Coordination ability of bradykinin with ZnII- and AgI-metal ions – Experimental and theoretical study

Inorganica Chimica Acta 392 (2012) 211–220

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Coordination ability of bradykinin with ZnII- and AgI-metal ions – Experimental and theoretical study Bojidarka Ivanova ⇑, Michael Spiteller Institut für Umweltforschung, Universität Dortmund, Otto-Hahn-Strasse 6, 44221 Dortmund, Germany

a r t i c l e

i n f o

Article history: Received 14 April 2012 Received in revised form 8 June 2012 Accepted 15 June 2012 Available online 29 June 2012 Keywords: Bradykinin ZnII- and AgI-complexes Physical properties Mass spectrometry Quantum chemistry Model ACE interactions

a b s t r a c t Electronic absorption (EAs), circular dichroic (CD), vibrational IR- and Raman-spectroscopy, nuclear magnetic resonance (NMR), electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS) were utilized to investigate the coordination ability of bradykinin (1) with the metal ions ZnII and AgI. The experimental data were supported by the theoretical quantum chemical calculations of the electronic structures and physical properties of the isolated complexes. Since the main aim of the study is related to the design of new artificial catalysts and angiotensin converting enzyme (ACE) inhibitors, the model interactions with the active center of ACE are also designed, taking into account the coordination mode of the ZnII-ion, crystallographically determinate in the enzyme. The role of the macromolecular conformation, the enzyme cavity around the metal ion, the coordination ability of the His383.A, His353.A and Glu411.A amino acid residues, and the competitive salt-bridged processes typical for the Arg-containing ligands are discussed with a view to explain the preferred untypical C-terminus coordination mode. The data were discussed and compared with those of the angiotensin I and II (Ang-I, Ang-II). Comprehensively were described the gas-phase stabilized complex species and fragmentation modes of the complexes by the MS methods, including the imaging mass spectrometric (IMS) techniques, representing a background for the further molecular design on the base of small synthetic, semi-synthetic and naturally occurred peptides, as well as discovery of new ACE-inhibitors, since the in vivo mapping using IMS methods, provided unique opportunity for the single experiment study of the localization of molecules, including metal complexes, achievement of the important semiquantitative, quantitative and structural information within the frame of cells, tissues, organs and whole systems. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction Angiotensin converting enzyme (ACE) was a ZnII-containing membrane bound ectopeptidase located on endothelial, epithelial and neuroepithelial cells, hydrolyzing small peptides by carboxypeptidase and aminopeptidase action [1]. It played an important role in generating the potent vasoconstrictor hormone Ang-II from Ang-I and in degrading the vasodilator peptide bradykinin (H-ArgPro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH) (1). The beneficial and wide usage of ACE inhibitors in the treatment of hypertension demonstrated its importance in cardiovascular homeostasis. The functional role of ACE in the brain, reproductive tract, gut and renal tubule, however, was not clear. The (1), injected intracerebroventricularly into the posterior region of the fourth ventricle, consistently produced an increase in the mean arterial pressure. So that a query about the mediator of this effect was an open question ⇑ Corresponding author. Tel.: +49 231 755 4089. E-mail addresses: (B. Ivanova).

[email protected],

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0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ica.2012.06.028

since no kinine has been identified as a mediator of any physiological function in the central nervous system (CNS), but (1) and its higher homologues and/or novel kinins were possible candidates [1]. The ACE in plasma was presumably formed by proteolytic cleavage of the carboxyl terminal hydrophobic anchor zone, but the substrates of ACE–Ang-I and ACE–(1) often has been found to have opposing actions on the cardiovascular system [1]. It has been suspected, but not yet proven, that an increase in the concentration of endogenous kinins also played an important role in the antihypertensive effect of this class of compounds. The crystallographic study of ACE [1g], provided the first investigation of the importance of the coordination manner the inhibitors, since the ZnIIion was joined bidentately via COO -group. The inner coordination sphere, contained additionally two N-coordinated His383.A and His353.A residues and a monodentately joined Glu411.A fragment by O-atom from COO -group (Scheme 1) [1g]. It has been hypothesized that the substrate hydrolysis by ACE was activated by chloride ion in a substrate-dependent manner at the C-domain, but not N-domain sites [1]. Thus, the reported series of comprehensive studies of the structure–activity relationships of ACE inhibitors

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Scheme 1. Crystal structure of the ACE according the PDB: 1O86 [1g] with the coordination sphere the ZnII and the surface accessible area of the inhibitor; chemical diagram the catalytic domain labeled as coordination mode I (a); the geometry of the ZnII-chromophore without the inhibitor labeled as coordination mode II (b); four different models of the coordination of Brad with the ZnII-center at the ACE labeled as coordination modes III–VI (c).

have established the crucial role of the: (a) terminal carboxyl, (b) amide carbonyl, and (c) ZnII-binding function. The hydrolysis of (1) resulted to isolation the H-Phe-Arg-OH and H-Ser-Pro-OH by cleavage the C-terminus, so that it would be of interest to study the coordination ability the ZnII with (1), since it is well known that the small peptides coordinated usually by the N-terminus. For the Ang-I and Ang-II have been reported the coordination preference both at N- and C-terminus depending of the experimental conditions (for example Refs. [1i,3g]). The model enzyme interactions are designed using the classical models via a reproducing the first coordination sphere of the ZnII according the crystallographic data for ACE (PDB 1O86, Protein Databank) [1g]. The modeling of the ACE–(1) system (Scheme 1) is performed evaluating chiefly the enzyme cavity, playing a major role in substrate recognition. A parallel with the catecholamine neurotransmission function, affected by aromatic amino acid hydroxylases, is performed [2]. Interestingly, in the tyrosine hydroxylase (TH) for example the crystallographic data (PDB 1T0H and 2T0H [2a–d], Protein Databank), showed that FeIII ion was coordinated similarly to the ZnII-ion in ACE, in a sixcoordinated manner, through N-nitrogen of His331.A and His336.A amino acids and O-centers of Glu376.A residue bidentately. Nonetheless that oxidation state of the metal ion FeII at high spin S = 2 was established by Mössbauer and XAS studies in TH [2a–d]. Therefore, the presented study contributed significantly for the understanding of the factors influenced the untypical coordination mode of the (1) and is of importance for the molecular design of new artificial catalysts and ACE-inhibitors, on the base of the naturally occurred [3,4], semi-synthetic and synthetic peptides. On the other site the above mentioned similarity of the coordination manner of the metal ions in the aromatic amino acid hydroxylases,

make the reported data of importance and interest to understand comprehensively as well the mechanisms of the catecholamine neurotransmission function [2]. Since the ZnII-possessed a completed d10-electronic configuration, a parallel study with the coordination ability the (1) with AgI-ion is performed [3a–e]. 2. Experimental 2.1. Synthesis Bradykinin stabilized as acetate salt, the anhydrous ZnCl2 and AgNO3 were Sigma–Aldrich products. The equimolar amounts of the peptide, and each of the metal salts were mixed in 25 ml solvent mixture water:ethanol at room temperature under stirring. The 0.5 ml 0.1 M NaOH was added dropwisely. The isolated precipitates were filtered off, dried washed with water and dried on P2O5. Yields of 93.4 ((1)–Zn) and 65.4% ((1)–Ag) were obtained. (1)–Zn, Found: C, 51.8; H, 6.3. Calc. [ZnC50H72N15O11Cl]: C, 51.8; H, 6.3%; (1)–Ag, Found: C, 51.4; H, 6.2. Calc. [AgC50H72N15O11]: C, 51.5; H, 6.2%. The obtained solid-state vibrational spectra (Fig. 1) of the neutral form of (1) were after the interaction of the starting bradykinin acetate salt with the 1 M NaOH in water. 2.2. Physical measurements HPLC–MS/MS measurements were made using TSQ 7000 instrument (Thermo Fisher Inc., Rockville, MD, USA). Two mobile phase compositions were used: (A) 0.1% v/v aqueous HCOOH and (B) 0.1% v/v HCOOH in CH3CN. Electrospray ionization mass

B. Ivanova, M. Spiteller / Inorganica Chimica Acta 392 (2012) 211–220

Fig. 1. IR-spectra of the (1), Ang-I, Ang-II and the complexes of (1) with ZnII and AgImetal ions in solid-state.

spectrometry (ESI): a triple quadrupole mass spectrometer (TSQ 7000 Thermo Electron, Dreieich, Germany) equipped with an ESI 2 source was used and operated at the following conditions: capillary temperature 180 °C; sheath gas 60 psi, corona 4.5 lA and spray voltage 4.5 kV. Sample was dissolved in acetonitrile (1 mg ml 1) and was injected in the ion source by an autosampler (Surveyor) with a flow of pure acetonitrile (0.2 ml min 1). Data processing was performed by Excalibur 1.4 software. A standard LTQ Orbitrap XL (Thermo Fisher Inc.) instrument was used for the MALDI-MS measurements, using the UV laser source at 337 nm. An overall mass range of m/z 100–1000 was scanned simultaneously in the Orbitrap analyzer. The ImageQuest 1.0.1 program package was used. Raman spectra in solid-state were recorded on: (i) Nicolet NXR 9610 FT-Raman spectrometer (both instruments were products of Thermo Electron Corporation, Baltimore, MD, USA), equipped with the semiconductor laser operating source at 976 nm. The resolution of 0.09 cm 1 was over the spectral range 100–3705 cm 1 and (ii) Horiba Jobin–Yvon Inc. (Edison, NJ, USA) 60000 triple monochromator spectrometer equipped with a Spectra-Physics Inc. (Mountain View, CA, USA) model 164 argon ion laser operated on the 514.5 nm line. The resolution of 1.0 cm 1 was over the spectral range 20–2500 cm 1. A triple integrated laser system was used for the variation of the excitation energy at 532, 633 and 785 nm, respectively. The laser power used was 100 mW, with a spectral band-space of 3 cm 1. The spectra were recorded at ambient conditions (T = 298 K, P = 1 atm). The Raman spectra of

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the solid crystal powders were measured in a glass capillary. The IR-spectra in the KBr-pellet were measurede using the Thermo Nicolet 6700 FTIR spectrometer within 4000–400 cm 1 at the resolution of the ±0.5 cm 1. Chromatographic confirmation about the purity of the studied compounds was performed with a Gynkotek (Germering, Germany) HPLC instrument, equipped with a preparative Kromasil 100 C18 column (250  20 mm, 7 lm; Eka Chemicals, Bohus, Sweden) and a UV detector set at 250 nm. The mobile phase was acetonitrile:water (90:10, v/v) at a flow rate of 4 mL/min. The analytical HPLC was performed on a Phenomenex (Torrance, CA, USA) RP-18 column (Jupiter 300, 150 mm  2 mm, 3 lm) under the same chromatographic conditions as above. The QA was performed on a Shimadzu UFLC XR (Kyoto, Japan) instrument, equipped with an auto sampler, PDA, an on-line degasser and column thermostat. As stationary phase a Phenomenex Luna Phenyl-Hexyl column (150 mm  3 mm i.d., 3 lm particle size) was used. The mobile phase consisted of 0.02% (v/v) TFA in water (solvent A) and acetonitrile methanol 75:25 (v/v; solvent B). Separation was achieved by a gradient analysis starting with 55A–45B, increasing the amount of solvent B in 30 min to 75% and 30.1 min to 100% B, stop time 40 min. For equilibration a post time of 15 min was applied. Other parameters: flow rate 0.30 ml/min, injection volume 5 ll, detection wavelength 280 nm; column temperature 35oC. The UV–Vis–NIR spectra between 200 and 800 nm, using solvent acetonitrile (Uvasol, Merck product) at a concentration of 2.5  10 5 M in 0.921 cm quartz cells were recorded on Tecan Safire Absorbance/Fluorescence XFluor 4 V 4.40 spectrophotometer. The thermogravimetric study was carried out using a Perkin-Elmer TGS2 instrument. The calorimetric measurements were performed on a DSC-2C Perkin Elmer apparatus under argon. The 1H and 13C NMR measurements were performed at 298 K with a Bruker DRX-600 spectrometer using 5 mm tubes and CD3CN as solvent. The chemical shift reference was sodium 3-(trimethylsilyl) tetradeuteriopropionate. 2.3. Computational methods 2.3.1. Quantum chemistry The calculations were performed with GAUSSIAN 09, DALTON 2011 and LSDALTON program packages [5a–c]. The visualization was performed using GAUSSVIEW3.0 [5d]. The geometries of the studied species were optimized at two levels of theory: second-order Moller– Plesset perturbation theory (MP2) and density functional theory (DFT) using the 6-31+G(d,p) basis set. The DFT method employed was B3LYP, B3PW91, CAM-B3LYP, and M06-2X functional [5].

Scheme 2. Chemical diagrams of the (1), Ang-I and Ang-II with the calculated qN(NBO) charges at the UB3PW91/SDD theoretical level, using the PCM. The obtained values were for the neutral forms of the studied peptides.

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The fully optimization procedure was presented in [5]. The electronic spectra in gas phase and solution were obtained, by timedependent density functional method (TD-DFT) method. The 6311++G(2d,p), aug-cc-pVDZ and aug-cc-pVTZ basis sets were used. The calculations in solution were carried out by the explicit supermolecule and microhydration approach, in which several water molecules were coordinated to the solute at the polarizable continuum approach (PCM). An explicit model, by a cluster of n solvent molecules surrounding the zwitterion Brad and its capable of building to FeII in ACE model was also applied. Series of model interactions (Scheme 1) were elucidated depending of the coordination mode the Brad by the triple-f quality TZVP, triple-f plus double polarization TZ2P, Los Alamos National Laboratory’s 2 double-f level [5]. The protonation ability of Brad was elucidated by the natural bond orbital (NBO) charges and analysis (Scheme 2) [5]. The ONIOM method was also employed, in which different parts of the supermolecule can be treated at different levels of accuracy, using different basis sets or even different quantum methods. Molecular mechanics calculations (MM) were performed, using DREIDING and UFF force fields. The crystallographic coordinates for ACE were used, obtained by the Protein Data Bank (PDB 1O86 [1g]). The charges were assigned to atoms using the DFT calculations and NBO values. 2.4. Chemometrics The R4Cal OpenOffice STATISTICs for WINDOWS 7 program package was used, applied the baseline correction, deconvolution, non-linear curve fitting and smoothing methods for treatment the experimental spectroscopic patterns [6]. The statistical significance of each regression coefficient was checked by the use of ttest. The model fit was determined by F-test. 3. Results and discussion 3.1. Electronic absorption and CD-spectroscopy Since the coordination processes affected significantly the molecular geometry of the peptides, the EAs and CD-spectroscopy were used studying the complexation ability of the (1), depending of the metal ions. The spectrum of (1), typical for an unstructured peptide, is characterized by a negative band at about 206 nm. The presence of the ZnII and AgI metal ions significantly changed the sign of the bands in the spectra. The positive band at 220 nm in the spectrum of (1) is perturbed as a results of the coordination and is observed at about 207–210 nm in the complexes. The band at 230 nm, often assigned to a charge-transfer band in the spectra of metaloproteins, which for (1) and its metal complexes, however, is difficult to attribute such origin, since the shown region of the electromagnetic spectrum coincide with the B-bands of the aromatic Phe-amino acid residues. The coordination mode of (1) would proposed by the EAs and CD spectroscopy, but no unambiguous evidences and structural was possible, due to the complex broad and overlapped spectroscopic profile of the curves. So, that only qualitative information for the induced complexation process is estimated. 3.2. Vibrational spectroscopy (IR and Raman methods) The vibrational characteristic data of (1) showed series of Amide I (mC@O, stretching vibrations) modes within 1700– 1600 cm 1 active both the in IR and Raman spectra, similar as well to the corresponding vibrations in Arg-I and Arg-II [7]. Within the 3500–3000 cm 1 a complex overlapped patterns, typical for the zwitterion peptides and the protonated guanydyl-fragments in

Arg-containing derivatives, difficult the assignment of the mNH stretching vibrations, so that a coordination manner via the deprotonation of the NH-amide groups is difficult to assigned unambiguously by vibrational spectroscopy in the case of (1). As could see (Fig. 1) the coordination of the ZnII- and AgI-ions resulted to the observation of the high frequency shifted Amide I bands at 1715 cm 1, which was an indirect evidence about the coordination the (1) via deprotonation of the NH-amide fragments, since the coordination through the amide N-centers leads to the distortion of the n–p conjugation within the frame of the NH–CO fragment resulted to the observed shifting the mC@O modes. The bands/frequencies at about 1740 cm 1 are typical for the mC@O stretching vibration of the COO -group where the monodentate coordination through the O-center leads to a reverse the C@O group in the initial zwitterion carboxylate fragment. 3.2.1. Mass spectrometric data The MS methods provided direct experimental evidences about the coordination ability of (1) studying the isotope ratios of ZnIIand AgI-complexes in gas-phase. The presented strategy included the analysis of the coordination ability the (1), Ang-I and Ang-II using theoretical NBO analysis (Scheme 2 and Table 1). Under the physiological conditions the His-containing peptides (including the catalytic domains in the enzymes [1,2,8]) usually formed the complexes through His-side chains, nevertheless that qN(NBO) values noted these centers as less preferred for coordination position/ s. Including as well the fact that His-as binding site provided additional complexities, by the basic nitrogen as a neutral donor and, on deprotonation of the weakly acidic N–H centers, since the qN(NBO) charges were within ( 0.233) to ( 0.132), values higher to those of the N-arginine nitrogen, and/or NH2-group at the N-terminus. The preference for the formation of less stable complexes, agreed to the hypothesized mechanisms of enzyme functions such for example the above mentioned activity the aromatic amino acid hydroxylases as well as the crystallographic data, where the catalytic domains were characterized with the metal ions joined to the macromolecule usually via an untypical coordination number for the corresponding ions [1,2], but the question why in (1), Ang-I, Ang-II the C-terminus was preferred for coordination the ZnII-ion remains? The qN(NBO) data clearly indicated the N-terminus as main coordination site, in accordance with the numerous studies on the complexation processes with small peptides [8–10]. The highest basicity of the Arg-residues is obtained theoretically (Scheme 2), defining the ionic character of the Arg-side chains and contributed substantially to the shown structure of (1) (Scheme 3) itself and involved in the coordination to ZnII- and AgI-ions. So, that the capability of Arg to form salt bridged structures and strong interaction of ionic groups with the polar solvents, is predicted theoretically by the shown data in Scheme 2, confirming that this factor is major one in the protein folding. Moreover, recently, it has been studied gas-phase stabilized complex salts with alkali metal ions, where the Arg-containing ligands have been stabilized as guanidyl-protonated forms [10]. In (1), the closed disposition of Arg- to N-terminus and the most stable conformation, where strong to moderate intramolecular hydrogen bonds are found, lead to a steric prevention the –NH2 as first coordination center. The Arg-residue at the C-terminus, resulted to the most readily stabilized structure (Scheme 2), with deprotonated carboxylic and protonated guanydyl-fragment, thus making the C-terminus suitable first coordination site. Interestingly, were the obtained qN(NBO) values of N-amide nitrogen linked the Phe-residues, facilitating the chelating effects. To address the above arised question, regarding the coordination sides in (1), experimentally, we examined how the competitive binding modes towards the metal ions ZnII and AgI effected the fragmentation of the peptide. The studied complexes showed series of peaks MS–M–fj (M = ZnII, or AgI, j = 1–

Table 1 The population analysis and the natural electron configurations of the metal chromophores in the mass spectrometric ZnII- and AgI-complex species (MS–M–fj, M = Zn or Ag ions, j = 1–4) at the UB3PW91/SDD theoretical level and PCM; The free Gibbs energies (DG) (kcal/mol). Atom

AO

MS–Zn–f1 Occupancy

px py pz px py pz px py pz px py pz px py pz dxy dxz dyz dx2 dz2

y2

0.73334 0.74004 0.67146 0.67126 0.81069 0.61738 0.81828 0.87132 0.77286 0.83819 0.76269 0.89841 0.89688 0.80897 0.75235 0.99187 0.99788 0.99450 0.98513 0.99629

0.37371 0.35963 0.33044 0.32157 0.35157 0.30398 0.40506 0.39442 0.40646 0.43983 0.45026 0.42923 0.30781 0.33203 0.28914 0.61747 0.61888 0.61556 0.61497 0.61687

Natural electron configuration N 2p(2.14)3p(0.01) N 2p(2.11)3p(0.01) O 2p(2.46) O 2p(2.46) O 2p(2.46) M 3d(4.97)4d(0.01)5p(0.02)

Occupancy 0.67175 0.81442 0.60400 0.66427 0.65956 0.79551 0.80347 0.88580 0.77310 0.81432 0.76353 0.89518 0.89296 0.80387 0.72991 0.94994 0.98340 0.96798 0.91927 0.98179

MS–Zn–f2 Energy 0.43313 0.46081 0.41787 0.42027 0.42494 0.41488 0.52556 0.51179 0.52053 0.52869 0.54168 0.52637 0.41253 0.43451 0.39301 0.58357 0.57686 0.57277 0.57805 0.57244

2p(2.12)3p(0.01) 2p(2.09)3p(0.01) 2p(2.46) 2p(2.46) 2p(2.43) 4d(4.80)5d(0.01)6p(0.01)7p(0.01)

Occupancy 0.68957 0.80650 0.61977 0.74383 0.72887 0.69385 0.88065 0.83520 0.63326 0.83216 0.80120 0.81169 0.79803 0.85679 0.77448 0.99340 0.99830 0.99191 0.98661 0.99542

MS–Ag–f2 Energy 0.44277 0.49852 0.45957 0.51723 0.49521 0.47455 0.49450 0.50535 0.45871 0.38038 0.38943 0.36558 0.53815 0.53162 0.54427 0.77052 0.77221 0.76909 0.76841 0.77084

2p(2.12)3p(0.01) 2p(2.17) 3p(0.01) 2p(2.43) 2p(2.45) 2p(2.35) 3d(4.97)4d(0.01)5p(0.02)

Occupancy 0.67874 0.81654 0.58856 0.74712 0.72711 0.64366 0.79106 0.76007 0.89544 0.79486 0.88722 0.77881 0.87535 0.84315 0.63095 0.95277 0.98327 0.96012 0.92827 0.98062

MS–Zn–f3 Energy 0.40905 0.46381 0.42462 0.51336 0.49371 0.47022 0.52574 0.53567 0.52603 0.52374 0.51522 0.52199 0.49627 0.50327 0.45806 0.58703 0.58126 0.57409 0.58472 0.57632

2p(2.08)3p(0.01) 2p(2.12)3p(0.01) 2p(2.46) 2p(2.45)3p(0.01) 2p(2.15) 4d(4.81)5d(0.01)7p(0.01)

Occupancy 1.58845 1.40973 1.22371 1.28856 1.65730 1.35948 1.59157 1.49459 1.69674 – – – – – – 1.99852 1.99942 1.99855 1.99789 1.99596

MS–Ag–f3 Energy 0.49290 0.48874 0.47436 0.50007 0.55125 0.49048 0.42228 0.44543 0.42525 – – – – – – 0.74332 0.74647 0.74859 0.74269 0.74836

2p(4.22)3p(0.03) 2p(4.31)3s(0.01)3p(0.03) 2p(4.68)3p(0.01) – – 3d(9.99)4p(0.01)5p(0.03)

Occupancy 0.78724 0.71338 0.58523 0.64430 0.80423 0.64806 0.55056 0.69244 0.82523 – – – – – – 0.99327 0.99730 0.99148 0.93066 0.98952

MS–Ag–f4 Energy 0.44895 0.45325 0.43555 0.49267 0.53511 0.47958 0.41518 0.47137 0.46522 – – – – – – 0.53697 0.53357 0.53798 0.52443 0.53428

2p(2.09)3p(0.01) 2p(2.10)3p(0.01) 2p(2.33) – – 4d(4.90)

Occupancy 0.77126 0.73568 0.62489 0.65739 0.71401 0.88148 – – – – – – – – – 0.97317 0.98770 0.99066 0.99463 0.99112

Energy 0.48367 0.43819 0.44313 0.43419 0.46170 0.48217 – – – – – – – – – 0.62289 0.62060 0.61053 0.62699 0.61420

2p(2.13)3p(0.01) 2p(2.25)3p(0.01) – – – 4d(4.94)7p(0.01)

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N N N N N N O O O O O O O O O M M M M M

MS–Ag–f1 Energy

215

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Scheme 3. Molecular structure and chemical diagrams of the (1) (a) and its (1)–ZnII ([ZnIIC50H72N15O11]Cl) and (1)–AgI ([AgIC50H72N15O11]) complexes (b); the metal ions were shown as the gray ball (b).

Fig. 2. ESI-MS spectra of (1)–Zn (a) and (1)–Ag (b) complexes; Chemical diagrams and fragmentation scheme of the gas-phase stabilized complex species (c); the MALDI-MS and IMS data for the (1)–(Ag) and the 3D image by logarithm plotting of Dx, Dy vs. the signal amplitude (grid) of selected ion range as a function of the location of the surface for the system (d).

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4) characterizing with significant DG values (Table 1). These data explained the relatively high intensities in the MS spectra (Fig. 2). The peaks at m/z 1124.66 and 1167.90 corresponded to [MC50H72N15O11]+ cations, where M = ZnII- or AgI-ions. The fragmentation scheme, was proposed studying the isotope ratios of the peaks shapes at m/z 359.90, 264.06 and 190.98 of [ZnC12H12O6N3]+, [ZnC7H7O5N2]+ and [ZnC4H3O3N2]+ ions (Fig. 2). Nevertheless, that MS studies on ZnII-protein systems reported the detection of complex species of the stable 67Zn isotope [10i,j], in (1)–Zn, the peak shapes are relatively less complicated, than those of proteins. The observed peaks at m/z 359.90/361.90/363.90; 264.06/266.06/268.06 and 190.98/192.98/ 194.98 are assigned to complex [64ZnC12H12O6N3]+/[66ZnC12 H12O6N3]+/[68ZnC12H12O6N3]+, [64ZnC7H7O5N2]+/[66ZnC7H7O5N2]+/ [68ZnC7H7O5N2]+, and [64ZnC4H3O3N2]+/[66ZnC4H3O3N2]+/ [68ZnC4H3O3N2]+ ions, taking into account the relative abundances (RA,%) of the Zn-isotopes of 48.89%, 27.81% and 18.57% [10i,j]. The peaks at m/z 191.98, 360.90 and 265.06 are assigned to complex cations of 67Zn isotope. The observed intensities, however were not corresponded to the expected RA of 4.11% (Fig. 2). The MS data of (1)–Ag showed peaks at m/z 403.07/405.07; 308.22/ 310.22 and 235.55/237.55 of [107AgC12H13O6N3]+/ [109AgC12H13O6N3]+, [107AgC7H8O5N2]+/[109AgC7H8O5N2]+ and [107AgC4H4O3N2]+/[109AgC4H4O3N2]+ complexes. Only in the MS spectrum of AgI-complex, were found as well peaks at m/z 190.04/192.04 of [107AgC3H4ON2]+/[109AgC3H4ON2]+ with intensities related the RA of 51.82% and 48.18% (Fig. 2). The formation of MS–Ag–f4 is explained with the ability of AgI-ion to form the complexes with the larger variation of the coordination number [3]. The obtained population analysis and electronic configurations of these complex species (Table 1) agreed to the experimental data, and indicated a metal-to-ligand charge transfer effect (MLCT). For the complexes with completed d10-electronic configuration, this phenomenon appeared crucial for their stabilization [3], moreover it has been observed for coordination compounds with d8 configuration the metal ion [3f]. The data are interpreted as experimental evidences that the metal ions bind to the C-terminus, nevertheless those theoretical values indicated that, no strong preference for the binding manner of (1). Thus, the proposed configuration of the peptide and the shown backbone conformation with the sterically blocked N-terminus ((Ser)NH


O@C(Arg), 2.938; (Phe)NH


O@C(Ser), 2.483; (Ser)OH


NH2(Arg), 3.408 and (Arg)HNH


O@C(Gly), 2.890 Å, Scheme 2), the two protonated Arg-residues at both the sites of the peptide, as well as the presence of Phe-amino acid close to the C-terminus resulted to the formation of mononuclear complexes of ZnII and AgI with (1). The ions were coordinated in a pentadentate manner forming a MN3O2 chromophore with the Cs symmetry. The ligands were joined through the O-atom from COO -group, two deprotonated N-amide nitrogen from Arg- and Phe-residues as well as O(@C) and OH-centers of Ser-fragment. Notably the protonated guanidine groups were projected, always observed in the studied complexes in gas-phase. The obtained data were agreed as well with the reported data on the coordination ability the ZnII with Ang-I, where clearly the peptide nitrogen deprotonation of His-residue has been evidenced and the coordination was not adjacent to the N-terminus [3g]. 3.2.2. Nuclear magnetic resonance data The resonances in the 1H NMR spectra of (1) and its complexes with ZnII and AgI were assigned by the standard combination of 2D COSY and NOESY experiments. Thus, was possible to assign the complete spin system of the aliphatic residues. The observation of the broad chemical shift signals for the OH (Ser) and amideNH protons within the studied pH range of 4.4–7.1 was not possible. Therefore, the assignment of these signals is not unambigu-

217

ously. The CH signals of the aromatic protons of the Phe-amino side chains showed an overlap resonances within d values of 7.13–7.56 (m, 10H) ppm. Only the NH-from the mixing time NOESY spectrum showed the proton signal for NH region of Phe at the C-terminus at about 8.34 ppm, which absent in the 1H NMR spectrum of the corresponding complexes. Significantly were effected the signals from the CaH(Arg), CaH(Phe), CaH(Ser) and CbH2(Ser) protons in (1), observed at about 4.42, 4.47, 3.76 and 2.92 ppm, respectively. The corresponding data for the complexes were 3.77, 4.45, 3.60 and 2.71 ((1)–Zn) and 3.70, 4.15, 3.56 and 2.67 ppm ((1)–Ag), respectively. As would expected the protons signals for the CbH2, CcH2 and CdH2 of Arg-residues were effected weakly from the coordination manner the peptide and were found at about 1.80, 1.57 and 3.22 ppm. A difference of about Dd 0.11 ppm was found for the CbH2(Phe) at about 3.11 ppm, respectively. The obtained data agreed reasonably well with the mass spectrometric results about the coordination mode the (1) with the ZnII- and AgI-ions as well as the theoretical NBO analysis, since the obtained chemical proton signals shifting in the AgI-complex corresponded to the most stable complex (Table 1). 3.2.3. Model ACE-interactions The molecular modeling of interactions of (1) to the ZnII-ion in the ACE was performed using the backbone structure of the peptide and the crystallographic coordinates for the enzyme (Schemes 1 and 2). The coordination mode I is described in the presence of inhibitor, where the metal ion is joined in a bidentate manner to the O-atoms from the COO -group (Zn–O, 2.141, 2.605 Å) [1g]. The ZnII-ion is coordinated additionally by two N-atoms from His353.A, His838.A residues (Zn–N, 2.071, 2.038 Å) and an O-center from the COO of the Glu411.A amino acid (Zn–O, 2.996 Å). The geometry of the chromophore ZnIIN2O3 is in Cs symmetry (Table 2). The perturbed C2v⁄ symmetry of the coordination ZnIIN2O chromophore without inhibitor was found (Mode II). Modes III– VI corresponded to different manner the coordination the (1) to the catalytic domain, i.e. via bi-(Mode III) and tridentately (Modes IV–VI), where the His353.A, His383.A and Glu411.A amino acid residues of the protein were joined. Since, under the physiological conditions as well as by the obtained theoretically NBO data (Table 1) the Arg-residues were protonated, the performed calculations were carried out using a guanydyl-cations. Thus, the modeled complexes were neutral in III, V and VI, and negatively charged in IV, respectively. The obtained DG values clearly indicated as most stable the model interaction of (1) via mode-IV, where the peptide was joined to the ZnII ion in a tridentate manner through the Oatom from the COO -group and a two deprotonated N-amide nitrogen from the Arg- and Phe-amino acid residues. The His253.A and Glu411.A are coordinated additionally in a monodentate manner by the N-, respectively, O-center (Zn–O, and Zn–N, 2.456, 2.803 Å). These data are in good agreement to the obtained results about the preferred cleavage of (1), resulting to the isolation of the H-Phe-Arg-OH via the C-terminus interaction with the domain [1]. On the other side, since the coordination manner I consisted on the positively charged ZnII-complex in the presence of the inhibitor [1g], than the role of the Cl -ions was only a charge-neutralization. In all of the obtained AgI-complexes and species of type MS–Zn–f1 and MS–Zn–f2, the MLCT effect appeared crucial for the complex stability (Table 2). Only in MS–Zn–f3 the obtained natural electron configuration (NEC) data would described in terms of the classical crystal and ligand field theory. As could see the data are in agreement the completed d10 configuration of the metal ion and an absence of the MLCT effect. These results would explain with the untypical for the ZnII-ion coordination T-shape with perturbed C3v⁄ (coordination number = 3) of the metal ZnN2O chromophore, but arised the question about the factors defining the observed relatively intensive peaks of the isotope shape in the MS (Fig. 2). The

218 Table 2 The population analysis and the natural electron configurations of the metal chromophores in the modeled ZnII–complexes with the ACE depending of the coordination modes shown in Scheme 1 at the UB3PW91/SDD theoretical level. Atom

AO

Mode I Occupancy

px py pz px py pz px py pz px py pz px py pz px py pz dxy dxz dyz dx2 dz2

y2

0.87407 0.72955 0.72932 0.73348 0.76573 0.83307 – – – 0.72286 0.91996 0.95288 0.70763 0.64241 0.58405 0.67249 0.69446 0.86143 0.99652 0.99690 0.99772 0.99644 0.99714

0.35523 0.29027 0.29441 0.27944 0.30010 0.32048 – – – 0.09527 0.20918 0.18433 0.09668 0.08398 0.08690 0.37208 0.37092 0.49050 0.45071 0.45553 0.45302 0.45099 0.45519

Natural electron configuration N 2p(2.33)3p(0.01) N 2p(2.33)3p(0.01) N – N – O 2p(2.60) O 2p(1.93) O 2p(2.23)3s(0.01) Zn 3d(4.98)4p(0.07)5s(0.01)4d(0.01)5p(0.01)

Occupancy 0.82544 0.76273 0.75985 0.84982 0.73269 0.76499 – – – 0.29450 0.64924 0.90082 – – – – – – 0.99255 0.99765 0.99820 0.99508 0.99623

Mode III Energy 0.49804 0.47526 0.46928 0.52390 0.47051 0.48300 – – – 0.29845 0.36221 0.39227 – – – – – –

2p(2.35)3p(0.01) 2p(2.35)3p(0.01) – – 2p(1.84) – – 3d(4.98)4p(0.04)

0.67167 0.67895 0.67768 0.67282 0.67925

Occupancy 0.50160 0.79657 0.18951 0.76608 0.85663 0.70076 0.52084 0.61161 0.69984 0.69060 0.90907 0.73220 0.96016 0.80823 0.75361 – – – 0.97728 0.88752 0.97271 0.97427 0.95640

Mode IV Energy 0.40646 0.44387 0.33011 0.34954 0.37083 0.33240 0.40736 0.41197 0.41555 0.64102 0.64880 0.63149 0.47621 0.45847 0.43267 – – – 0.89397 0.94701 0.90971 0.89293 0.93010

2p(1.49) 2p(2.32)3p(0.01) 2p(1.83)3p(0.01) – – – – 3d(4.77)4p(0.04)5d(0.01)

Occupancy 0.84545 0.76329 0.78252 0.01300 0.36113 0.50212 – – – 0.65688 0.72857 0.69446 0.75905 0.88988 0.72224 0.40568 0.82723 0.41073 0.95193 0.98335 0.95715 0.99454 0.98599

Mode V Energy 0.79716 0.78892 0.77902 0.49287 0.56615 0.59833 – – – 0.78826 0.76566 0.74704 0.85743 0.87575 0.83781 0.73001 0.79614 0.71965 1.06027 1.06114 1.04995 1.06704 1.05672

2p(2.39)3p(0.01) 2p(0.88) 2p(1.64)3p(0.01) – 2p(2.08) 2p(2.37) – 3d(4.87)5p(0.02)5d(0.01)

Occupancy 0.54511 0.18044 0.70915 0.60062 0.56071 0.32362 0.74559 0.81564 0.77226 0.79843 0.69079 0.69804 – – – – – – 0.99023 0.99242 0.99478 0.93777 0.97828

Mode VI Energy 0.41591 0.35330 0.43197 0.40167 0.40705 0.37655 0.64915 0.56774 0.55135 0.51171 0.49355 0.50580 – – – – – – 0.78252 0.78670 0.77404 0.76789 0.77476

2p(1.43) 2p(1.48) 2p(2.33)3p(0.01) p(2.24)3p(0.01) 2p(2.19) – – 3d(4.89)5p(0.02)5d(0.01)6p(0.01)

Occupancy 0.03830 0.13442 0.02286 0.73198 0.80826 0.79786 0.75799 0.66458 0.97077 0.78143 0.63432 0.95295 – – – – – – 0.99065 0.99381 0.99649 0.96382 0.98223

Energy 0.31364 0.33260 0.31517 0.66521 0.59822 0.60268 0.51671 0.52109 0.56085 0.54820 0.50464 0.53978 – – – – – – 0.83945 0.85158 0.83963 0.83753 0.84173

2p(0.20) 2p(2.34)3p(0.01) 2p(2.39) – 2p(2.37) – – 3d(4.93)5p(0.02)5d(0.01)6p(0.01)

B. Ivanova, M. Spiteller / Inorganica Chimica Acta 392 (2012) 211–220

N N N N N N N N N O O O O O O O O O Zn Zn Zn Zn Zn

Mode II Energy

B. Ivanova, M. Spiteller / Inorganica Chimica Acta 392 (2012) 211–220

answer of this question assumed further detail MS study of the series of ZnII- and AgI-complexes with N and O-containing ligands and different dentate character, geometry of the metal chromophore, and more. If we discussed the ACE system only in this term, than the data for the NEC for the metal ions in III and IV, means that we must expected a lowest DG value for I (Scheme 1). So that, of one side the NEC data agreed to the reported ACE-activity and proposed mechanisms of the cleavage of (1) [1], but on the other side only evaluating the DG values, we can assumed that an additional important role played the enzyme cavity, in terms, the environment around the catalytic center. In this respect the obtained DG quantities, contributed to ACE–(1) stability of the molecular complex system via coordination mode IV.

4. Conclusions In this paper, we investigate in details the fragmentation scheme and the isotope peak shapes of the isolated ZnII- and AgImetal complexes with bradykinin (1) as an effort to explain the untypical coordination ability of the peptide via the C-terminus, evaluating the factors such as salt-ridged ability the Arg-amino acid side chains, the conformational preference of the peptide, the macromolecular cavity, i.e. the environment around the metal ion in the angiotensin converting enzyme (ACE), taking into account as well the specifically competitive coordination ability of the His and Arg-residues in the enzyme and the peptide. Theoretical calculations are carried out of the initial ZnII-containing complex using the crystallographic data for the ACE with and without the presence of inhibitor as well as four model interactions of the (1) to the metal center. The candidate structure of the peptide–metal ion complex in ACE molecular system as well as the corresponding isolated ZnII- and AgI-complexes provided theoretical evidences to support our experimental mass spectrometric observations that the peptide molecule preferred to coordinate in a tetradentate manner to the metal ions via the C-terminus. A deprotonation the N-amide fragments of the Arg and Phe amino acids, a monodentate coordination of the O-atom from the COO group of the peptide and a OH-coordination of the Ser-side chain is proposed. Contrast, the interactions in ACE showed that (1) acted as a tridentate ligand by the C-terminus via the O-atom from the COO -group and the two deprotonated N-amide Arg- and Phefragments. Additionally, the ZnII-ion is joined to the N-heterocyclic nitrogen from the His353.A and a O-atom from the Glu411.A amino acid fragments in the enzyme. As main factors contributing to the stability of the isolated species in gas phase were the metalto-ligand charge transfer effect and protein environment around the catalytic domain in ACE, evaluated by the cavitation energy. The obtained data on the coordination and protonation ability of (1) were discussed and compared with the available ones for the coordination ability the Ang-I and Ang-II, which would be of interest and related the main aim of the study in the field of the molecular design and searching of the of new ACE-inhibitors on the base of small synthetic, semi-synthetic and naturally occurred peptides.

Acknowledgments The authors thank the Deutscher Akademischer Austausch Dienst (DAAD), the Deutsche Forschungsgemeinschaft (DFG), the central intrumental laboratories for structural analysis at Dortmund University (Germany) and the analytical and computational laboratory clusters at the Institute of Environmental Research (INFU) at the same University.

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