Copper(II) ions interactions in the systems with triamines and ATP. Potentiometric and spectroscopic studies

Journal of Inorganic Biochemistry 177 (2017) 89–100

Contents lists available at ScienceDirect

Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio

Copper(II) ions interactions in the systems with triamines and ATP. Potentiometric and spectroscopic studies

MARK

S.K. Hoffmanna,⁎, J. Goslara, R. Bregier-Jarzebowskab,⁎, A. Gasowskab, A. Zalewskab, L. Lomozikb a b

Institute of Molecular Physics, Polish Academy of Sciences, Mariana Smoluchowskiego 17, 60179 Poznan, Poland Faculty of Chemistry, A. Mickiewicz University, Umultowska 89b, 61614 Poznan, Poland

A R T I C L E I N F O

A B S T R A C T

Keywords: Copper(II)-ATP-triamine 3,3-tri Spermidine EPR NMR UV–Vis Potentiometry

The mode of interaction and thermodynamic stability of complexes formed in binary and ternary Cu(II)/ATP/ triamines systems were studied using potentiometric and spectroscopic (NMR, EPR, UV–Vis) methods. It was found that in binary metal-free systems ATP/HxPA species are formed (PA: Spd = spermidine or 3,3-tri = 1,7diamino-4-azaheptane) where the phosphate groups from nucleotides are preferred negative centers and protonated amine groups of amines are positive centers of reaction. In the ternary systems Cu/ATP/Hx(PA) as well as Cu/(ATP)(PA) species are formed. The type of the formed Cu(II) complexes depends on pH of the solution. For a low pH value the complexation appears between Cu(II) and ATP molecules via oxygen atoms of phosphate groups. For a very high pH value, where ATP is hydrolyzed, the Cu(II) ions are bound to the nitrogen atoms of polyamine molecules. We did not detect any direct coordination of the N7 nitrogen atom of adenosine to Cu(II) ions. It means that the CueN7 interaction is an indirect type and can be due to noncovalent interplay including water molecule. EPR studies were performed at glassy state (77 K) after a fast freezing both for binary and ternary systems. The glassy state EPR spectra do not reflect species identified in titration studies indicating significant effect of rapid temperature decrease on equilibrium of Cu(II) complexes. We propose the molecular structure of all the studied complexes at the glassy state deduced from EPR and optical spectroscopy results.

1. Introduction Aliphatic polyamines (PA) - biogenic amines: putrescine (Put) = NH2(CH2)4NH2, spermidine (Spd) = NH2(CH2)3NH(CH2)4NH2 and spermine (Spm) = NH2(CH2)3NH(CH2)4NH(CH2)3NH2 occur in relatively large concentrations in cells of all living organisms [1–4] and take part in many biochemical processes [1,3,5]. Polyamines, compounds of high basicity, react in the form of polycations with negative fragments of such important biomolecules as DNA, RNA and protein, which affects the cell growth and the proliferation process. The presence of polyamines leads to changes in the structure of nucleic acids on several levels of their organization, which determines gene expression processes and genetic information transfer. The inhibition of the synthesis of polyamines in cells results in the retardation of proliferation [6,7]. Ribosomes with PA deficiency are less active compared to those with normal amine concentration, as well as unstable even at the appropriate level of magnesium [8,9]. The concentration of amines in the organism depends on the kind of tissue and cell age. A relatively high polyamine concentration, equal to about 2 mM spermidine or 4 mM

⁎

spermine has been found in the pancreas [10]. The PA level varies even in different parts of the same organ. For instance, an average concentration of polyamines in human leukocytes is over a dozen times higher than in erythrocytes [11]. Of particular importance is the information that PA level increases in cancerous cells, which enables to monitor the development of illness and potentially paves the way for the search of chemotherapeutic drugs [12]. Polyamines also take part in the development of Alzheimer's disease and other transmissible diseases [13]. Despite research conducted for many years, we are still far from unequivocal determination of the PA role in the processes occurring in the organisms [14]. For example, a clearer elucidation is required which of the factors: polycation charge or its structure, plays the crucial role in noncovalent interactions in the systems of living organisms, taking into account the reactions of complex formations with metal ions present in the living cells. Among the first convincing reports on the formation of molecular complexes as a result of noncovalent interactions involving protonated polyamines are those published almost 40 years ago [15,16]. Manning's polyelectrolyte theory suggests that the charge of reactants exerts the main effect on the mode of the interactions [17,18]. However, such an

Corresponding authors. E-mail addresses: [email protected] (S.K. Hoffmann), [email protected] (R. Bregier-Jarzebowska).

http://dx.doi.org/10.1016/j.jinorgbio.2017.09.004 Received 15 May 2017; Received in revised form 6 September 2017; Accepted 6 September 2017 Available online 09 September 2017 0162-0134/ © 2017 Elsevier Inc. All rights reserved.

Journal of Inorganic Biochemistry 177 (2017) 89–100

S.K. Hoffmann et al.

water molecules are not closely considered as an active coordination species. Except various experimental techniques used in investigations of interactions of Cu(II) with biomolecules the theoretical calculations of molecular geometry and possible binding sites were performed by Molecular Orbital (MO) theory for nucleic acid bases [25], by DFT calculations for compounds of aminoacids and nucleobases [26], and by Ligand Field Molecular Mechanics method for Cu(II) amine complexes [27]. However, a consistent picture of Cu(II) coordination obtained from various methods has been not achieved. In this paper we describe interactions between ATP and polyamines (PA) spermidine (Spd = 1,8-diamino-4-azaoctane) and between shorter analogue of Spd, i.e. 1,7-diamino-4-azaheptane = 3,3-tri in water solutions. Their molecular formulae are schematically presented in Scheme 1. We have analyzed potentiometry and NMR results for identification of intermolecular noncovalent interactions, which are responsible for stacking of ATP and PA biomolecules. When Cu(II) is added, a competition between intermolecular interaction and coordination interaction appears and various Cu(II) complexes are formed depending on component concentrations and pH value of the studied solution. Because of controversial results of the published data we present electron paramagnetic resonance (EPR) and optical spectra of Cu(II) first for binary systems Cu-polyamine and Cu-ATP (i.e. separately for Cu-phosphate, Cu-adenosine and Cu-ribose). The analysis of the spectra indicates possible Cu binding sites in the ternary Cu/ATP/PA system. It should be stressed that EPR measurements were performed after rapid freezing at liquid nitrogen temperature (77 K) i.e. in a solid glassy state, where anisotropic EPR spectra are well resolved. It is generally assumed that the rapid freezing allows an observation of an instantaneous state of the solution. However, we have observed a significant discrepancy between a picture observed by potentiometry at liquid state and a simpler picture observed by EPR spectroscopy at glassy state. This problem is still very weakly recognized.

approach does not explain the specificity of some reactions. Polyamines cannot be regarded as a point charge as it was assumed in the description of reactions of these biomolecules with metal ions, e.g. with magnesium [19]. The structure and spatial matching between polyamine and another bioligand should be taken into consideration when analyzing experimental results. Metal ions present in living cells and the formation of coordination compounds influence the character of noncovalent interactions between bioligands. Indeed, the centers of such interactions are at the same time the sites of metal–ligand bonds [5]. Metal ions influence structure and activity of biomolecules significantly affecting the charge distribution pattern and intermolecular interactions. The action of metal ions is different for various ions and depends primarily on their binding sites and molecular conformation. Alkali metal ions as Mg(II) or Ca(II) bind preferentially to the phosphate groups and stabilize the DNA double helix. Transition metal ions like Cu (II) or Zn(II) bind to nucleic acid basis leading to DNA denaturation by destroying the hydrogen bond structure. Copper(II) ion is an essential trace element being the internal component of many enzymes. This ion is required for growth and development of many organisms [20]. However, a binding of Cu(II) to some bioactive molecules can result in enhancing of their toxicity [21]. It is generally assumed, that metal ions are preferably coordinated by oxygen atoms of phosphate groups. However, the enhanced stability of Cu(II) complexes with adenosine nucleoside and NMR spectra suggest that nitrogen N7 of purine residue can be directly involved in the coordination [22]. Knowledge of the binding sites, complex geometry and its electronic structure are essential for any microscopic model of ATP activity. It is generally known that nucleosides are polydentate ligands with various potential binding sites, which are related to: negatively charged oxygen atoms of the phosphate group, nitrogen atoms N1 and N7 of the adenine, and hydroxyl groups of the ribose sugar [5]. When polyamine molecules appear in a solution then three additional Cu(II) coordination sites appear at NH2 and NH groups of 3,3-tri and spermidine (see Scheme 1). These polyamines are of tridentate type. We have recently studied interactions and Cu(II) coordination properties in ternary systems with linear bidentate polyamines tn = 1,3-diaminopropane and putrescine [23]. Yet, our understanding of the modes of action of Cu(II) ions at the molecular level is still not satisfactory. This is due to the complexity of the studied systems with various competing intermolecular interactions. There exist different coordination sites with different binding properties and a competition between aggregation of large biomolecules and Cu(II) coordination. Various type complexes of different stability are formed in various conditions depending on pH-value, relative concentration of components, metal charge compensation mechanism [24] and temperature. Most of studies have been performed in water solutions, but usually the

2. Experimental 2.1. Materials Adenosine-5′-triphosphate sodium salt (ATP) (purity 99%), 1,7diamino-4-azaheptane (3,3-tri) – C6H17N3 (purity 98%) and 1,8-diamino-4-azaoctane - spermidine (Spd) – C7H19N3 (purity 97%) was purchased from Sigma-Aldrich. Polyamine nitrates were prepared by dissolving appropriate amounts of free amine in methanol followed by adding equimolar amounts of HNO3. The obtained white precipitates were recrystallized, washed with methanol and dried in air. The ligands were subjected to elemental analysis, results of which (3,3-tri 22.51% C, 25.94% N, 6.10% H and Spd 25.32% C, 24.87% N, 6.61% H) were in

Scheme 1. Chemical formulae of the bioligands studied.

90

Journal of Inorganic Biochemistry 177 (2017) 89–100

S.K. Hoffmann et al.

13 C and 31P NMR spectra were measured for samples prepared by dissolving suitable amounts of ligands in D2O. The solutions of DNO3 and NaOD were employed to adjust pD and the correction of readings (a pH meter N517 made by Mera Tronik) was made according to the formula: pD = pHreadings + 0.40 [33]. The ligand concentration in the samples studied was 0.1 M at the concentration ratio of L:L’ = 1:1. The 13 C NMR spectra were recorded on a Varian Gemini 300VT NMR spectrometer with dioxane as an internal standard. The positions of 13C NMR signals were converted on TMS scale. The 31P NMR spectra were recorded on a Varian Unity 300 NMR spectrometer with H3PO4 as a standard. The optical absorption spectra in the visible region were recorded on a UV/Vis Thermo Fisher Scientific Evolution 300 spectrophotometer. Samples were prepared in H2O at ligand concentrations analogous to those used in the case of samples for potentiometric titrations at the metal-to-ligand ratio of M:L:L’ = 1:4.6:4.6, using a Plastibrand PMMA cell with 1 cm path length. The EPR spectra were recorded on a SE/X 2547 Radiopan X-band spectrometer at liquid nitrogen temperature. Samples were prepared in a water:glycol solution (1:1) to avoid water crystallization during freezing. The metal to ligand ratio was 1:4.6:4.6 and the resulting concentration of the Cu(II) ions was 1·10− 3 M. Rapid freezing down to 77 K prevents water crystallization and produces homogeneous glassy state. EPR spectra were computer simulated using WIN-SimFonia Bruker routine.

Table 1 Overall stability constants (logβ), equilibrium constants(logKe) for complexes formed in ATP/triamine systems. System

Complex

Reaction

logβ

logKe

ATP/3,3-tri

(ATP)H3(3,3tri) (ATP)H4(Spd)

ATP + H33,3-tri ⇆ (ATP) H3(3,3-tri) HATP + H3Spd ⇆ (ATP) H4(Spd) ATP + H3Spd ⇆ (ATP) H3(Spd)

31.06(11)

3.03

38.87(7)

2.76

32.94(6)

3.33

ATP/Spd

(ATP)H3(Spd)

Overall protonation constants of ligands: H33,3-tri = 28,03, H23,3-tri = 20.22, H3,3tri = 10.48. H3Spd = 29.61, H2Spd = 21.03, HSpd = 10.97; H2ATP = 10.88, HATP = 6.50.

a good agreement with theoretically calculated values (3,3-tri: 22.51% C, 26.24% N, 6.29% H; Spd: 25.15% C, 25.14% N, 6.63% H) ( ± 0.3% error apparatus). The elemental analysis was performed on an Elemental Analyzer CHN 2400, Perkin-Elmer. Cu(NO3)2 ∙ 3H2O (p.a.) was purchased from POCh, Poland and purified by recrystallization from water. The concentration of Cu(II) ions was determined by the method of inductively coupled plasma optical emission - mass spectrometry (ICP MS) on a Varian ICP-MS spectrometer. Carbonate-free NaOH solution (used as a titrant) was prepared from Sørensen solution and analyzed by the ICP MS method. D2O, NaOD and DCl were purchased from the Institute for Nuclear Research – Świerk, Poland.

3. Results and discussion

2.2. Potentiometric measurements

3.1. Interactions in the triamine/ATP systems

Potentiometric titrations were carried out on a Methrom 702 SM Titrino autotitrator. The Methrom 6.0233.100 glass electrode was calibrated in terms of hydrogen ion concentration [28] after a preliminary calibration using phthalate buffer (pH = 4.002) and borax buffer (pH = 9.225). The concentration of ligands was 1·10− 2 M in the metalfree systems and 2·10− 3 M in the ternary systems containing copper(II) ions. In the metal-free systems the concentration ratio L:L’ (where L = ATP and L’ = triamine) in the studied samples was 1:1, whereas in the systems with Cu(II) the ratios L:L’ were from 1:3.5:3.5 to 1:4.6:4.6. Potentiometric titrations were conducted at the constant ionic strength μ = 0.1 M (KNO3) at 20 ± 1 °C in an inert gas (helium) atmosphere using a CO2-free NaOH solution (~ 0.2 M) as a titrant. The addition of subsequent portions of NaOH solution did not change the ionic strength because the measurements were conducted beginning from totally protonated polyamine, hence –NHx+ cations were replaced by equivalent amounts of Na+. The initial volume of a sample was 30 cm3. The choice of a model and the determination of stability constants (logβ) of complexes formed in the analyzed systems were performed using a computer program HYPERQUAD [29]. Computer-aided analysis of each system was based on 6 titrations containing 100–250 data points. The verification has started from the simplest hypothesis, and in the next steps the models were gradually up-sized by adding more species and the results were verified until the rejected species were eliminated in the upgraded model. A correctness of the model has been verified in [30]. The aforementioned procedure has been employed both in the case of analysis of the ATP-triamine systems and in that of the complex compounds formed in the ternary systems containing metal ions. Hydrolysis constants of Cu(II) ions published the literature [31] were used in the calculations.

It is observed that the noncovalent interactions between triamines 3,3-tri or Spd and ATP produce molecular complexes in the binary systems. The overall stability constants (β) and the stoichiometric composition of the species (ATP)Hx(PA) were calculated on the basis of the potentiometric results subjected to computer analysis, Table 1. The experiment is possible because the formation of adducts is accompanied by realise of hydrogen ions and a shift in the acid-base equilibrium of the substrates, which permits determination of the equilibrium parameters. Because of the difference in the number of protons in particular complexes (e.g. the value of β has different dimension for (ATP)H3(PA) and (ATP)H4(PA) species), the analysis of thermodynamic stability of complexes was made without a direct comparison of logβ, as has been sometimes made in the discussion of results. The calculated equilibrium constants for the general reaction Hx(ATP) + Hy(PA) ⇆ (ATP) Hx + y(PA) are logKe = logβ(ATP)Hx + y(PA) − logβHx(ATP) − logβHy(PA), correspond to the energy of bioligand bonding in the formed complex. The subsequent β values are the overall stability constant of the complex, overall protonation constant of ATP and overall protonation constant of PA, respectively. In the entire pH range the studied (from 2.5 to 10.5) complexes of both amines (ATP)H3(PA) are formed above pH of about 4. The species (ATP)H3(Spd) reaches the maximum concentration at pH of about 8 at which about 80% of the system components are bound, whereas the species (ATP)H3(3,3-tri) predominates at pH of about 7 at which about 70% bioligands are bound (Fig. 1). A higher concentration of the complex of biogenic amine Spd corresponds to a higher value of equilibrium constant of formation (compared to its shorter analog 3,3-tri). In the system ATP/Spd also (ATP)H4(Spd) species occur already at pH ≈ 2, whereas at pH = 5 they bind of about 60% of substrates. Differences in the mode of interactions in complexes (ATP)H4(Spd) and (ATP)H3(Spd) were also found from an analysis of shifts in NMR signals (Table 2) ascribed to carbon atoms located near sites of noncovalent interactions (which is associated with the change in the electron density at reaction centers). In the range of (ATP)H4(Spd) formation, pH ≈ 5, there are no shifts in 31P NMR signals originated from Pγ –atoms, which suggests the exclusion of this phosphate group from

2.3. Spectroscopic measurements The mode of interactions was determined on the basis of spectroscopic measurements performed in the pH ranges in which particular species were predominant, as found by equilibrium studies. The distribution of particular species was determined using the program HALTAFALL [32]. 91

Journal of Inorganic Biochemistry 177 (2017) 89–100

S.K. Hoffmann et al.

Fig. 1. Distribution diagrams for (a) ATP/3,3-tri and (b) ATP/Spd systems; the percentage of the species refers to total ligand, cATP = 1 ∙ 10− 2 M, cPA = 1 ∙ 10− 2 M; (a) H2ATP, 2. HATP, 3. ATP, 4. H33,3-tri, 5. H23,3-tri, 6. H3,3-tri, 7. 3,3-tri, 8. (ATP)H3(3,3-tri); (b) H2ATP, 2. HATP, 3. ATP, 4. H3Spd, 5. H2Spd, 6. Spd, 7. Spd, 8. (ATP)H4(Spd), 9. (ATP)H3(Spd).

amine binding (the shift occurs in the case of Pα and Pβ). With the increase in pH-value and the formation of (ATP)H3(Spd), this group (besides Pα and Pβ) becomes included in the interaction (Table 2). This results in the rise in Ke value, compared to four-proton species (Table 1). In the conditions of formation of both adducts, changes are observed in the location of 13C NMR signals ascribed to C(2), C(6), C(5) and C(8) atoms from ATP It suggests that also endocyclic nitrogen atoms N(1) and N(7) from the nucleotide take part in the reaction as the negative centers. Protonated amine groups Spd are the positive centers, located mainly at the shorter methylene chain of the amine (small changes in NMR spectra, Table 2). The possibility of employing changes in the spectrum for the determination of interaction centers has been confirmed by the experiment performed for the system ATP/3,3-tri at pH = 3. The distribution curves (Fig. 1a) shows that the complexes are not formed in such conditions, and no significant shifts in NMR signals are observed, indeed (Table 2). The complex (ATP)H3(3,3-tri) is formed from pH of about 4 and predominates at pH = 7. In contradiction to the complex (ATP)H3(Spd), the endocyclic nitrogen atoms from nucleotide in the adduct (ATP)H3(3,3-tri) are most likely not involved in the interactions (the absence of clear signal shifts in the 13C NMR spectrum, Table 2). The phosphate groups are the main negative centers, whereas protonated amine groups 3,3-tri are the positive centers. Usually, changes in the location of NMR signals observed at such interactions appear in the case of amines clearly smaller than in nucleotides. A proposed mode of the interaction in (ATP)H3(3,3-tri) complex is presented in Fig. 2.

Table 2 13 C NMR and Systems

ATP/3,3-tri

ATP/Spd

31

Fig. 2. Tentative mode of interaction in (ATP)H3(3,3-tri) adduct.

3.2. Interactions in the ternary Cu/ATP/triamine systems In Cu/ATP/triamine ternary systems, a formation of heteroligand complexes was detected. On the basis of the computer analysis of potentiometric data, a coordination model was proposed. It was found that in the system Cu/ATP/3,3-tri two complexes are formed: Cu(ATP) H(3,3-tri) dominant at pH about 6.5 and Cu(ATP)(3,3,-tri) dominant at

P NMR signal positions for ATP/triamine systems and their changes in relation to those of single ligands (Δ), [ppm]. pH

3.0 Δ 5.0 Δ 7.0 Δ 5.0 Δ 7.0 Δ

ATP

PA

C(2)

C(6)

C(5)

C(8)

Pα

Pβ

Pγ

C(1)

C(2)

C(3)

C(4)

C(5)

C(6)

C(7)

149.071 0.011 152.59 0.028 153.671 0.041 153.665 0.102 154.061 0.431

153.671 0.007 155.574 0.029 156.382 0.012 156.699 0.154 156.008 0.372

119.168 0.026 119.308 0.038 119.382 0.027 119.355 0.085 119.449 0.089

142.989 0.042 140.966 0.230 140.679 0.099 140.693 0.073 140.635 0.055

− 10.675 0.041 − 5.870 0.022 − 6.381 0.499 − 5.699 0.191 − 6.513 0.317

− 22.713 0.010 − 21.670 0.957 − 21.777 0.295 − 22.726 0.191 − 22.377 0.307

− 11.087 0.004 − 10.933 0.345 − 10.946 0.083 − 11.290 0.010 − 11.243 0.213

45.475 0.030 45.435 0.084 45.321 0.305 37.310 0.073 37.373 0.097

37.357 0.004 37.343 0.023 37.330 0.210 23.721 0.027 23.759 0.037

25.545 0.013 24.531 0.028 24.591 0.359 45.201 0.067 45.295 0.041

47.599 0.057 47.798 0.048

22.756 0.041 22.723 0.031

23.984 0.020 23.998 0.027

39.620 0.008 39.653 0.019

92

Journal of Inorganic Biochemistry 177 (2017) 89–100

S.K. Hoffmann et al.

Fig. 3. Distribution diagrams for: (a) Cu(II)ATP/3,3-tri and (b) Cu(II)ATP/Spd systems; the percentage of the species refers to total metal, cCu2 + = 4.4 ∙ 10− 4 M, cATP = 2 ∙ 10− 3 M, cPA = 2 ∙ 10− 3 M; (a). 1. H2ATP, 2. HATP, 3. ATP, 4. H33,3-tri, 5. H23,3,-tri, 6. H3,3-tri, 7. 3,3-tri, 8. Cu2 +, 9. CuH(ATP), 10. Cu(ATP), 11. Cu(3,3-tri), 12. Cu(3,3-tri)2, 13. Cu(ATP)H(3,3tri), 14. Cu(ATP)(3,3-tri); (b). 1.H2ATP, 2. HATP, 3. ATP, 4. H3Spd, 5. H2Spd, 6. HSpd, 7. Spd, 8. Cu2 +, 9. CuH(ATP), 10. Cu(ATP), 11. Cu(ATP)H3(Spd), 12. Cu(ATP)H2(Spd), 13. Cu (ATP)H(Spd), 14. Cu(ATP)(Spd).

Table 3 Overall stability constants (logβ), equilibrium constants (logKe) for complexes formed in ATP/triamine systems, including positions of d-d transmission. Complex

pH

Reaction

logβ

logKe

λmax

Cu(ATP)H(3,3-tri) Cu(ATP)(3,3-tri) Cu(ATP)H3(Spd) Cu(ATP)H2(Spd) Cu(ATP)H(Spd) Cu(ATP)(Spd)

6.5 8.0 5.0 7.0 7.5 9.0

CuATP + H3,3-tri ⇆ Cu(ATP)H(3,3-tri) CuATP + 3.3-tri ⇆ Cu(ATP)(3,3-tri) CuATP + H3Spd ⇆ Cu(ATP)H3(Spd) CuATP + H2Spd ⇆ Cu(ATP)H2(Spd) CuATP + HSpd ⇆ Cu(ATP)H(Spd) CuATP + Spd ⇆ Cu(ATP)(Spd)

24.01(6) 18.13(4) 39.45(6) 33.49(7) 25.78(8) 18.58(6)

7.91 11.50 3.42 6.03 8.38 11.95

– 630 nm 751 nm 736 nm – 630 nm

3.3.1. Cu-polyamine complexes Polyamines are well coordinated molecules via nitrogen atoms at suitable conditions. In water solutions at low pH-value the triamines exist in protonated form, with two NH3+ groups in terminal positions and central NH2+ group. At these conditions the electrostatic repulsion between positively charged Cu(II)-ion and amines precludes Cu(II) coordination. With the increasing pH-value amines are progressively deprotonated and Cu(II) can be coordinated. At physiological conditions (pH ≈ 7) amines are fully protonated. The composition and stability of the formed complexes have been already studied by potentiometric and optical spectroscopy and EPR methods [34,35]. Potentiometric measurements indicate that below pH = 6 the Cu(II) is coordinated by water molecules [34]. Optical spectroscopy and EPR are complementary methods since anisotropic properties of the EPR spectra are directly related to d-d band positions in visible spectra being the orbital energies of Cu(II) ion. Optical absorption spectra of Cu(3,3-tri) and Cu (Spd) at pH > 8 are identical with absorption peak at 16000 cm− 1 (625 nm) as presented in Fig. 4. This indicates that Cu-complexes in the both systems are identical. Unfortunately, the UV–Vis spectra have not resolved d-d bands and consist of a single slightly asymmetrical broad line without a shoulder expected for non-distorted octahedral and square–planar copper(II) complexes. The single optical absorption band indicates that the excited orbital levels are very close in energy. Such the case can appear for tetrahedral distortion of four-coordination complexes with C2v-symmetry [36–38]. The increasing distortion from square–planar toward tetrahedron results in a reverse of energy level sequence as it is schematically plotted in the inset of Fig. 4. A closer analysis is possible using EPR results. EPR spectra of the glassy state at 77 K confirm that the CuN4chromophores are identical in Cu(3,3-tri) and Cu(Spd) at pH > 7

pH in the range 7–9 (Fig. 3a). A crucial difference in the relative concentrations of both species and differences in the values of formation constants (Table 3) suggest that the interaction type is different in the both complexes. The position of d-d band maximum of the complex Cu(ATP)(3,3-tri) suggests the coordination via the phosphate group from ATP and metalation of polyamine with the participation of 2 or 3 nitrogen atoms, whereas in the case of Cu(ATP)H(3,3-tri) the value of logKe = 7.91, that is by almost four orders of magnitude smaller in comparison to Cu (ATP)(3,3-tri), suggests that it can be a complex with a smaller number of nitrogen donor atoms involved. The values of formation constants and the position of d-d band for the system Cu/ATP/Spd suggest that at pH < 7 a molecular complex between Cu(ATP)H3(Spd) and amine is formed in the external coordination sphere, but the logKe values indicate that with increasing pH spermidine enters into the metalation process. The coordination mode is discussed below in details on basis of the results of EPR and UV–Vis measurements.

3.3. Molecular and electronic structure of binary Cu(II)-triamine and CuATP complexes at frozen solution. EPR and UV–Vis spectra Before a discussion of optical and EPR results for ternary Cu-ATP-PA systems we will present results of measurements and interpretation of data for binary systems formed from polyamines and ATP and its components after rapid freezing at 77 K. EPR results will be collected in a single table and presented on a correlation diagram of EPR parameters A|| vs. g||, which allows evaluation of the complex geometry. The resulting diagram for the studied systems, numbered according to the summarizing Table 4 will be discussed below. 93

Journal of Inorganic Biochemistry 177 (2017) 89–100

S.K. Hoffmann et al.

Table 4 EPR g-factors and hyperfine splitting (in 10− 4 cm− 1) of Cu(II)-complexes. No

Binary systems 1 1 2 3a 3b 4 5 6a 6b 7a 7b 7c Ternary systems 8a 8b 9a 9b 10a 10b 11a 11b

System

Cu(3,3-tri) Cu(Spd) Cu (diphosphate) Cu(triphosphate) Cu(ribose) Cu(adenine) Cu-adenosine Cu(ATP)

Cu(ATP)(3,3-tri) Cu(ATP)(Spd) Cu(ATP)(Put) Cu(ATP)(tn)

Reference systems * Cu(H2O)62 + − CuO6 * Cu(acac)2 − CuO4 * Zn(imO6(ClO4)2 − CuN6 * Cu-BSA − CuN4

pH

g-factors

Hyperfine splitting

Ref.

g||

g⊥

A||

A⊥

> 6.0 >7 > 4.5 4–7 10.5 > 11 3–8 6.5 11.6 3.0 6.5 11.0

2.226 2.226 2.358 2.400 2.244 2.235 2.31 2.36 2.235 2.390 2.351 2.235

2.045 2.045

− 19 − 19

– – 2.07 2.67 2.04

− 194 − 194 144 135 195 193 167 148 193 137 140 193

5.5 8.0 4–6 7.5 3.2 8.0 5.0 8.0

2.39 2.230 2.42 2.275 2.400 2.380 2.375 2.274

2.07 2.045 2.08 2.059 2.072 2.071 2.073 2.055

137 185 121 177 138 140 144 180

5 5 5 29 <5 <5 <5 20

7

2.400 2.225 2.306 2176

2.099

142 187 158 221.8

10

9.2

2.04

2.07

10

5 10

12.8

This paper This paper [48] [48] [48] [47] [51] [47] [47] This paper This paper This paper This paper This paper [23] [23] [23] [2] [39,40] Our data [59] [58]

Remarks: a) 4, 6b and 7c = ribose spectra at high pH. b) 7a, 8a = Cu(ATP) spectra from phosphate coordination. c) 3a and 10a = Cu(H2O)62 +. The reference systems are marked by astrisk in Fig. 7. Errors: g|| ± 0.002; g⊥ ± 0.004; A|| ± 3; A⊥ ± 5. Abbreviations: tn = 1,3 diaminepropane; Put = putrescine, acac = acetyloacetonate, im – imidazole, BSA = bovine serum albumine.

ground state. These parameters are collected, with some reference data, in summarizing Table 4. At low pH-values (below pH = 5) only the [Cu(H2O)6]2 + complexes exist in the studied water solution, as it is proved by the

(Fig. 5) with single type species having an axial symmetry of the electric crystal field at Cu(II) site. EPR parameters show a relatively low g|| value (about 2.226) and a high value of hyperfine splitting (A|| = − 194 × 10− 4 cm− 1) characteristic for dx2-y2 (or dxy) orbital

Fig. 4. Optical spectrum of Cu-polyamine complex for Cu(Spd) and Cu(3,3-tri) with three Gaussian components (dashed lines) with position evaluated from EPR data The inset shows d-orbital splitting variation from four-coordinated complex deformation from teterahedral to distorted planar C2v symmetry.

94

Journal of Inorganic Biochemistry 177 (2017) 89–100

S.K. Hoffmann et al.

Fig. 5. Comparison of EPR spectra of Cu(3,3-tri) and Cu(Spd) complexes in glassy state at 77 K (spectra No.1 in Table 4). Dashed line is a simulated spectrum with parameters collected in Table 4.

Fig. 7. EPR parameters correlation diagram of A∣∣ vs g∣∣. Dashed lines are theoretical plots according to the equation A∣∣/Pd = − κ − 4α2/7 + (g∣∣ − 2.0023) + 3(g⊥ − 2.0023)/7 with orbital reduction factor α2, 3d–function parameter Pd = 0.036 cm− 1, Fermi constant κ = 0.43 and g⊥ = 2.05. The straight dashed lines are plot for α2 = 0.8 and α2 = 0.7. Points refer to the numbers in Table 4 with stars as reference spectra.

written as 2 2 ψanti A1 = α[c1d x ‐y + c 2 pz] + α1ψ A1 (L)

ψanti A2 = βd xy + β1 ψ A2 (L) ψanti B1 = γ[c3 dxz + c4 px ] + γ1ψB1 (L) ψanti B2 = γ[c3 d yz + c4 py ] + γ1ψB2 (L)

(1)

The corresponding bonding orbitals are obtained by changing of the sign of each ligand coefficient αi, βi, γi. The g-factors are calculated [42] (in the “hole”-notation), as

g = 2.0023 +

8λ Cu 2 2 2 α β c1 anti Exy

g⊥ = 2.0023 +

2λ Cu 2 2 β γ c1 c3 anti Exz , yz

(2)

−1

is the spin-orbit coupling parameters. The ΔEi.j where λCu = 829 cm are the d-orbital energies calculated with respect to the ground state. Thus, a relatively low g|| must be related to the low value of the productαβc1 describing delocalization in the ground state dx2-y2 diminished by pz-orbital of nitrogen atom contribution. Hyperfine splitting parameters are

Fig. 6. EPR spectrum of Cu(Spd) in water solution at acidic and neutral conditions. At low pH only Cu(H2O)6 complexes exist. The spectrum is marked by *.

4 4 Pp 2 2 6 P β c2 + Δg − Δg⊥ = −κ ⎛⎜c12 + d c22⎞⎟ − β 2c12 + 7 5 14 P P p d ⎝ ⎠ 2 4 Pp 2 2 11 A⊥ P β c2 + Δg⊥ = −κ ⎛⎜c12 + d c22⎞⎟ + β 2c12 − 7 5 P 14 Pd P p d ⎝ ⎠ A

characteristic EPR spectrum (Fig. 6) [40]. When pH value grows a signal from Cu-polyamine complexes appears and coexists with aqueous complexes signal at pH = 6.5. The latter complex quickly disappears. The both studied polyamines form identical complexes with Cu(II) in the water solution. The position of the complexes (No. 1 in Table 4) in the correlation diagram of Fig. 7 suggests their four-coordination. Closer information on molecular structure of the complexes can be derived from analysis of the EPR parameters in terms of MO-theory supported by UV–Vis spectra for C2v symmetry. Orbital symmetry at C2v geometry is given in the inset of Fig. 4. Two effects can complicate EPR parameters calculations at this relatively low symmetry. The dx2-y2 and dz2 orbitals having A1-symmetry can be mixed influencing significantly the EPR parameters [41] with lowering the g⊥-value below 2.04. This effect is small in our spectra and can be neglected. Another effect can result from a mixing of the Cu(II) 4p–orbitals into the d-orbitals. The antibonding molecular orbitals having mainly copper d-character with mixed 4p–orbitals for CuN4 complex of C2v symmetry can be

Pd

−1

(3)

is a parameter depending on average where Pd = 0.036 cm extension of the radial wave function of Cu(II) 3d–orbital, Pp = 0.0402 cm− 1 is the similar parameter for 4p orbitals of Cu(II). First term in Eq. (3) for A|| is the isotropic Fermi contribution κ reduced by delocalization via 4pz state of Cu(II). Taking into account that every MO-coefficient should be in the range (0.5–1), with appropriate normalization condition, as c12 + c22 = 1, and considering possible orbital energies within the observed optical line and possible signs of hyperfine splitting, we evaluated unknown parameters from Eqs. (2) and (3) as Exy = 13,000(200) cm− 1, Exz,yz = 15,500(200) cm− 1, Ez2 = 17,400(200) cm− 1, α2 = 0.86(3), c12 = 0.97(2),β2 = 0.85(14) andγ2 ≈ 1 (the errors are given in parentheses). The orbital energies well reproduce observed UV–Vis spectrum as shown by the dashed line 95

Journal of Inorganic Biochemistry 177 (2017) 89–100

S.K. Hoffmann et al.

Fig. 8. Model of Cu(II) coordination with spermidine molecules. The distorted squareplanar configuration is formed by two Spd molecules.

Fig. 9. Proposed octahedral structure of Cu-triphosphate complex in water solution at pH = 4.5–7.0.

in Fig. 4. The α2-parameter shows typical unpaired electron density delocalization from the ground state onto ligands with a very small contribution from 4p admixture (about 3%). A relatively low β2-coefficient value results from the density delocalization via dxy state suggesting a π-type interaction with coordinating polyamine molecules, whereas no interaction along the z-axis (γ2 ≈ 1). Optical absorption and EPR results show that copper(II) ion is coordinated by two chelating polyamine molecules via two nitrogen atoms. The studied polyamines have three possible coordinating sites: two terminal nitrogen atoms of NH2-groups and central nitrogen of NH-group. They form sixand seven member chelating ring for 3,3-tri and Spd, respectively, with tridentate coordination in the single crystal structure [43]. However, the EPR spectra and optical spectra of studied polyamines are identical with those of Cu-putrescine complex [23], thus the same mode of coordination by two terminal nitrogen atoms can be assumed. The proposed model of Cu-spermidine complex is presented in Fig. 8 with two Spd molecules trans‑coordinating Cu(II) in tetrahedrally distorted square planar environment. Molecular structure of spermidine is taken from the single crystal data [43].

complex structure appears producing increase in the d-d band energy to 13,900 cm− 1 (719 nm). A new EPR spectrum is formed with parameters g|| = 2.244 and A|| = 195 × 10− 4 cm− 1, indicating a change from distorted octahedral CuO6 to a pseudotetrahedral CuO4 stereochemistry as suggested in [49] (No. 3b in the correlation diagram of Fig. 7). 3.3.3. Cu-ribose and Cu-adenine complexes The ribose is a relatively poor ligand and is able to coordinate Cu(II) at high pH-values (above pH = 11) only, giving an axial symmetry EPR spectrum with parameters g|| = 2.235 and A|| = − 193 × 10− 4 cm− 1 (Table 4). This indicates a planar four coordination CuO4 (No. 4 in diagram of Fig. 7). Adenine is a nucleobase and essential part of ATP with four nitrogen atoms 1,3,7 and 9 (see numbering in Scheme 1) being potential binding sites for Cu(II). The EPR spectrum of axial symmetry with parameters g|| = 2.310 and A|| = 167 × 10− 4 cm− 1 (Table 4) [54] exists in the range pH = 3–8 indicating stable structure of Cu-adenine complex with planar chromophore CuN4 (see the correlation diagram: No. 5 in Fig. 7).

3.3.2. Cu-polyphosphate complexes Monophosphate anion PO43‐ and polyphosphates (pyrophosphate, triphosphate) are relatively strong Cu(II) coordinating ligands. The Cuphosphate complexes were studied in crystalline form [44,45] and in water solutions [46–48]. The copper monophosphate complexes are not formed at basic conditions where copper hydroxide precipitates. Diphosphates copper(II) complexes show EPR spectrum in pH range 4.5–10.5 with parameters g|| = 2.359 and A|| = 144 × 10− 4 cm− 1 [48] characteristic for dx2-y2 orbital ground state in a strongly elongated octahedral stereochemistry, as it is visible in correlation diagram (No. 2 in correlation diagram of Fig. 7). Earlier EPR investigation of diphosphate copper(II) complexes in water solution were not very informative and it was suggested that at least two different copper complexes exist. Our EPR and UV–Vis studies of triphosphates [48] have shown that at very low pH (below 2.0) the copper(II) ions are not coordinated by phosphate groups, which are fully protonated and only aqueous complexes Cu(H2O)62 + exist. A further increase in pH-value results in a partial ionizing of the phosphate groups which allows Cu(II) coordination. At neutral pH (4.5–7) the EPR spectrum exists with parameters g|| = 2.400 and A|| = 135 × 10− 4 cm− 1 characteristic for (4 + 2) distorted octahedral coordination (No. 3a in the correlation diagram of Fig. 7). A corresponding optical absorption spectrum confirms the EPR conclusion. The spectrum consists of a very asymmetrical broad band suggesting an existence of non-resolved low-energy shoulder with maximum absorption at about 12,400 cm− 1 (807 nm). The most probable complex structure with chelating phosphate groups is Cu(phosphate)2(H2O)2 as it is shown in Fig. 9. A less probable structure can be formed by four water molecules in the basal coordinating plane and axial coordination by oxygen atoms of phosphate groups. At a very high pH-value (above 10.5) a transformation of Cu-

3.3.4. Cu-adenosine complexes Adenosine is formed by a bonding of adenine and ribose. Coordination properties of both components were studied separately, as described above. Cu(II) binding to adenosine in a frozen water solution has been studied by EPR [47] and by UV–Vis spectroscopy [50]. EPR spectra depend on the pH-value. Below pH = 5 only Cu(H2O)62 + complexes exist giving characteristic spectrum at glassy state marked by asterisk in Fig. 6. At pH ≈ 6 a new EPR spectrum appears with parameters g|| = 2.360 and A|| = 148 × 10− 4 cm− 1 (Table 4). Its position in correlation diagram (No.6a in Fig.7) suggests that the complex is a distorted octahedral and the corresponding visible spectrum band has the maximum at 12700 cm− 1 (790 nm). This position is very close to the Cu-phosphate complex (No. 2), thus the complex is formed by two ribose molecules coordinating by oxygen atoms in the basal plane and two water molecules at apical positions. At pH > 11 the Cu-complex EPR parameters are identical with those of the Cu-ribose complex (No. 4) and the d-d band position is shifted to higher energy 15,800 cm− 1 (633 nm) indicating that water molecules are not further coordinating at strongly basic conditions and Cu(II) ion in Cuadenosine complex is not bound by adenine nitrogen atoms at any pH at glassy state, but exclusively by oxygen atoms of the ribose moiety. This confirms conclusions of the paper [47]. 3.3.5. Cu-ATP complexes at 77 K When triphosphate is attached to an adenosine molecule the ATP molecule is formed with additional potential binding sites being oxygen atoms of the phosphate groups. An equilibrium in the water solution is a result of competition between Cu(II) coordination by ATP (phosphates, ribose or adenine), Cu(II) coordination by H2O, noncovalent 96

Journal of Inorganic Biochemistry 177 (2017) 89–100

S.K. Hoffmann et al.

Fig. 12. Proposed structure of the Cu-ATP complex at low pH with coordination by oxygen atoms of the phosphate groups.

3.3.2). The proposed structure of Cu-APT complex in glassy state at 77 K is presented in Fig. 12. When pH-value of the solution is in the neutral range a new Cucomplex appears and coexists with the still existing complex No. 7a. The new spectrum has slightly lower g|| -value and slightly higher A|| value (Table 4). Its position in the correlation diagram (position No. 7b in Fig. 7) indicates still octahedral symmetry but with a weaker axial interaction. This indicates that water molecules coordinating in apical direction are involved in some intermolecular interactions with outersphere molecules. Simultaneously, a small shift of the absorption band toward higher energy appears, confirming the EPR spectrum behavior. This indicates an important role of water molecules. Hydration properties of ATP are known from studies of high-resolution microwave dielectric relaxation at neutral pH [52]. An ATP molecule is surrounded by one layer of constrained water, and triphospahate group is additionally surrounded by 3–4 layers of hyper-mobile water molecules. Hydration layers can play an important role during Cu(II) complexation and during ionization of the phosphate groups. The situation is complicated by the fact that simultaneously the N7 atom of the adenine ring is deprotonated being potentially well coordinating site and/or an acceptor for hydrogen bonding. Often it is assumed that CueN7 bond or a macrochelate involving N7 are formed [5,53,54]. This assumption has been deduced from experiments showing indirect and usually small effects of coupling (contact) between Cu and N7. However, it was stated that such effects are caused rather by noncovalent interactions between coordinating water molecule and N7 atom as observed by NMR [22] and Raman spectroscopy [55]. Our EPR results also exclude the direct coordination by N7 nitrogen atom and only a small influence of axially bonded H2O can be recognized. In an intermediate pH range ATP becomes unstable in unbuffered water and begins dissociate, i.e. the PO42 − groups are successively detached when charge balance is shifted. It is due to electrostatic repulsion between negatively charged PO4− groups and noncovalent bonds to the surrounding water molecules [56]. As a result the Cu(phosphate)2(H2O)2 complexes are destroyed, the observed EPR spectrum becomes weak because various new Cu (hydroxo)-compounds are formed. These are mostly not soluble in water and precipitate in a form of gels (pH = 5–11) producing a cloudy solution [47,57]. A further increase in pH value to the basic range produces a continuous deprotonation of the protonated species. At pH ≈ 11 the solution becomes clear again due to coordination of Cu(II) to ribose oxygen atoms, which are deprotonated in this pH range. In fact, the observed EPR spectrum (Fig. 10) is the same as Cu-ribose spectrum (Table 4, No. 7c = No.4 in the diagram Fig. 7) and confirmed by visible absorption spectrum (Fig. 11). This explicitly indicates coordination of Cu(II) by the ribose oxygen atoms in square-planar configuration. The problem of Cu(II) binding by ATP in water solution is not satisfactorily solved. There is a general agreement that at low pH, at acidic conditions, Cu(II) is coordinated by phosphate groups whereas at very basic conditions (pH > 10) Cu(II) is coordinated by ribose oxygen

Fig. 10. EPR spectrum of Cu(ATP) complexes recorded in the glassy state (77 K) for three pH-values. The three types of Cu(II) complexes can be recognized with parameters collected in Table 4. Computer simulated spectrum is shown by the dashed lines.

interaction between ATP and water molecules, and between ATP molecules (self-aggregation through π-stacking of the rings). The equilibrium depends on the pH-value. At a very low pH value the phosphate groups and adenosine are fully protonated and Cu(II) is coordinated by H2O forming Cu(H2O)62 + octahedral complexes. Its position in the correlation diagram (Fig. 7) is marked by asterisk. The further increase in pH-value results in a successive deprotonation of ATP components what is observed as changes in EPR and optical absorption spectra. The EPR spectra recorded at acid conditions (pH = 3), neutral conditions (pH = 6) and highly basic conditions are shown in Fig. 10, and the corresponding optical absorption spectrum is presented in Fig. 11. The EPR spectrum at pH = 3 (No. 7a) is nearly the same as that for the Cu-phosphate complex (see Table 4). Thus, we can conclude that at low pH-value, when a partial ionization of the phosphate groups of ATP occurs, the Cu(II) is coordinated exclusively by oxygen atoms of phosphate groups forming square-planar complex. Additionally, at the axial positions the water molecules can coordinate as it is indicated by the position in the correlation diagram (Fig. 7). This assignment is consistent with the optical spectrum having absorption band at low pH identical with that of Cu-phosphate in the same pH-range (see Section

Fig. 11. Absorption spectrum of Cu(ATP) in the visible range for low and high pH-values.

97

Journal of Inorganic Biochemistry 177 (2017) 89–100

S.K. Hoffmann et al.

Fig. 14. UV–Vis absorption spectrum of Cu(ATP)(Spd) system at various pH values.

asymmetry, which was not essential in water solution of Cu(Spd), influences the Cu(II) coordination in the ternary system as it is visible in potentiometric distribution diagram, in EPR and UV–Vis spectra. A difference in behavior of 3,3-tri and Spd is clearly seen when compare the UV–Vis spectra. For Cu/ATP/3,3-tri the spectrum consists of a single band at 16000 cm− 1 above pH = 6.5 and is identical with the spectrum of Cu/ATP/Spd/at high pH. However, for Cu/ATP/Spd an evolution the spectrum with increasing pH is observed as it is shown in Fig. 14. At pH < 5 the spectrum shows coordination by phosphate groups of ATP and at high pH (above pH = 9) the spectrum shows coordination by the polyamine. At intermediate pH range a new complex is formed. This is reflected in EPR spectrum behavior presented in Fig. 15. Octahedral Cu(ATP) complexes with EPR spectrum characteristic for Cu (II) coordination by phosphate groups exist in low pH range. At pH

Fig. 13. EPR spectra of Cu(ATP)(3,3-tri) system at intermediate and high pH value. The Cu(ATP) and Cu(3,3-tri) spectra (dashed lines) are added for a comparison. Spectra are numbered according to the Table 4.

atoms. In an intermediate range, in physiological conditions, the situation is still not well understood. Possible interactions between all components of the system are not well identified and charge distribution and charge compensation are not sufficiently considered. Moreover, a role of water molecules in not taken into account and a role of Na+ ions (introduced with sodium ATP salt) and anions (like NO3−) introduced with Cu(II) salt are usually omitted. 3.4. Molecular and electronic structure of ternary Cu/ATP/triamine complexes Having separately discussed the results of Cu-coordination by various parts of ATP and by polyamines we can now consider copper(II) coordination in ternary system in water solution with biomolecules concentration 2·10− 3 M and order of magnitude lower Cu(II) concentration (4.4·10− 4 M). One can expect a more complicated situation because of a competition between ATP and 3,3-tri and Spd polyamines. However, we have found it rather simple. It is surprising, that the polyamines which display identical behavior when coordinate Cu(II) in water solution (see Section 3.3.1) shows a different behavior in ternary solution with ATP. In Cu/ATP/3,3-tri system at pH < 3.5 Cu(II) is coordinated by water molecules forming Cu(H2O)62 + complexes. At higher pH the Cu (II) is coordinated by ATP molecule forming complex with phosphate groups as shown by EPR spectrum No. 8a identical with Cu(ATP) spectrum No.7a (Fig. 13). Around pH = 5.5, where the spectrum No. 8a was recorded, the Cu(II) coordinated by phosphate groups dominates, but coexist with small amount of Cu(polyamine) complexes. At higher pH the Cu(ATP) complex is destroyed and Cu(II) is coordinated by 3,3tri molecules as it is shown by a comparison the spectrum at pH = 8 (spectrum No. 8b) with spectrum Cu(3,3-tri) (spectrum No. 1, Fig. 13). This is confirmed by the UV–Vis spectrum which is identical with that for Cu(3,3-tri) at pH > 7 (see Fig. 4). EPR parameters of the Cu/ATP/ 3,3-tri system are collected in Table 4. Thus, in the ternary systems at high pH the Cu(II) ion is coordinated by 3,3-tri in the same manner as in binary Cu(3,3-tri) in water solution. The 3,3-tri molecule is a flexible symmetrical molecule with central NH-group and two terminal coordinating NH2-groups. Spd is asymmetrical molecule with central NH-group separated by three CH2-gropus from one side and four CH2-groups from the other side (Scheme 1). This

Fig. 15. EPR spectrum Cu(ATP)(Spd) at various pH-values of the water solution. The dashed line spectra of binary systems Cu(ATP) (spectrum 7b) and Cu(Spd) (spectrum 1) are added as reference spectra. The dotted lines spectrum 9b is a simulated spectrum with parameters listed in Table 4. Two EPR spectra coexist in pH range 7.5–9.5.

98

Journal of Inorganic Biochemistry 177 (2017) 89–100

S.K. Hoffmann et al.

and geometry of Cu(II) complexes formed in ternary system Cu/ATP/ polyamines (spermidine, or 3,3-tri) in water solution at different pH. The system is rather complex, thus for simplification we studied separately all components in binary and ternary systems. In binary system ATP/polyamine the noncovalent intermolecular interactions and possible molecular aggregation was detected by pH titration. In the ATP/Spd adducts preferred centers of interaction are protonated amine groups of Spd and phosphate groups of nucleotide. Endocyclic nitrogen atoms are also involved in the interplay. In the ATP/3,3-tri system only the phosphate groups from the nucleotide take part in interaction with polyamine. In the ternary Cu/ATP/PA systems, MLHxL’ complexes occur, where metal ions are coordinated by phosphate groups of ATP. The indirect noncovalent interaction involving of polyamine molecules also appears. Potentiometric measurements identified various type Cu(II) complexes in water solution and determined their protonation state in dynamical equilibrium state at a constant room temperature. Simpler picture is found in spectroscopic UV–Vis and EPR studies in frozen glassy state. UV–Vis and EPR are not sensitive to the ligand protonation determined by titration method. Potentiometric results show that most Cu (II) complexes form asymmetric unit, whereas all EPR studied Cucomplexes are axially symmetrical in glassy state with dx2 − y2 orbital ground state. This allows structural analysis and discussion of their local magnetic properties using the EPR parameters correlation diagram (Fig.7). The diagram shows two groups of Cu(II) complexes (in the elliptical frames): complexes with distorted octahedral symmetry having high g||-value and small A||, and distorted four coordinated complexes having small g||-value and high A||. The former exist at low pH-value whereas the latter exist in high pH solution. Thus, the diagram allows identification of a transformation of Cu-complexes from octahedral structure into distorted planar structure when basicity of the water solution increases. Axial symmetry of Cu(II) complexes at glassy state strongly restricts the possible complex structure. We propose the molecular structure for all studied frozen complexes consistent with EPR and optical spectroscopy. We did not detect by EPR any direct coordination of the N7 nitrogen atom of adenosine to Cu(II) ions, although such binding is often assumed as essential in metal complexes formation at physiological conditions. It means that the interaction between Cu(II) and N7 in glassy state is rather of indirect type realized via noncovalently bonded water molecule. Most essential discrepancy between low-temperature glassy EPR and room-temperature potentiometry is that in the liquid state the single ligand CuLHx type complexes dominate, whereas at glassy state all EPR studied complexes are CuL2-type as clearly indicated by axial crystal field symmetry. Moreover, EPR spectra do not show simultaneous coordination of Cu(II) by ATP = L and polyamine = L’, although the CuLL’ complexes dominate in solution at room temperature. These essential differences can be related to the dynamical equilibrium in solution and its changes after freezing. Usually the metal complexes in a solution are treated as static rigid structures. However, their structure is dictated by dynamical equilibrium and molecular dynamics [54]. It is known that ATP can oscillate between quite different conformations [26,60] and this is facilitate the ATP hydrolysis destroying of the formed Cu(II) complexes with phosphate groups [61]. It seems that when dynamics stops under freezing the considerable changes in the concentration distribution complex of various complexes appear. Such behavior of Cu(II) complexes with organic ligands was already reported [62] but, generally, effect of freezing is underestimated in literature. It is known that pH-value decreases under freezing and enthalpy of multicomponent solution favors the species with high coordination number. As the result the frozen solution at 77 K can be dominated by CuL2-type species although they exist in residual concentration (a few percent only) at 298 K [62].

Fig. 16. Optical absorption band of Cu(ATP)(Spd) at pH = 7.5 decomposed into three Gaussian component.

about 7.5 a new EPR spectrum No. 9b appears with parameters listed in Table 4. This spectrum coexists with Cu(II) coordination by phosphate groups spectrum, which disappears for higher pH value. At pH = 9.5 the spectrum No. 1 appears and coexists with the intermediate spectrum No. 9b. The EPR parameters of the spectrum No. 9b on the correlation diagram of Fig. 7 shows that octahedral 7b spectrum is transformed into a four-coordinated chromophore spectrum 9b with a possible tetrahedral distortion. It means, that the water molecules coordinating in apical positions of Cu(ATP) complex are successively affected above pH = 6.5. It can be due to noncovalent interactions between coordinated water and Spd molecules. All observed spectra show axial complex symmetry D4h-type with dx2 − y2 ground orbital state of Cu(II) ion. For this symmetry we can apply Eqs. (2) and (3) for g-factors and A-splitting neglecting p-orbital mixing (c1 = c3 = 1 and c2 = 0). The spin delocalization parameter α2 and Fermi contact interaction contribution to the hyperfine splitting can be calculated from the following expressions

7 ⎛ A⊥ − A 5 (g − 2.0023) ⎞ + (g − 2.0023) − 6⎝ 14 ⊥ Pd ⎠ 1 ⎛ A 4 2 6 (g − 2.0023) ⎞ κ= 2 − − α + (g − 2.0023) + 7 14 ⊥ α ⎝ Pd ⎠

α2 =

⎜

⎜

⎟

⎟

(4)

which gives α2 = 0.78 and κ = 0.43. Using Eqs. (2) and (3) with experimental g-factor and hyperfine splitting the other delocalization parameters and orbital energies were found as Exy = 13,200(200) cm− 1, Exz,yz = 15,100(200) cm− 1, Ez2 = 16,000(200) cm− 1, α2 = 0.78(2), β2 = 0.85(5) and γ2 = 0.65(5) (the errors are given in parentheses). Moreover, the signs of A-splitting were found as negative both for parallel and perpendicular A-value. A decomposition of optical band into Gaussian components is shown in Fig. 16. The delocalization of unpaired electron spin density onto ligands in the main coordination plane, described by MO coefficients α2 and β2, is practically the same for the complex having EPR spectrum 9b and for binary complex Cu(Spd) having EPR spectrum 1. However, the γ2 -coefficient describing delocalization at apical complex position is smaller in the complex 9b suggesting a tetrahedral deformation. A similar behavior of optical spectrum of Cu/ATP/Spd system has been observed in paper [35]. 4. General remarks and conclusions The goal of the paper was a determination of molecular structure 99

Journal of Inorganic Biochemistry 177 (2017) 89–100

S.K. Hoffmann et al.

[34] A. Wojciechowska, L. Bolewski, L. Lomozik, A study of polyamine complex formation with H+, Cu(II),Zn(II), Pb(II) and Mg(II) in aqueous solution, Monatsh. Chem. 122 (1991) 131–138. [35] J.A. da Silva, J. Felcman, A.L.R. Merce, A.S. Mangrich, R.S.C. Lopes, C.C. Lopes, Study of binarey and ternary complexes of copper(II) with some polyamines and adenosine 5′ triphosphate, Inorg. Chim. Acta 356 (2003) 155–166. [36] R.J. Dudley, B.J. Hathaway, P.G. Hodgson, The single-crystal electronic and electron spin resonance spectra of bis-(2,2′-bipyridylamine)copper(II) diperchlorate, J. Chem. Soc. Dalton Trans. (1972) 882–886. [37] S.K. Hoffmann, J. Goslar, Crystal field theory and EPR parameters in D2d and C2v distorted tetrahedral copper(II0 complexes), J. Solid State Chem. 44 (1982) 343–353. [38] J. Gouteron, S. Jeannin, Y. Jeannin, J. Livage, C. Sanchez, X-ray, ESR, and optical absorption studies of tetrakis(cyclohexylamine)copper(II) nitrate: an example of a flattened-tetrahedral copper(II) complex, Inorg. Chem. 23 (1984) 3387–3393. [39] W.B. Lewis, M. Alei, L.O. Morgan, Magnetic resonance studies on copper(II) complex ions in solution. I. Temperature dependences of the 17O NMR and copper(II) EPR linewidths of Cu(H2O)62 +, J. Chem. Phys. 44 (1966) 2409–2417. [40] S.K. Hoffmann, J. Goslar, S. Lijewski, K. Basiński, A. Gąsowska, L. Łomozik, EPR and potentiometric studies of copper(II) binding to nicotinamide adenine dinucleotide (NAD+) in water solution, J. Inorg. Biochem. 111 (2012) 18–24. [41] S.K. Hoffmann, J. Goslar, K. Tadyszak, Electronic structure and dynamics of low symmetry Cu2 + complexes in kainite-type crystal KZnClSO4·3H2O: EPR and ESE studies, J. Magn. Reson. 205 (2010) 293–303. [42] S.K. Hoffmann, J. Goslar, S. Lijewski, A. Zalewska, EPR and ESE of CuS4 complex in Cu(dmit)2: g-factior and hyperfine splitting correlation in tetrahedral Cu-sulfur complexes, J. Magn. Reson. 236 (2013) 7–14. [43] R. Barbucci, M.J.M. Campbell, M. Canna, G. Marongiu, Seven membered chelating ring in copper(II) complexes with spermidine, Inorg. Chim. Acta 46 (1980) 135–138. [44] S. Neeraj, T. Loiseau, C.N.R. Rao, A.K. Cheetham, Synthesis ans structure of [C3N2H5][Cu(H2PO4)2Cl]·H2O with a chain structure: the first example of an organically tempated coppr(II) phosphate, Solid State Sci. 6 (2004) 1169–1173. [45] C. Gunther, D. Stachel, L.D. Bogomolova, Spectral investigation of crystalline (CuHPO4, Cu2P4O12, and Cu2P8O22) and glassy copper phoshates, Adv. Condens. Matter Phys. (2013) 297504(7 pp.). [46] H. Csopak, K.E. Falk, The specific binding of copper(II) to alkaline phosphate of E. coli, FEBS Lett. 7 (1970) 147–150. [47] Y.Y.H. Chao, B.R. Kearns, Magnetic resonance studies of copper(II) interaction with nucleosides and nucleotides, J. Am. Chem. Soc. 99 (1977) 6425–6434. [48] R. Jastrzab, L. Lomozik, Stability and coordination mode of complexes of polyphosphates and polymetaphosphates with copper(II) ions in aqueous solution potentiometric, spectral and theoretical studies, J. Solut. Chem. 39 (2010) 909–919. [49] K.E. Allen, Metal chelators as antioxidants for food preservation, in: Fereidoon Shahidi (Ed.), Handbook of Antioxidants for Food Preservation, Elsevier, Amsterdam, 2015, p. 87 (ch. 4.5). [50] G. Onori, D. Blidaru, Optical spectroscpopic study of the Cu(II) complexes with adenosine and adenine nucleotides I. Cu(II)-adenosine and Cu-D-ribose systems, Nuovo Cimento 5 (1985) 339–347. [51] B.M. Weckhuysen, H. Leeman, R.A. Schoonheydt, Synthesis and spectroscopy of clay intercalated Cu(II) bio-monomer complexes: coordination of Cu(II) with purines and nucleotides, Phys. Chem. Chem. Phys. 1 (1999) 2875–2880. [52] G. Mogami, T. Wazawa, N. Morimoto, T. Kodama, M. Suzuki, Hydration properties of adenozine ophodphate series as studied by microwave dielectric spectroscopy, Biophys. Chem. 154 (2011) 1–7. [53] L. Palladino, S. Della Longa, A. Reale, M. Belli, A. Scafati, G. Onori, A. Santucci, Xray absorption near edge structure (XANES) of Cu(II)-ATP and related compounds in solution: quantitative determination of the distortrion of the Cu site, J. Chem. Phys. 98 (1993) 2720–2726. [54] H. Sigel, A.A. Massoud, N.A. Corfu, Comparison of the extent of mscrochelate formation in complexes of divalent metal ions with guanosine (GMP2 −), inosine (IMP2 −), and adenosine 5′-monophosphare (AMP2 −). The crucial role of N-7 basicity in metal ion-nucleic base recognition, J. Am. Chem. Soc. 116 (1994) 2958–2971. [55] A. Lamir, N.T. Yu, A Raman spectroscopic study of the interaction od divalent metal ions with adenine moiety of adenosine 5′-triphosphate, J. Biol. Chem. 254 (1979) 5882–5887. [56] D.G. Nicholls, S.J. Ferguson, S. Ferguson, Bioenergetics, 3rd edition, Academic Press, San Diego, 2002. [57] L. Ciani, S. Branciamore, M. Romanelli, G. Martini, et al., Appl. Magn. Res. 24 (2003) 55–71. [58] T. Sawada, K. Fukumaru, H. Samurai, Coordination-dependent ESR spectra of copper(II) complexes with a CuN4 type coordination mode: relationship between ESR parameters and stability constants or redox potentials of the complexes, Chem. Pharm. Bull. 44 (1996) 1009–1016. [59] V.S.X. Anthonisamy, R. Anantharam, R. Murugesan, Cu(II)-doped hexakis(imidazole)cadmium(II) perchlorate, Spetrochim. Acta A 55 (1998) 135–142. [60] E. Kobayashi, K. Yura, Y. Nagai, Distinct conformation of ATP molecule in solution and on protein, Biophysics 9 (2013) 1–12. [61] Y. Mizukura, S. Maruta, Analysis of the conformational changes of myosin during ATP hydrolysis using fluorescence resonance energy transfer, J. Biochem. 132 (2002) 471–482. [62] R. Sipos, T. Szabo-Planka, A. Rockenbauer, N.V. Nagy, J. Sima, M. Melnik, I. Nagypal, Equilibria of 3-pyridylmethanol with copper(II). A comparative electron spin resonance study by the decomposition of spectra in liquid and frozen solution, J. Phys. Chem. A 112 (2008) 10280–10286.

References [1] V. Zappia, A.E. Pegg (Eds.), Progress in Polyamine Research, Plenum Press, New York, 1988. [2] L. Lomozik, Handbook of metal-ligand interaction in biological fluids, in: G. Berthon (Ed.), Metal Complexes with Polyamines, vol. 1, Marcel Dekker Inc., New York, 1995, pp. 686–697 (Basel, Hong Kong). [3] S.S. Cohen, A Guide to the Polyamines, Oxford University Press, Oxford, 1998. [4] C.W. Tabor, H. Tabor, Polyamines, Annu. Rev. Biochem. 53 (1984) 749–790. [5] L. Lomozik, A. Gasowska, R. Bregier-Jarzebowska, R. Jastrzab, Coordination chemistry of polyamines and their interactions in ternary systems including metal ions, nucleosides and nucleotides, Coord. Chem. Rev. 249 (2005) 2335–2350. [6] K. Samal, P. Zhao, A. Kendzicky, L.P. Yco, H. McClung, E. Gerner, M. Burns, A.S. Bachmann, G. Sholler, AMXT-1501, a novel polyamine transport inhibitor, synergizes with DFMO in inhibiting neuroblastoma cell proliferation by targeting both ornithine decarboxylase and polyamine transport, Int. J. Cancer 133 (2013) 1323–1333. [7] D. Ramani, J.P. De Bandt, L. Cynober, Aliphatic polyamines in physiology and diseases, Clin. Nutr. 33 (2014) 14–22. [8] K. Igarashi, K. Kashiwagi, K. Kishida, T. Kakegawa, S. Hirose, Decrease in the S1 protein of 30-S ribosomal subunits in polyamine-requiring mutants of Escherichia coli grown in the absence of polyamines, Eur. Biochem. 114 (1981) 127–131. [9] K. Igarashi, K. Kashiwagi, K. Kishida, Y. Watanabe, A. Kogo, S. Hirose, Defect in the split proteins of 30-S ribosomal subunits and under-methylation of 16-S ribosomal RNA in a polyamine-requiring mutant of Escherichia coli grown in the absence of polymines, Eur. J. Biochem. 93 (1979) 345–353. [10] A. Raina, J. Janne, Physiology of the natural polyamines putrescine, spermidine and spermine, Med. Biol. 53 (1975) 121–147. [11] O.M. Rennert, J.B. Shukla, Polyamines in health and disease, in: R.Q.A. Campbel (Ed.), Advances in Polyamine Research, vol. 2, Raven Press, New York, 1978, pp. 195–211. [12] N. Minois, D. Carmona-Gutierres, F. Madeo, Polyamines in aging and diseases, Aging 3 (2011) 716–732. [13] H.M. Wallace, A.V. Fraser, Inhibitors of polyamines metabolism, Amino Acids 26 (2004) 53–365. [14] H. Bachrach, The early history of polyamine research, Plant Physiol. Biochem. 48 (2010) 490–495. [15] S. Bunce, E.S.W. Kong, The interactions between nucleic acids and polyamines. I. High resolution carbon-13 and hydrogen-1 nuclear magnetic resonance studies of spermidine and 5′-AMP, Biophys. Chem. 8 (1978) 357–368. [16] C. Nakai, W. Glinsmann., interactions between polyamines and nucleotides, Biochemistry 16 (1977) 5636–5641. [17] G.S. Manning, Limiting laws and counterion condensation in polyelectrolyte solutions. 8. Mixtures of counterions, species selectivity, and valence selectivity, J. Phys. Chem. 88 (1984) 6654–6661. [18] G.S. Manning, Is the counterion condensation point on polyelectrolytes a trigger of structural transition? J. Phys. Chem. 85 (1988) 3772–3777. [19] E. Kimura, M. Kodama, T. Yatsunami, Macromonocyclic polyamines as biological polyanion complexons. 2. Ion-pair association with phosphate and nucleotides, J. Am. Chem. Soc. 104 (1982) 3182–3187. [20] M.C. Linder, Biochemistry of Copper, Ch.4, Plenum Press, New York, 1991, pp. 73–134. [21] W. Szczepanik, J. Ciesiolka, J. Wrzesinski, J. Skala, M. Jezowska-Bojczuk, Interaction of aminoglycosides and their copper(II) complexes with nucleic acids: implication to the toxicity of these drugs, Dalton Trans. (2003) 1488–1494. [22] Z. Szabo, Multinuclear NMR studies of the interactions of metal ions with adenine nucleotides, Coord. Chem. Rev. 252 (2008) 2362–2380. [23] R. Bregier-Jarzebowska, A. Gasowska, S.K. Hoffmann, L. Lomozik, Interactions of diamines with adenosine-5′-triphosphate (ATP) in the systems including copper(II) ions, J. Inorg. Biochem. 162 (2016) 73–82. [24] Z. Guo, Y. Wang, A. Yang, G. Yang, The effect of pH on charge inversion and condensation of DNA, Soft Matter 12 (2016) 6669–6674. [25] V. Kothekar, S. Dutta, Molecular orbital calculations of metal ion interaction with nucleic acid bases I. Binding of Cu(II) with adenine, guanine, uracil and cytosine, Int. J. Quantum Chem. 12 (1977) 505–514. [26] M. Jaworska, P. Lodowski, A. Mucha, W. Szczepanik, G. Valensin, M. Cappannelli, M. Jezowska-Bojczuk, Characterization of copper(II) interactions with sinefungin a nucleoside antibiotic: combined potentiomeric, spectroscopic and DFT studies, Bioinorg. Chem. Appl. (2007) 12 (article ID 53521). [27] R.J. Deeth, J.A. Hearnshaw, Molecular modeling for coordination compounds: Cu (II)-amine complexes, Dalton Trans. (2005) 3638–3645. [28] H.M. Irving, M.G. Miles, L.D. Pettit, A study of some problems in determining the stoichiometric proton dissociation constants of complexes by potentiometric titrations using a glass-electrode, Anal. Chim. Acta 38 (1967) 475–488. [29] P. Gans, S. Sabatini, A. Vacca, Investigation of equilibria in solution. Determination of equlibrium constants with the HYPERQUAD suite program, Talanta 43 (1996) 1739–1753. [30] L. Lomozik, M. Jaskolski, A. Wojciechowska, A multistage verification procedure for the selection of models in the studies of complex formation equlibria, Pol. J. Chem. 65 (1991) 1797–1807. [31] R.N. Sylva, M.R. Davidson, The hydrolysis of metal ions. Part 1. Copper(II), J. Chem. Soc. Dalton Trans. 232 (1979) 232–236. [32] N. Ingri, W. Kakolowicz, L.G. Sillen, B. Warnqwist, High speed computers as a supplement to graphical methods, Talanta 14 (1967) 1261–1269. [33] P.K. Glasoe, F.A. Long, Use of glass electrodes to mesaure acidities in doxide, J. Phys. Chem. 64 (1960) 188–189.

100