations between the ir spectral changes, metal ion binding mode, DNA condensation, and denaturation, as well as conformational features, were established. Spectroscopic evidence has shown that at low metal/DNA (P) molar rations 1/80 and 1/40, copper and lead ions bind mainly to the PO of the backbone, resulting in increased base-stacking interaction and duplex stability. The major copper ion base binding via G-C base pairs begins at r > 1/40, while the lead ion base binding occurs at r > 1/20 with the A-T base pairs. The denaturation of DNA begins at r = 1/10 and continues up to r = 1/2 in the presence of copper ions, whereas a partial destabilization of the helical structure was observed for the lead ion at high metal ion concentration (r = 1/2). Metal-DNA binding also results in DNA condensation. No major departure from the B-family structure was observed, upon DNA interaction with these metal ions. © 1993 John Wiley & Sons, Inc.     

Quantum chemical investigations on the complexes of Ca2+ and Zn2+ with aliphatic dipeptides

Several 1:1 complexes of Zn²⁺ with glycylglycine and of Ca²⁺ and Zn²⁺ with prolylglycine and glycylproline have been calculated within the Hartree-Fock method using a minimal GLO basis set. It was found that in spite of the fact that there are large differences in complex binding energy the relative stabilities of the different binding sites are the same in the case of Ca²⁺ and Zn²⁺ ions. Electronic density diagrams have been produced to illustrate the changes in electronic distribution caused by complex formation.     

Spectroscopic and molecular modeling studies of caffeine complexes with DNA intercalators.

Recent studies have demonstrated that caffeine can act as an antimutagen and inhibit the cytoxic and/or cytostatic effects of some DNA intercalating agents. It has been suggested that this inhibitory effect may be due to complexation of the DNA intercalator with caffeine. In this study we employ optical absorption, fluorescence, and molecular modeling techniques to probe specific interactions between caffeine and various DNA intercalators. Optical absorption and steady-state fluorescence data demonstrate complexation between caffeine and the planar DNA intercalator acridine orange. The association constant of this complex is determined to be 258.4 +/- 5.1 M-1. In contrast, solutions containing caffeine and the nonplanar DNA intercalator ethidium bromide show optical shifts and steady-state fluorescence spectra indicative of a weaker complex with an association constant of 84.5 +/- 3.5 M-1. Time-resolved fluorescence data indicate that complex formation between caffeine and acridine orange or ethidium bromide results in singlet-state lifetime increases consistent with the observed increase in the steady-state fluorescence yield. In addition, dynamic polarization data indicate that these complexes form with a 1:1 stoichiometry. Molecular modeling studies are also included to examine structural factors that may influence complexation.     

CHIH-DFT determination of the molecular structure and IR and UV spectra of solanidine

Solanidine is the steroidal aglycon of some potato glycoalkaloids and a very important precursor for the synthesis of hormones and some pharmacologically active compounds. In this work, we make use of a new chemistry model within Density Functional Theory, called CHIH-DFT, to calculate the molecular structure of solanidine, as well to predict its infrared and ultraviolet spectra. The calculated values are compared with the experimental data available for this molecule as a means of validation of our proposed chemistry model. Figure Molecular structure of solanidine calculated with the CHIH(small) model chemistry     

DNA interaction with Mn2+ ions at elevated temperatures: VCD evidence of DNA aggregation

Interaction of natural calf thymus DNA with Mn(2+) ions was studied at room temperature and at elevated temperatures in the range from 23 degrees C to 94 degrees C by means of IR absorption and vibrational circular dichroism (VCD) spectroscopy. The Mn(2+) concentration was varied between 0 and 1.3M (0 and 10 [Mn]/[P]). The secondary structure of DNA remained in the frame of the B-form family in the whole ion concentration range at room temperature. No significant DNA denaturation was revealed at room temperature even at the highest concentration of metal ions studied. However at elevated temperatures, DNA denaturation and a significant decrease of the melting temperature of DNA connected with a decrease of the stability of DNA induced by Mn(2+) ions occurred. VCD demonstrated sensitivity to DNA condensation and aggregation as well as an ability to distinguish between these two processes. No condensation or aggregation of DNA was observed at room temperature at any of the metal ion concentrations studied. DNA condensation was revealed in a very narrow range of experimental conditions at around 2.4 [Mn]/[P] and about 55 degrees C. DNA aggregation was observed in the presence of Mn(2+) ions at elevated temperatures during or after denaturation. VCD spectroscopy turned out to be useful for studying DNA condensation and aggregation due to its ability to distinguish between these two processes, and for providing information about DNA secondary structure in a condensed or aggregated state.     

Association of antitumor antibiotic Mithramycin with Mn2+ and the potential cellular targets of Mithramycin after association with Mn2+

Mithramycin (MTR), an aureolic acid group of antitumor antibiotic is used for the treatment of several types of tumors. We have reported here the association of MTR with an essential micronutrient, manganese (Mn²⁺). Spectroscopic methods have been used to characterize and understand the kinetics and mechanism of complex formation between them. MTR forms a single type of complex with Mn²⁺ in the mole ratio of 2:1 [MTR: Mn²⁺] via a two step kinetic process. Circular dichroism (CD) spectroscopic study indicates that the complex [(MTR)₂ Mn²⁺] has a right-handed twist conformation similar in structure with the complexes reported for Mg²⁺ and Zn²⁺. This conformation allows binding via minor groove of DNA with (G, C) base preference during the interaction with double-stranded B-DNA. Using absorbance, fluorescence, and CD spectroscopy we have shown that [(MTR)₂ Mn²⁺] complex binds to double-stranded DNA with an apparent dissociation constant of 32?μM and binding site size of 0.2 (drug/nucleotide). It binds to chicken liver chromatin with apparent dissociation constant value 298?μM. Presence of histone proteins in chromatin inhibits the accessibility of the complex for chromosomal DNA. We have also shown that MTR binds to Mn²⁺ containing metalloenzyme manganese superoxide dismutase from Escherichia coli.     

Extended X-ray absorption fine structure of Mn2+ and Mn2+ X ATP complex bound to coupling factor 1 of the H+-ATPase from chloroplasts

The spinach chloroplast ATPase, coupling factor 1, contains three tight Mn2+-binding sites which interact cooperatively. The bound manganese coordinations were studied by x-ray absorption fine structure analysis. Mn2+ was found to be bound to the enzyme with an average Mn-O bond length of 2.15 +/- 0.15 A, significantly shorter than the 2.15 +/- 0.15 A of the Mn-O bond of the average first hydration shell for Mn2+ in aqueous solution. On adding ATP to the manganese-enzyme mixture, a tertiary complex of Mn2+ X ATP X enzyme was formed as indicated by the appearance of a second shell. Mn-P bond distances were estimated at 4.95 +/- 0.15 A in the tertiary Mn2+ X ATP X enzyme complex, which was considerably longer than the Mn-P bond distance of 3.36 +/- 0.15 A for the Mn2+ X ATP complex in aqueous solution. The Mn-P bond distance in the tertiary Mn2+ X ATP X enzyme complex decreased to 4.32 +/- 0.15 A when selenite, a potent effector of ATPase activity, was added. Based on these results, it is suggested that the tertiary complex is required for catalysis. The stimulation of ATP hydrolysis by anions such as selenite may be the result of shortening the distance between Mn2+ and the ATP phosphates in the enzyme active site.     

Comparison of the reaction progress of calcineurin with Mn2+ and Mg2+

Activation of calcineurin by Mn2+ and Mg2+ was compared using a heavy atom isotope analogue of the substrate p-nitrophenyl phosphate (pNPP). Heavy atom isotope effects were measured for Mg2+ activation and compared to published results of the isotope effects with Mn2+ as the activating metal. Isotope effects were measured for the kinetic parameter Vmax/Km at the nonbridging oxygen atoms [18(V/K)nonbridge]; at the position of bond cleavage in the bridging oxygen atom [18(V/K)bridge]; and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The isotope effects increased in magnitude upon changing from an optimal pH to a nonoptimal pH; the 18(V/K)bridge effect increased from 1.0154 (+/-0.0007) to 1.0198 (+/-0.0002), and the 15(V/K) effect increased from 1.0018 (+/-0. 0002) to 1.0021 (+/-0.0003). The value for 18(V/K)nonbridge is 0. 9910 (+/-0.0003) at pH 7.0. As with Mn2+, the 18(V/K)nonbridge isotope effect indicated that the dianion was the substrate for catalysis, and that a dissociative transition state was operative for the phosphoryl transfer. Comparison to results for Mn2+ activation suggested that chemistry was more rate-limiting with Mg2+ than with Mn2+. Changing the activating metal concentration showed opposite trends with increasing Mg2+ increasing the commitment factor and seemingly making the chemistry less rate-limiting. The influence of viscosity was evaluated as well to gauge the role of chemistry. The activation of calcineurin-catalyzed hydrolysis of pNPP1 by Mg2+ or Mn2+ at pH 7.0 was compared in the presence of viscogens, glycerol and poly(ethylene glycol). Increasing glycerol caused different effects with the two activators. With Mn2+ as the activator, calcineurin activity showed a normal response with kcat and kcat/Km decreasing with viscosity. There was an inverse response with Mg2+ as the activator as values of kcat/Km increased with viscosity. From values of the normalized kcat/Km with Mn2+, the chemistry was found to be partially rate-limiting, consistent with previous heavy atom isotope studies (22). The effect observed for Mg2+ seems consistent with a change in the rate-limiting step for the two different metals at pH 7.0.     

Mn 2+ , and Zn 2+ 1:1 complexes with biochemically significant thioether carboxylic acids and some of the sulfoxide and sulfone derivatives

The 1:1 complexes of Mn²⁺, Cu²⁺, and Zn²⁺ with S-carboxymethyl alkyl and S-carboxymethyl aryl mercaptans were studied in water containing 50% dioxane (I = 0.1; t = 25 °). The determination of the stability constants and a comparison with simple carboxylate complexes reveals that the complexes of Cu²⁺ (and slightly also of Zn²⁺) with the S-carboxymethyl alkyl mercaptans are more stable than expected from only basicity of the carboxylate groups. This suggests that the thioether group participates in complex formation, i.e., chelates are formed. The Mn²⁺ complexes of both kinds of ligands, and the Cu²⁺ or Zn²⁺ complexes with S-carboxymethyl aryl mercaptans have the stability expected according to the basicity of the carboxylate groups. NMR experiments with S-carboxymethyl ethyl mercaptan confirm the formation of chelates with Cu²⁺ and suggest simple carboxylate complexes with Mn²⁺. Analogous experiments with (S-carboxymethyl phenyl mercaptan do not allow an unequivocal statement about the distribution between simple carboxylate complexes and chelates for both metal ions. Also, as the thioether acids are biologically oxidized, the complex stabilities of several of such oxidized derivatives were measured.