Gabi Drochioiu
email: gabidr@uaic.ro

Marilena Manea

Mihaela Dragusanu

Ecaterina Manuela Mu- Rariu

Brandusa Stela Dragan

Alina Petre

Gabor Mezo

Michael Przybylski

Manuela Murariu

Ecaterina Stela Dragan

Alina Brandusa

Petre

Interaction of β-amyloid(1-40) peptide with pairs of metal ions: An electrospray ion trap mass spectrometric model study

Keywords: amyloid β-peptide, metal ion-peptide complex, Alzheimer's disease, ESI ion trap MS

The stoichiometries and the affinity toward simple and paired metal ions of synthetic amyloid-β(1-40) peptide (Aβ1-40) were investigated by electrospray ion trap mass spectrometry (ESI-MS), circular dichroism (CD), and atomic force microscopy (AFM). The results lead to the working hypothesis that pHdependent metal binding to Aβ1-40 may induce conformational changes, which affect the affinity toward other metals. A significant copper and zinc binding to Aβ1-40 peptide at pH 5.5 was found, whereas nickel ions commonly bind to each molecule of β-amyloid peptide. Some complexes of Aβ1-40 with more than one nickel ion were identified by ESI-MS. In addition, nickel ions proved to enhance Aβ oligomerization. On increasing pH, up to 12 ions of zinc may bind to a single Aβ molecule. Under the same pH and concentration conditions, the binding pattern of the independent copper and silver ions to Aβ1-40 was different from that of the equimolecular mixture of the two metal ions. One might assume that some conformational changes due to water loss altered the capacity of Aβ peptide to bind certain heavy metal ions. As a consequence, copper-silver interaction with the binding process to Aβ1-40 became highly complex. A competition between silver and nickel ions for Aβ1-40 binding sites at high pH was also observed. New strategies were proposed to identify the characteristic signals for some important metal ion-peptide complexes in the spectra recorded at high pH or high concentrations of metal ions. To explain the formation of such a large number of high metal ion-Aβ complexes, we took into consideration the participation of both histidine residues and free amino groups as well as carboxylate ones in the binding process. Finally, CD and AFM studies supported the mass spectrometric data.

Please cite this article as: Gabi Drochioiu, Marilena Manea, Mihaela Dragusanu, Manuela Murariu, Ecaterina Stela Dragan, Brandusa Alina Petre, Gabor Mezo, Michael Przybylski, Interaction of β-amyloid peptide with pairs of metal ions: An electrospray ion trap mass spectrometric model study, Biophysical Chemistry (2009), doi: 10.1016/j.bpc.2009.05.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Electrospray-ionization mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopic investigations have confirmed that some sites of β-amyloid peptide (Aβ) are able to specifically bind metal ions [START_REF] Hut | Gas-phase coordination properties of Zn 2+ , Cu 2+ , Ni 2+ , and Co 2+ with histidinecontaining peptides[END_REF][START_REF] Zirah | Zinc binding properties of the amyloid fragment Abeta(1-16) studied by electrospray-ionization mass spectrometry[END_REF]. Metal ions may interact with both amyloid precursor protein (APP) and Aβ to potentate Alzheimer's disease by participating in the aggregation of these normal cellular proteins and in the generation of reactive oxygen species [START_REF] Adlard | Metals and Alzheimer's disease[END_REF][START_REF] Bush | The metallobiology of Alzheimer's disease[END_REF][START_REF] Barnham | Neurodegenerative diseases and oxidative stress[END_REF]. These metals are normally found at high concentrations in the regions of the brain most susceptible to AD neurodegeneration. Lowering Cu concentrations can downregulate the transcription of APP, showing that APP and Aβ form part of the Cu homeostatic machinery in the brain [START_REF] Maynard | Metals and amyloid-beta in Alzheimer's disease[END_REF]. Aβ peptide, which is over accumulated in the AD brain, can efficiently generate reactive oxygen species in the presence of the transition metals [START_REF] Smith | The redox chemistry of the Alzheimer's disease Amyloid-β peptide[END_REF]. In fact, increased brain metal levels have been associated with normal aging and a variety of diseases, including Alzheimer's disease (AD) [START_REF] Lahiri | Amyloid, cholinesterase, melatonin and metals, and their roles in aging and neurodegenerative diseases[END_REF]. Aβ has anti-oxidant properties, and at low levels, it has trophic rather than toxic properties and these protective effects have been attributed to the antioxidant properties of metallated Aβ [START_REF] Scheibe | Ecto-alkaline phosphatase activity identified at physiological pH range on intact P19 and HL-60 cells is induced by retinoic acid[END_REF]. The βamyloid plaques that are one of the pathological features of Alzheimer's disease are composed of amyloidβ peptides and are highly enriched in metal ions such as Zn, Fe and Cu [START_REF] Lau | Cholesterol and Clioquinol modulation of Aβ(1-42) interaction with phospholipid bilayers and metals[END_REF][START_REF] Atwood | Characterization of copper interactions with Alzheimer amyloid β peptides: identification of an attomolar-affinity copper binding site on amyloid β1-42[END_REF]. High concentrations of Zn 2+ and Cu 2+ are released during neurotransmission, which might explain why Aβ precipitation into amyloid commences in the synapse [START_REF] Lee | Proc. Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant APP transgenic mice[END_REF][START_REF] Terry | The pathogenesis of Alzheimer disease: an alternative to the amyloid hypothesis[END_REF]. Contrary, the addition of low levels of zinc induces proteaseresistant aggregation and precipitation of synthetic Aβ [START_REF] Huang | Zincinduced Alzheimer's Abeta(1-42) aggregation is mediated by conformational factors[END_REF]. Besides, zinc ions afford protection against Aβ toxicity at low concentrations and enhance toxicity at high concentrations [START_REF] Lovell | Protection against amyloid beta peptide toxicity by zinc[END_REF]. Copper and iron also induce peptide aggregation that is increased at acidic pH [START_REF] Atwood | Dramatic aggregation of Alzheimer A_ by Cu(II) is induced by conditions representing physiological acidosis[END_REF]. The involvement of metal ions as a potential key factor towards conformational modifications and aggregation of amyloid is widely recognized [START_REF] Drago | Potential pathogenic role of beta-amyloid(1-42)-aluminum complex in Alzheimer's disease[END_REF]. Circular dichroism (CD) studies indicated quite minimal ordering of the main-chain with Zn 2+ or Cd 2+ addition and that Zn 2+ coordination is distinct from that of Cu 2+ ions [START_REF] Syme | Solution 1 H NMR investigation of Zn 2+ and Cd 2+ binding to amyloid-beta peptide (Aβ) of Alzheimer's disease[END_REF]. Copper, iron, and zinc bind Aβ, generate an allosterically ordered membrane-penetrating oligomer, induce aggregation and give rise to reactive oxygen species [START_REF] Curtain | Alzheimer's disease amyloid-b binds Cu and Zn to generate an allosterically ordered membrane-penetrating structure containing SOD-like subunits[END_REF]. Iron(II) or iron(III) block Cu-mediated neurotoxicity in a highly reducing environment [START_REF] White | Iron inhibits neurotoxicity induced by trace copper and biological reductants[END_REF]; lipophilic ferrous chelators are effective agents for moderating neuronal

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ACCEPTED MANUSCRIPT 4 damage in conditions such as AD where intracellular oxidative stress plays a significant role in disease pathology [START_REF] Jayasena | Membrane permeability of redox active metal chelators: An important element in reducing hydroxyl radical induced NAD + depletion in neuronal cells[END_REF].

Nevertheless, less is known how toxic and essential metals may influence each other in regard to their retention by the peptides. We considered that a basic treatment of Aβ peptide containing two heavy metals (copper(II) and silver(I) or silver and nickel(II) might reveal some interactions which take place between these metal ions and proteins within the living bodies under various pH conditions. Therefore, the aim of the present study is to detail the stoichiometries and binding properties of multiple cooperative metal ion-binding sites on synthetic Aβ1-40. By the introduction of additional methodological strategies the obtained spectra were simplified and some extra information was obtained. It is our also intention to shed some light on the relationship between various metal ions and amyloid peptide, as well as its impact on AD pathology.

EXPERIMENTAL

Reagents:

Triethanolamine, methyl alcohol, silver nitrate, nickel chloride, ferric chloride, zinc chloride, copper sulfate, and mercury chloride were purchased from Merck (Darmstadt, Germany). All stock solutions of metal ions were prepared using MilliQ grade water (18 MΩ). the coupling steps by ninhydrin assay to assess the presence of free amines. In the case of negative results a new cycle was started. The cycle was repeated from the neutralization step when the colorimetric test indicated ineffective coupling. After assembling the peptide chain on the resin, the N-terminal Bocprotecting group was removed prior to the final cleavage with HF.

Synthesis of

Deprotection and cleavage of the peptide from the resin with HF

After removal of the N-terminal Boc-protecting group, the peptide-resin was neutralized and washed with DCM (4 times) and ethanol (2 times, 0.5 min). Before HF cleavage the resin was dried in a desiccator overnight. The cleavage of the side chain protecting groups and the detachment of the peptide from the resin by HF was performed in a Teflon apparatus (Peptide Institute, Osaka, Japan). For 1 g of peptideresin 10 mL of liquid HF was used in the presence of 1 g p-cresol, 0.1 g DTT and 0.1 g L-Cys. The cleavage was carried out for 90 min at 0 °C. After evaporation of HF (captured in a CaO-filled tower) the peptide was precipitated with cooled diethyl ether. The mixture of precipitated peptide and resin was filtered and washed 3-4 times with diethyl ether (30-40 mL) to remove scavengers. The unprotected peptide was dissolved in 10% acetic acid solution and then lyophilized.

Reverse phase high performance liquid chromatography (RP-HPLC)

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ACCEPTED MANUSCRIPT with eluent A (0.1% TFA in water) and eluent B (0.1% TFA in acetonitrile-water (80:20 v/v)) was used at a flow rate of 1 mL/min at ambient temperature. Peaks were detected at 220 nm.

ESI-ion trap mass spectrometry

Electrospray ionization mass spectrometric experiments and tandem mass spectrometry were carried out on a Bruker Daltonics Esquire 3000Plus (Bremen, Germany) ion trap mass spectrometer. Spectra were acquired in the 200-2000 m/z range. Samples were dissolved in a methanol/water 1:1 (v/v) solvent mixture, containing 0.1% acetic acid, and the pH was maintained at 5.5 with acetic acid or triethanolamine. Binding of individual metal ions to β-amyloid peptides was studied by ESI-ion trap MS at pH 5.5, at a peptide concentration of 10 pmol/µL and 1:10 peptide: metal ion ratio. The competition of various metal ions towards the binding sites of Aβ peptide was investigated at a 1: 5: 5 molar ratio (Aβ1-40 peptide: metal 1: metal 2), pH 5.5, Aβ1-40 concentration being 10 pmol/µL. In order to study the binding of the individual metal ions to peptide at higher pH, triethanolamine was added instead of acetic acid to obtain pH 8.00, 9.00, and 9.20, respectively. Besides, the binding of metal ions to Aβ1-40 was studied at pH 7.4 or 6.6 using 5 mM ammonium acetate as a solvent. All masses were calculated using the charge state ruler tools provided with Bruker Daltonics DataAnalysis software of MS instrument. This soft is able to select all of the various charged molecular ions, corresponding to the same peptide or metalpeptide complex.

In order to identify the characteristic signals for some important metal ion-peptide complexes in more complicated spectra we applied two main strategies. First, the spectra of complexes with low metal ion-peptide ratios were carried out. Next, ESI-mass spectra of higher metal ion-peptide ratios complexes

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or those at high pH were recorded. The signals found in the first spectra were identified in the more complicated spectra, using also the charge state ruler.

Circular Dichroism (CD) Spectroscopy

CD spectra were recorded using a Jasco spectropolarimeter model J-710 (Labor and Datentechnik GmbH, Germany). All measurements were performed in quartz cells with a path length of 0.5 mm. Data were collected at the wavelengths from 180 to 260 nm in 0.2 nm increments. Four individual measurements were averaged in order to obtain the reported CD spectrum. The measurements were carried out at 25.0 ± 0.2 °C. Calculations of the fractional percentage of secondary structures were carried out using the computer programs Spectra-Manager (Jasco), CD-Deconvolution and Dicroprot [START_REF] Wang | Escherichia coli CorA periplasmic domain functions as a homotetramer to bind substrate[END_REF].

Atomic Force Microscopy (AFM)

Images were taken at room temperature (22 °C) on a SPM Solver PRO-M AFM (NT-MTD Co.

Zelenograd, Moscow, Russia) using the tapping mode. All images were acquired using a high resolution no contact "Golden" silicon NSG10/Au/50 cantilever with Au conductive coating. The cantilever was 100 μm in length, 35 μm in width, and with a 2 μm thickness and had a typical tip radius of 10 nm. A resonant frequency of 254 kHz and a force constant of 11.5 N/m were applied. All AFM images were obtained at a resolution of 256 × 256 pixels on a scale of 10 μm × 10 μm. Image analysis was done with scanning probe microscopy software, WSxM 4.0 Develop 10.0 (Nanotec Electronica S. L.). No complex between two or more molecules of peptide and copper ions was identified in the mass spectrum at acidic pH.

RESULTS

Synthetic

The relative intensity of [M+Cu+4H] 6+ was 83.8% as compared to that of [M+6H] 6+ , whereas the corresponding ion [M-H 2 O+Cu+4H] 6+ was only 27.7% of the [M-H 2 O+4H] 6+ intensity. These data showed that possible conformational changes due to water loss diminished the capacity of the amino terminal area of Aβ peptide to bind certain heavy metal ions.

Zinc binding to Aβ1-40

At pH 5.5, Aβ1-40 molecules may bind up to two zinc ions with different intensities. Whereas the seven charged ion of the peptide did not appear in the mass spectrum, the zinc-Aβ peptide complex showed an intense signal at m/z 628.3 (Figure 1,c), which was assigned to [M+Zn+5H] 7+ ion. Aβ-Zn complex was also confirmed by the peaks at m/z 732.8 (6+), 879.0 (5+; a less intensive signal), and 549.9 (3.5% of the intensity of 722.3 peak, [M+Zn+6H] 8+ ).

The Zn-Aβ complex with Zn:Aβ molar ratio 2:1 appeared in the spectrum at m/z 743.

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9

The loss of a molecule of water reduced the zinc binding capacity of Aβ1-40 probably due to conformational changes. Nevertheless, a small peak at m/z 751.9 was noted, which was assigned to [M-H 2 O+3Zn] 6+ . Although iron binding to peptides will be discussed elsewhere, the presence of high molecular complexes such as that containing four molecules of Aβ and six iron ions should be outlined.

Nickel binding to

Metal-metal interactions during the binding process

The previous results demonstrated that potential target sites for the evaluation of metal binding need to be carefully analyzed for interfering metal-metal interactions [START_REF] Curtain | Alzheimer's disease amyloid-b binds Cu and Zn to generate an allosterically ordered membrane-penetrating structure containing SOD-like subunits[END_REF][START_REF] Schlosser | Copperinduced oligomerization of peptides: a model study[END_REF][START_REF] Murariu | Synthesis and mass spectrometric characterization of a metal-affinity decapeptide: copper-induced conformational alterations[END_REF][START_REF] Popa | Effect of radioactive and non-radioactive mercury on wheat germination and the anti-toxic role of glutathione[END_REF][START_REF] Drochioiu | Novel UV assay for protein determination and the characterization of copper-protein complexes by mass spectrometry[END_REF][START_REF] Drochioiu | Synthesis and characterization of a nonapeptide with specific Cu-binding properties[END_REF][START_REF] Gradinaru | Protein determination based on the biuret absorbtion in the UV range and copper binding to peptides[END_REF]. In this study, decreases of bound silver by Aβ, and an increase in bound copper were the most pronounced interactions between the two toxic metals (Figure 3). Under the same pH and concentration conditions, the binding pattern of the The picture of copper-silver interaction to Aβ1-40 was highly complex and had a completely unexpected pattern (Figure 3,c). In addition to the peaks in the individual spectra of copper and silver, Within these experiments, the charge state ruler proved to be quite effective in identifying the peaks in the complicated spectra of non-covalent complexes of peptides.

characteristic

Metal ion binding to Aβ1-40 at basic pH

Relatively small pH changes may affect the metal ion binding capacity of the peptides and proteins. On increasing pH due to the use of triethanolamine, the solution containing Aβ peptide may provide rather complicated mass spectra in the presence of individual metal ions. The high pH ESI MS spectra of proteins or even simple peptides are far too complex for interpretation as most of the signals overlap heavily. Under the same conditions, the spectra of metals ion-peptide adducts become rather complicated but not impossible to be deciphered. By the introduction of additional methodological strategies, such as the comparison of complicated spectra with those obtained at low pH and lower metal ion concentration, as well as the use of the charge state ruler the complex spectra were much simplified and some extra information was obtained.

Copper binding to amyloid-β peptide at pH 8.0. A slight increase in pH dramatically stimulated copper ion binding to Aβ1-40 (Figure 4). Thus, Aβ1-40 peptide bound only one copper ion at pH 6.6 ) as well as by the peaks at m/z 424.9 (12+), 636.9 (8+), or (727.8 (7+). Although the m/z 203.9 signal was associated with Aβ1-40+2Zn complex (22+), it also belongs to [M-H 2 O+12Zn+H] 25+ ion, and therefore this signal was found highly intensive, being the sum of two independent signals.

The most intense signal at m/z 211.9 was assigned to [M +5Zn+12H] 22+ , so that, of so many peaks in the spectrum, the majority of them belongs to monomolecular Aβ complexes with more than one ion of zinc. Nevertheless, one molecule of Aβ proved to bind more than five ions of nickel. For example, the peak at m/z 203.9 could be assigned to six nickel ions bound to Aβ1-40. However, nickel ions seem to be able to form oligomers when bound to peptides; the highest peak in the spectrum, m/z 355.1, was assigned to a metal-peptide complex containing three molecules of Aβ1-40 and two nickel ions. The existence of

Nickel

[3M+2Ni] complex was also confirmed by the signals at m/z 655.9 (20+), 546.9 (24+) and others. Even a single ion of nickel may induce oligomerization of Aβ peptide. An Aβ1-40 trimer bound to a nickel ion was identified at m/z 207.9, 221.9, 225.8, 622.0, etc. On increasing the pH and metal ion:peptide ratio, the spectra pattern became complicated dramatically. To avoid the assignment of some m/z values to false species, we referred only to the most evident complexes of Aβ with the investigated metal ions as revealed by the significant signals in the spectra. In addition, we increased stepwise both pH and metal ion concentration and followed the appearance of the new peaks that prove the formation of new species. Finally, the highest peak in the spectrum at m/z 651.2 was assigned to [M+2Ag+5H] 7+ . Hence, treating Aβ1-40 molecules with silver ions in the 1:10 molar ratio, numerous species of silver-peptide complex were found, whereas Aβ adduct with eight silver ions was prevalent. To explain the formation of such a large number of silver-Aβ complexes, we took into consideration the participation of both histidine residues and free amino group as well as carboxylate ones in the binding process.

Nickel-silver competition for Aβ1-40 binding sites at basic pH

Treating Aβ1-40 in the presence of triethanolamine with a solution containing both silver and nickel ions, a competition among these ions was observed (Figure 6). Apparently, the spectrum of silver-nickel-Aβ1-40 peptide system became simplified as compared to those of both silver-Aβ and nickel-Aβ complexes.

Indeed, the main signals in this spectrum were only found at 355.0 (100%), 203.9 (77.8%), 205.9 (72.2%), 411.0 (40.4%), 357.0 (39.1%), 413.0 (33.0%), 337.1 (21.7%), 618.0 (18.9%), and 653.9 (10.9%), respectively. However, contrary to current spectra of peptides or metal ion-peptide complexes at neutral pH, each group of signals in a given area of the spectrum of metal ion-peptide complexes could not be assigned to an isotopic distribution but many individual complexes. For example, the major signal at m/z .0 (11+), and 250.9 (18+). We also found 4Ni:Aβ140 complex at m/z 651.9 (7+), 506.9 (9+), 414.9 (11+), and 207.9 (22+) as well as species containing five nickel ions in a molecule at 771.1 (6+), 660.9 (7+), 385.9 (12+), and 201.9(23+), respectively.

No other signals for nickel ions bound to Aβ were found except those characteristic to 6Ni:Aβ1-40 molar ratio: 780.9 (6+), 586.0 (8+), 361.0 (13+), and 223.9 (21+). We also searched for signals according to their calculation using masses for the expected complexes. Thus, we found characteristic signals for Ag:Aβ, 2Ag:Aβ, or for Ag:Ni:Aβ, 2Ag:Ni:Aβ, etc.

Nevertheless, each signal in a small range of m/z values consists of many signals, which can be assigned to different silver-Aβ complexes, or silver-nickel-Aβ ones. We found a complex with MW 4919.4 composed of one molecule of Aβ1-40, five silver ions and one nickel ion. Its characteristic signals were also found at m/z 820.9 (6+), 616.0 (8+), 448.5 (11+), 411.0 (12+), and 205.9 (24+), respectively.

The main signal in the area from m/z 201.9 to 209.9 was that at m/z 203.9, which was assigned to

[M+11Ag+3Ni+11H] 28+ , corresponding to a silver-nickel-Aβ1-40 complex with the molecular weight of 5678.0 (calcd. 5678.6). Its existence was also supported by the other peaks at m/z 710.9 (8+), 632.0 (9+), 356.0 (16+), and 247.9 (23+), respectively. We clearly stated that generally high molecular mass complexes give signals at lower m/z values due to their high charged ions, whereas the low molecular peptides and metal ion-peptide complexes at higher ones.

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Circular dichroism investigation

The influence of metal ions on the secondary structure of the Aβ1-40 peptide was followed by CD spectroscopy. Metal ion complexation has previously been shown to be a strong trigger for a secondary structure switch from an α-helix to a β-sheet [START_REF] Murariu | Synthesis and mass spectrometric characterization of a metal-affinity decapeptide: copper-induced conformational alterations[END_REF]. The CD spectra of Aβ1-40 peptide (180 μM) in aqueous solutions or in the presence of copper ions, sodium dodecylsulfate and nickel ions at two pH values are shown in Figure 7. The CD spectrum of the peptide showed a low degree of ordering (49.1%

unordered forms and 34.3% β-sheet conformers), which was not drastically modified by the addition of copper ions. However, a slight increase in the pH value enhanced the proportion of β-sheet conformers (up to 40.2%). In the presence of SDS, the conformational equilibrium changed from unordered and βturn structures to β-sheet (46%) and α-helix conformers (Table 2, Figure 7). Some other measurements showed that SDS produces similar conformational equilibriums of Aβ1-40 species that are independent on the pH values and copper ion concentrations. SDS induced a complete abolition of β-turn conformers and an increase in the population of β-sheet conformers (Table 2). To explain the atomization of Aβ aggregates by SDS, even if it produces an increasing in β-sheet proportion of Aβ1-40 molecules, we considered the hydrophobic interactions between SDS molecules and the hydrophobic amino acid residues of Aβ1-40 molecule, which may induce a shift toward α-helical conformers which behave different from fibrillogen β-sheet ones.

Nickel ions changed dramatically the secondary structure of Aβ1-40 from a large proportion of random coil (49.1%) to one of α-helix conformers (25.3%). In addition, nickel induced a slow decrease in the population of β-turn conformers, and a slight increase in that of β-sheet species (38.5%). These findings are concordant with the MS measurements, which showed the formation of various nickel-Aβ1-40 complexes containing variable molar ratios between Aβ and nickel.

By addition of equimolecular amounts of iron ions, the conformational equilibrium was almost unchanged (43.7% unordered conformers and 37.5% β-sheet ones). Iron induced a very slow increase in

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the population of beta conformers, that are related to Aβ toxicity and Alzheimer's pathogenesis. In contrast to copper ions, which increase the proportion of β-sheet conformers, or nickel ions, which induce the formation of β-turn species, Fe(III) had little effect on the conformational equilibrium. However, hydrophobic environments, such as that generated by SDS, increased α-helical proportion, which is supposed to reduce β-sheet toxicity.

Atomic force microscopy investigation

The self-assembled nanostructures of peptides in aqueous solution were significantly influenced by the presence of metal ions (Figure 8). Fresh solution of Aβ1-40 formed a smooth film on the glass plate of the microscope. Copper ion binding to Aβ1-40 peptide plays an important role in the formation of nanostructures as shown in Figure 8,b. Copper ions thus generated a rough surface containing numerous big structures as high as 200 nm. It is also clear from this image that copper ions are not able to induce large nanstrucutres, but small hills. However, the strong contrast between the AFM images recorded before and after copper treatment confirmed a morphological change in the surface packing of the peptide shell. Although SDS do not form non-covalent complexes with Aβ1-40, the high roughness of the surface in Figure 8,c showed an evident interaction of the two molecules, which results in the formation of supramolecular structures. On treating copper-Aβ1-40 complex with SDS, similar nanostructures are created from these adducts. Similarly, silver-Aβ1-40 film differed dramatically from that of Aβ1-40 one, as well as that of the solution containing silver ions, Aβ1-40 molecules and SDS (10:1:10 molar ratio;

Figure 8,d).

DISCUSSION

The stoichiometry and binding mode of Aβ1-40 peptide showed a great variety depending on the metal ion to ligand ratio, and pH. These results are concordant with those obtained previously on working with individual metal ions [START_REF] Smith | Copper-mediated Amyloid-Toxicity Is Associated with an Intermolecular Histidine Bridge[END_REF][START_REF] Syme | Copper Binding to the Amyloid-{beta} (A{beta}) Peptide Associated with Alzheimer's Disease: Folding, Coordination Geometry, pH Dependence, Stoichiometry, and Affinity of A{beta}-(1-28): Insights from a Range of Complementary Sectroscopic Techniques[END_REF][START_REF] Karr | N-Terminal Deletions Modify the Cu 2+ Binding Site in Amyloid-β[END_REF][START_REF] Karr | Amyloid-ß Binds Copper(II) in a Mononuclear Metal Ion Binding Site[END_REF]. Although blood and internal biochemical environment is characterized by a

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18 pH close to 7.39 (7.35 -7.45), most fluids inside the body as well as body tissues function at different pH ranges [START_REF] Lagadic-Gossmann | Alterations of intracellular pH homeostasis in apoptosis: origins and roles[END_REF][START_REF] Chappell | Metabolic alkalosis despite hyperlactatemia and hypercapnia: Interpretation and therapy with help of the Stewart concept[END_REF]. Under various physiological conditions, acutely dissociated neurons were also found to have a wide range of pHi values (6.3-7.7) [START_REF] Aoki | Effects of pH on brain energetics after hypothermic circulatory arrest[END_REF][START_REF] Yates | Enzyme activities in relation to pH and lactate in postmortem brain in Alzheimer-type and other dementias[END_REF]. During near-complete rat forebrain ischemia, cerebral intracellular pH decreased from 7.01 to 6.17 [START_REF] Anderson | Is intracellular brain pH a dependent factor in NOS inhibition during focal cerebral ischemia?[END_REF]. This was the incentive to investigate the interaction of metals with peptides within a large pH range. The coordination of metal ions to Aβ is the same in both aqueous solution and lipid environments, with His 6 , His 13 , and His 14 all involved [START_REF] Curtain | Alzheimer's disease amyloid-b binds Cu and Zn to generate an allosterically ordered membrane-penetrating structure containing SOD-like subunits[END_REF]. However, the peptide backbone becomes more important in metal ion binding process on increasing pH. Nickel ions bind strongly to Aβ1-40 even in acidic solutions. Nevertheless, silver ions enhance copper binding to Aβ in acidic solutions, whereas they compete with nickel ions at higher pH.

The generation of the Aβ toxic species is modulated by the Cu 2+ concentration and the ability to form an intermolecular His bridge as demonstrated by EPR spectroscopy [START_REF] Smith | Copper-mediated Amyloid-Toxicity Is Associated with an Intermolecular Histidine Bridge[END_REF]. Several spectroscopic studies have indicated that the copper-binding site of Aβ consists of the three histidine residues His6, His13 and His14 and an as yet undefined fourth ligand; proposed ligands include Tyr10 [START_REF] Curtain | Alzheimer's disease amyloid-b binds Cu and Zn to generate an allosterically ordered membrane-penetrating structure containing SOD-like subunits[END_REF][38][START_REF] Tickler | Amyloid-b as a 'difficult sequence' in solid phase peptide synthesis[END_REF][START_REF] Antzutkin | Amyloidosis of Alzheimer's Abeta peptides: solid-state nuclear magnetic resonance, electron paramagnetic resonance, transmission electron microscopy, scanning transmission electron microscopy and atomic force microscopy studies[END_REF], the Nterminal nitrogen [START_REF] Syme | Copper Binding to the Amyloid-{beta} (A{beta}) Peptide Associated with Alzheimer's Disease: Folding, Coordination Geometry, pH Dependence, Stoichiometry, and Affinity of A{beta}-(1-28): Insights from a Range of Complementary Sectroscopic Techniques[END_REF] or an as yet unidentified carboxylate side chain [START_REF] Karr | N-Terminal Deletions Modify the Cu 2+ Binding Site in Amyloid-β[END_REF][START_REF] Karr | Amyloid-ß Binds Copper(II) in a Mononuclear Metal Ion Binding Site[END_REF].

Our study revealed a high affinity of Aβ toward iron(III) ions, with which Aβ forms both ironbound oligomers and high molecular weight polymeric species. They might have a high pathophysiological significance because pH and reduction-oxidation balance of certain cells may alter alongside many pathological processes.

Depending on the pH values, zinc ions may bind to Aβ peptide in various proportions as our investigation revealed. Although protein chelation of zinc is typically mediated by histidines, cysteines and carboxylates at low pH [START_REF] Yang | Examining the zinc binding site of the amyloid-beta peptide[END_REF], amino groups may play an important role under slight alkaline conditions.

Further investigation should be done to clarify the effect of metal ions on the conformational changes of Aβ as well as their interference with the other metal ions within the binding process. Aβ [START_REF] Hut | Gas-phase coordination properties of Zn 2+ , Cu 2+ , Ni 2+ , and Co 2+ with histidinecontaining peptides[END_REF][START_REF] Zirah | Zinc binding properties of the amyloid fragment Abeta(1-16) studied by electrospray-ionization mass spectrometry[END_REF][START_REF] Adlard | Metals and Alzheimer's disease[END_REF][START_REF] Bush | The metallobiology of Alzheimer's disease[END_REF][START_REF] Barnham | Neurodegenerative diseases and oxidative stress[END_REF][START_REF] Maynard | Metals and amyloid-beta in Alzheimer's disease[END_REF][START_REF] Smith | The redox chemistry of the Alzheimer's disease Amyloid-β peptide[END_REF][START_REF] Lahiri | Amyloid, cholinesterase, melatonin and metals, and their roles in aging and neurodegenerative diseases[END_REF][START_REF] Scheibe | Ecto-alkaline phosphatase activity identified at physiological pH range on intact P19 and HL-60 cells is induced by retinoic acid[END_REF][START_REF] Lau | Cholesterol and Clioquinol modulation of Aβ(1-42) interaction with phospholipid bilayers and metals[END_REF][START_REF] Atwood | Characterization of copper interactions with Alzheimer amyloid β peptides: identification of an attomolar-affinity copper binding site on amyloid β1-42[END_REF][START_REF] Lee | Proc. Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant APP transgenic mice[END_REF][START_REF] Terry | The pathogenesis of Alzheimer disease: an alternative to the amyloid hypothesis[END_REF][START_REF] Huang | Zincinduced Alzheimer's Abeta(1-42) aggregation is mediated by conformational factors[END_REF][START_REF] Lovell | Protection against amyloid beta peptide toxicity by zinc[END_REF][START_REF] Atwood | Dramatic aggregation of Alzheimer A_ by Cu(II) is induced by conditions representing physiological acidosis[END_REF] was previously shown to bind up to three cations upon increasing the Zn 2+ concentration [START_REF] Zirah | Zinc binding properties of the amyloid fragment Abeta(1-16) studied by electrospray-ionization mass spectrometry[END_REF]. Under CID,

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

19 zinc binding induces specific cleavages after the three histidines of the Aβ(1-16) sequence (H 6 , H 13 and H 14 ), showing their simultaneous implication in the Zn 2+ coordination sphere [START_REF] Zirah | Zinc binding properties of the amyloid fragment Abeta(1-16) studied by electrospray-ionization mass spectrometry[END_REF]. In addition, Glu11 residue has been identified as providing the carboxylate side chain when Zn 2+ is bound to Aβ1-16 [START_REF] Zirah | Structural changes of region 1-16 of the Alzheimer disease amyloid beta-peptide upon zinc binding and in vitro aging[END_REF].

Circular dichroism spectroscopy and electron microscopy were used as well to monitor the effects of metal ions on the peptide β-sheet conformation and fibril aggregation. These observations indicated a specific zinc binding site within Aβ that involves these central histidine residues. Complementary studies with Cd 2+ ions cause perturbations to 1 H NMR spectra of Aβ that are strikingly similar to that observed for Zn 2+ [START_REF] Syme | Solution 1 H NMR investigation of Zn 2+ and Cd 2+ binding to amyloid-beta peptide (Aβ) of Alzheimer's disease[END_REF]. The binding of divalent metals like Zn II , Cd II and Co II to a synthetic peptide, APP 170-188 induces the dimerisation of the peptide [START_REF] Ciuculescu | Metal-binding properties of the peptide APP170-188: A model of the ZnII-binding site of amyloid precursor protein (APP)[END_REF].

Metal ions have been suggested to induce aggregation of amyloid beta-peptide, which is a key event in Alzheimer's disease [START_REF] Miura | Copper selectively triggers beta-sheet assembly of an N-terminally truncated amyloid beta-peptide beginning with Glu3[END_REF]. Although, histidine-containing peptide fragments of prion protein are efficient ligands to bind various transition metal ions with the following order of metal affinity [START_REF] Jószai | Transition metal complexes of terminally protected peptides containing histidyl residues[END_REF]:

Pd(II) >> Cu(II) >> Ni(II) ≥ Zn(II) > Cd(II) ~ Co(II) > Mn(II), our study showed the nickel was bound by Aβ with higher affinity than copper ions. The high selectivity of metal binding is connected to the involvement of both imidazole and amide nitrogen atoms in metal binding for various metal ions [START_REF] Jószai | Transition metal complexes of terminally protected peptides containing histidyl residues[END_REF].

Our results showed that Aβ peptide is helical in hydrophobic environments and becomes mostly β-sheet in aqueous media or in the presence of some metal ions, which is concordant with Lau et al. [START_REF] Lau | Magnetic Resonance Studies of -Amyloid Peptides[END_REF].

Glycine residues in Aβ1-40 may be important in metal binding because they allow an appropriate folding of the peptide. Indeed, the GxxxG motifs of Aβ is a key structural and functional determinant [START_REF] Xu | Conformational transition of amyloid {beta}-peptide[END_REF].

Besides, β-Amyloid protein oligomers induced by metal ions and acid pH are distinct from those generated by slow spontaneous ageing at neutral pH, as previously shown [START_REF] Klug | ] 6+ [Aß1-40+6H] 6+ [Aß1-40+Ni+3H] 5+ [Aß1-40+Ni+5H] 7+ [Aß1-40+5H] 5+ [Aß1-40+2Ni+H] 5+ [Aß1-40+2Ni+3H] 7+ [Aß1-40+3Ni+H] 7+ [2Aß1-40+3Ni+6H] 12+ [Aß1-40+3Ni] 6+ [2Aß1-40+3Ni+6H] 12+ 627[END_REF].

The three techniques used here (ESI-MS, CD, and AFM) revealed the complex interaction between Aβ1-40 peptide with various metal ions under different pH conditions, with possible impact on the research methodology in various pathologies. to bind certain heavy metal ions. As a consequence, copper-silver interaction with the binding process to Aβ1-40 was highly complex. A competition between silver and nickel ions at high pH for Aβ1-40 binding sites was also observed. New strategies were proposed to identify the characteristic signals for some important metal ion-peptide complexes in the spectra recorded at high pH or high metal concentrations.

Unlike the current spectra of peptides or metal ion-peptide complexes at neutral pH, each group of signals in a given area of the spectrum of high metal ion-peptide complexes at higher pH cannot be assigned to an isotopic distribution but many individual complexes. To explain the formation of such a large number of high metal-Aβ complexes, we took into consideration the participation of both histidine residues and free amino group as well as carboxylate ones in the binding process. Circular dichroism and atomic force microscopy studies supported the mass spectrometric data. Oligomerization is also possible (m/z 727.1). Table 1. The main molecular ions identified in Figure 3,c.

[M+2Cu+2H] 6+ [M+Ag+Na+4H] 6+ [M+Cu+Ag+3H] 6+ [2M+3Cu] 6+ [M-H 2 O+2Ag+4H] 6+ [M-H 2 O+4Cu-2H] 6+ [M+4Cu-2H] 6+

5 OH

 5 β-amyloid(1-40) peptide Peptide H-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV-NH 2 (Aβ1-40) was synthesized manually, by SPPS, according to the Boc/Bzl strategy using MBHA resin (0.5 mmol/g coupling capacity) as a support. The following Boc-protected amino acid derivatives were used: Boc-Val-OH, Boc-Gly-OH, Boc-Met-OH, Boc-Leu-OH, Boc-Ile-OH, Boc-Ala-OH, Boc-Lys(ClZ)-OH, Boc-Asn-A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT , Boc-Ser(Bzl)-OH, Boc-Asp(OcHex)-OH, Boc-Glu(OcHex)-OH, Boc-Phe-OH, Boc-Gln-OH, Boc-His(Bom)-OH, Boc-Tyr(BrZ)-OH and Boc-Arg(Tos)-OH. The protocol of the synthesis was as follows: 1) DCM washing (3 x 0.5 min); 2) Boc cleavage using 33% TFA in DCM (2+20 min) for the first 5 cleavage steps and 33%TFA, 3% anisole in DCM containing 50 mg L-methionine and 10 mg DTT/ 10 mL cleavage solution (2+20 min) in order to avoid methionine oxidation; 3) DCM washing (5 x 0.5 min); 4) Neutralization with 10% DIEA in DCM (4 x 1 min); 5) DCM washing (4 x 0.5 min); 6) Coupling 3 or 5 equiv of Boc-amino acid derivative-DCC-HOBt in DCM-DMF mixture (60 min); 7) DMF washing (2 x 0.5 min); and 8) DCM washing (2 x 0.5 min). 8) Monitor

  Aβ1-40 peptide showed characteristic peaks at m/z 722.3 ([M+6H] 6+ , the highest peak), and 866.5 for [Aβ+5H] 5+ . The molecular ions [M-H 2 O+6H] 6+ and [M-H 2 O+5H] 5+ also appeared in the spectrum and displayed relative intense signals at m/z 719.4 and 863.0, respectively (Figure 1,a). In A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 8 addition, the aspartic acid-lacking Aβ1-40, namely y39 fragment, with MW 4211.1 was identified, being more visible in the spectra of metal ion-peptide complexes.

3. 1 5

 15 Copper binding to Aβ1-40 at pH 5.Our study revealed a significant copper binding to Aβ1-40 peptide at pH 5.5 and a 1:1 Cu: Aβ molar ratio (Figure1,b). The Cu-Aβ complex appeared in the spectrum at m/z 628.1 (7+), 732.5 (6+), 878.8 (5+), and 1098.3 (4+), respectively. The second copper atom was present in the following copper-peptide ions: [M+2Cu+3H] 7+ (m/z 636.8), [M+2Cu+2H] 6+ (m/z 742.9), [M+2Cu+H] 5+ (m/z 891.0), and [M+2Cu] 4+ (m/z 113.5).

Aβ1- 40 Aβ1- 40 3 . 4 10 ratio

 40403410 peptide displayed a high affinity toward nickel ions even at low pH (Figure 1,d). Up to three nickel ions bound to each molecule of β-amyloid peptide as demonstrated by the peaks at m/z 722.2, 731.7, 741.2, and 750.6, which were assigned to [M+6H] 6+ , [M+Ni+4H] 6+ , [M+2Ni+2H] 6+ , and [M+3Ni] 6+ , respectively. On the other hand, the signals of five-charged ions at m/z 877.8 and 888.9 clearly confirmed the formation of Ni-Aβ1-40 complexes in the molar ratios 1:1 and 1:2. Low intensity signals, which were assigned to the complex of Aβ1-40 with four nickel ions, were also found at m/z 912.4 (5+), 760.6 (6+), 652.1 (7+), 570.7 (8+), 507.2 (9+), 380.8 (12+), and 304.9 (15+), respectively. Silver and iron binding to Aβ1-40 Both silver and iron ions displayed different binding patters as compared to the other investigated metal ions. The intense signal at m/z 740.1 was assigned to Ag-Aβ1-40 complex (Figure 1,e). Water loss from the Aβ molecule may result in conformational changes with lower metal binding capacity, which was evident from both Aβ complex with silver and the other metal complexes (Figure 1,b-e). The other signal for Ag-Aβ1-40 complex at m/z 887.8 with lower intensity as compared to [M+Ag+5H] 6+ signal was assigned to [M+Ag+4H] 5+ (Figure 1,e). Iron(III) ion produced both the oligomerization of the peptide and iron-rich high molecular mass complexes. Besides the above mentioned signals, iron ions produced many other Fe-Aβ species that were identified with the help of the charge state ruler (Figure 2). Because the main signals of Fe-Aβ complexes hindered the display of other lower signals, we had to investigate a narrower m/z range, close to m/z 722.3 (m/z of [M+6H] 6+ ). Here, 5+, 6+, and 7+ ions of Aβ1-40 together with the multi-charged ions of multi-A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Fe-Aβ1-40 complexes, including that of [M+3Fe-3H] 6+ ion, were found in the spectrum. In fact, a large group of Fe(III)-Aβ1-40 complexes were identified with the help of a single ESI ion trap mass spectrum, starting from that of Aβ1-40 pentamer with only one iron ion, [5M+Fe+32H] 35+ , and ending to the complex with nine iron ions bound to one Aβ1-40 molecule.

  independent copper and silver ions to Aβ1-40 was different from that of the equimolecular mixture of the two metal ions. First, Aβ1-40 bound easily one copper ion to form the [M+Cu+4H] 6+ complex, which was detected at m/z 732.5. Only a small part of Aβ1-40 molecules produced complexes containing two copper ions bound to one Aβ molecule (m/z 742.7; [M+2Cu+2H] 6+ ). Water-lacking Aβ1-40 had a lower affinity towards copper ions than the normal Aβ1-40 (Figure 3,a; m/z 729.7). Second, both Aβ1-40 and Aβ1-40-H 2 O molecules proved to have a high affinity toward silver ions (Figure 3,b), forming mono-silver complexes (m/z 737.3 and 740.1, respectively). The bi-silver complexes at m/z 757.9 corresponding to [M+2Ag+4H] 6+ was not considered because its low intensity.

12 ( 8 (

 128 A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Figure 4,a), whereas species with one, two and even three copper ions were determined at pH 8.0 (Figure 4,b). Zinc binding. Although the mass spectrum of Aβ peptide treated with 10:1 molar ratio zinc ions seems to be very simple, when enlarged each peak this one becomes a complex group of signals belonging to various Zn(II)-Aβ species. The characteristic signals of Aβ1-40 practically disappeared and specific signals of Aβ molecules complexes with from two to ten zinc ions appeared in the mass spectrum (Figure 5,a). For example, the major peak at m/z 638.0 was assigned to [M+2Zn+5H] 7+ , being accompanied by the other signals of this molecular complex at m/z 203.9 (22+), 248.8 (18+), 344.2 (13+) and 447.0 (10+). Searching for metal-richer complexes of Aβ peptide, we found a well expressed complex of one molecule of Aβ with six ions of zinc; its major peak was displayed at m/z 337.1 (14+), and the others at m/z 214.22+), 363.0 (13+), 428.9 (11+), 523.8 (9+), and 941.8 (7+), respectively. In addition, the signal at m/z 355.0 (14+) was assigned to [M+10Zn-6H] 14+ . Whereas the peak at m/z 638.0 was assigned to one Aβ1-40 molecule bound to two zinc ions, the signal at m/z 637.0 (8+) revealed the presence of Aβ peptide bound to 12 zinc ions. The existence of this complex was also confirmed by the most intense signal in the group around m/z 850.0 (m/z 848.9, [M+12Zn-18H] 6+

  binding. The characteristic signals for Aβ peptide practically disappeared and various groups of new signals revealed a complex pattern of nickel binding to peptides (Figure 5,b). In aqueous solutions of nickel salts and Aβ1-40 peptide found in a molar ratio of 10:1, either single molecules of Aβ bound one to more nickel ions or Aβ oligomers bound nickel in various proportions. First, we identified A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 13 the simplest complex of Aβ1-40 with nickel, [M+Ni+4H] 6+ , at m/z 732.0 and 209.9 (21+) or 232.0 (19+).

1 ( 2 ( 1 (

 121 Silver binding. Generally, silver ions bound to individual molecules of Aβ1-40, without forming oligomeric species. Aβ1-40 peptide without one molecule of water proved to bind easily eight silver ions to form an Aβ complex that displayed specific signals in the mass spectrum at m/z 1034.5 (5+), as well as at m/z 862.4 (6+), 739.2 (7+), 575.2 (9+), 370.1 (14+), 324.1 (16+), 305.1 (17+), 288.1 (18+), 273.19+), 247.0 (21+), and 236.0 (22+), respectively. Another peak at m/z 1035.4 was assigned to [M+8Ag-3H] 5+ , which was less intensive than that of [M-H 2 O+8Ag-3H] 5+ . This complex of Aβ1-40 with eight silver ions was also supported by characteristic signals at m/z 863.2 (6+), 518.2 (10+), 471.1 (11+), 432.12+), 226.0 (23+), and 208.0 (25+), respectively. The peak at m/z 216.0 (23+) also appeared at m/z 990.4 associated with [M-H 2 O+6Ag-H] 5+ complex, and consequently, it was assigned to Aβ1-40 complex with six silver ions, being present at m/z 413.1 (12+), and m/z 226.0 (22+). Looking for metal-richer complexes of Aβ1-40, we found a signal at m/z 689.3, which could be assigned to [M+11Ag-3H] 8+ . However, first we calculated the molecular weight of this complex and A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 14 looked for its five charged ion (m/z 1102.5). Following this procedure, we identified the other peaks of Aβ1-40 molecules, each of them containing eleven silver ions, such as m/z 788.3 (7+), 368.0 (15+), 345.16+), 325.1 (17+), 307.1 (18+), and 263.1 (21+), respectively. Aβ1-40 proved to bind also 12 Ag ions to each molecule as revealed by the relatively less intense signals at m/z 1124.4 (5+), 937.3 (6+), 703.3 (8+), 625.2 (9+), 469.2 (12+), 433.2 (13+), 402.1 (14+), 352.1 (16+), 313.1 (18+), 235.0 (24+), 217.0 (26+), and 209.0 (27+).

355. 0

 0 consists of a group of two intense peaks and three lower ones, ranging from m/z 355.0 to m/z 359.0. The peak at m/z 355.0 was assigned to [M+6Ag+8H] 14+ ion, that at m/z 356.0 to [M+11Ag+3Ni-H] 16+ , whereas the signal at m/z 357.0 (calcd. m/z 357.2) was assigned to [M+8Ag+3Ni+H] 15+ , and m/z 358 A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT could be assigned to [M+3Ag+10H] 13+ , although they are different charged and contain various ratios between the metal ions. Therefore, we searched first for the signals characteristic to 1:1 molar ratio Ni-Aβ complexes. Indeed, one can find low signals, at the detection limit, for one nickel ion bound to a single molecule of Aβ1-40 at m/z 731.9 (6+), 488.3 (9+), 338.3 (13+), 231.9 (19+), and 209.9 (21+) only. Nevertheless, trace signals at m/z 741.2 (6+), 635.5 (7+), 371.0 (12+), and 247.7 (18+) could be attributed to Aβ1-40 complex with two nickel ions. In addition, the existence of 3Ni:Aβ1-40 molar ratio complex was demonstrated by the signals at m/z 750.8 (6+), 563.3 (8+), 451.0 (10+), 410

  We have reported here a complex pattern of metal-metal competition for Aβ1-40 binding sites, which depend essentially on the involved metal ions, their concentration, and pH changes. Thus, metal ions bind specifically to Aβ1-40 peptide and change dramatically its conformation. Increasing pH results in increasing metal ions by Aβ. Significant copper and zinc bind to Aβ1-40 peptide at pH 5.5. However, up to three nickel ions commonly bind to each molecule of β-amyloid peptide, whereas the complex of Aβ1-40 with four nickel ions was also confirmed by low signals at m/z 760.6 (6+) and 652.1 (7+). In addition, nickel ions proved to enhance Aβ oligomerization. Nevertheless, with increasing pH, up to 12 ions of zinc may bind to a single Aβ molecule. Both silver and iron ions displayed different binding patters as compared to the other investigated metal ions. Aβ1-40 peptide without one molecule of water proved to bind easily eight silver ions to form an Aβ complex that displayed specific signals in the MS spectrum at m/z 1034.5. Iron(III) ions produced both the oligomerization of the peptide and the formation of iron-rich peptide complexes. Under the same pH and concentration conditions, the binding pattern of the independent copper and silver ions to Aβ1-40 was different from that of the equimolecular mixture of the two metal ions. Maybe, some conformational changes due to water loss altered the capacity of Aβ peptide

F

  Figures caption

Figure 2 .

 2 Figure 2. Species of iron(III) ion-Aβ1-40 complex at pH 5.5, as displayed in a truncated ESI ion trap mass

Figure 3 .

 3 Figure 3. Binding competition or synergism between copper ions and silver ones to Aβ1-40 sites

Figure 4 .

 4 Figure 4. Effect of pH on copper binding to Aβ1-40: a) ESI ion trap mass spectrum of copper ion-Aβ1-40

Figure 5 .

 5 Figure 5. Metal ion binding to Aβ peptide at pH 9.2: a) ESI ion trap mass spectrum of Ni(II)-Aβ1-40

Figure 6 .

 6 Figure 6. Competition and synergism between silver and nickel ions within the process of

Figure 7 .

 7 Figure 7. CD spectra of Aβ1-40 peptide and of its complexes with copper and nickel ions at pH 7 and 8

Figure 8 .

 8 Figure 8. AFM images (10 μm x 10 μm) of Aβ1-40 and its 1:10 molar ratio adducts: a) Aβ1-40; b) Aβ1-

Figure 1 ,Figure 1 40 AFigure 1 Figure 1 Figure 1 Figure 1 Figure 2 .

 114011112 Figure 1,a. ESI ion trap MS spectrum of Aβ1-40

Figure 7 .Figure 8 .

 78 Figure 7. CD spectra of Aβ1-40 peptide and its complexes with copper and nickel ions at pH 7.0 and 8.0, in the presence of sodium dodecyl sulfate or not (SDS:Aβ1-40, 1:1 molar ratio).

6

  Analytical RP-HPLC was performed on a Knauer system (H. Knauer, Bad Homburg, Germany) using an analytical Phenomenex Jupiter C 4 column (250 × 4.6 mm I.D.) with 5 µm silica (300 Å pore size) (Torrance, CA) as a stationary phase. Linear gradient elution (0 min 0% B; 5 min 0% B; 55 min 100% B)

  of silver complexes with water-loss Aβ1-40 from m/z 737.3. For example, [M-H 2 O+Ag+5H] 6+ and [M-H 2 O+2Ag+4H] 6+ displayed intense signals at m/z 737.3 and 755.5, respectively. Silver ions potentate the binding capacity of copper ions to Aβ, including Aβ1-40 without one molecule of water. Thus, the intense Aβ complexes in such a conformation that would enable them to react easily with copper ions. In this case, silver ions probably catalyze copper binding to Aβ peptides via a reaction mechanism based on conformational changes and replacing peptide-bound silver ions by copper ions.

	signals for peptide oligomerization by copper ions (m/z 738.3; [2M+3Cu] 6+ ) and those for hypothesized that silver binding to Aβ1-40 peptide and especially Aβ1-40-H 2 O one might result in the ternary complexes ([M+Ag+Na+4H] signals at m/z 763.6 and 761.7 were assigned to [M+4Cu-2H] 6+ and [M-H 2 O+4Cu-2H] 6+ . We formation of silver-

6+

, m/z 744.0; [M+Cu+Ag+3H] 6+ , m/z 749.3) were found (Table

1

).

Apparently, copper diminished the signal of [M+Ag+5H] 6+ ion at m/z 740.1, but stimulated the formation

A C C E P T E D

M A N U S C R I P T

Table 2 .

 2 Conformational changes of β-amyloid peptide (Aβ1-40 or Aβ) in the presence of copper ions (Cu), nickel ions (Ni), and sodium dodecyl sulfate (SDS) at pH 7.0 and pH 8.0.

	ACCEPTED MANUSCRIPT
	M A N U S C R I P T
	A C C E P T E D

Table 1 .

 1 The main molecular ions identified in Figure3,c.

	m/z	Molecular Ion	m/z	Molecular Ion
	719.4 722.2	[M+6H] 6+ [M-H 2 O+6H] 6+	742.9 740.1	[M+Ag+5H] 6+
	726.2	[M+Na+5H] 6+	744.0
	728.6	[M+K+5H] 6+	749.3
	729.9 732.9	[M-H 2 O+Cu+4H] 6+ [M+Cu+4H] 6+	754.0 755.5
	737.3 738.3	[M-H 2 O+Ag+5H] 6+ [2M+3Cu] 6+	761.7 763.6

Table 2 .

 2 Conformational changes of β-amyloid peptide (Aβ1-40 or Aβ) in the presence of copper ions (Cu), nickel ions (Ni), and sodium dodecyl sulfate (SDS) at pH 7.0 and pH 8.0.

	Conformation (%

) Aß1-40 Aß/Cu/pH 7 Aß/Cu/pH 8 Aß/Cu/SDS/pH 8 Aß/Ni/pH 7

						ACCEPTED MANUSCRIPT
	1 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV 40
	a)	[Aβ1-40+6H] 6+	722.3 [Aβ1-40+5H] 5+ 866.6	[M+H] + 4328.2 M A N U S C R I P T
					1082.8 A C C E P T E D [Aβ1-40+4H] 4+
	α-helix 200	400	0.0 600	0.0 800	0.0 1000	22.8 1200	1400	25.3 1600	1800	m/z
	ß-sheet		34.3	34.7	40.2	46.0		38.5
	ß-turn		16.6	17.0	16.6	0.0		9.1
	random coil	49.1	48.2	43.2	31.2		27.2
	Total		100.0	100.0	100.0	100.0		100.0


Rastislav Varhač

Nataša Tomášková

Marián Fabián

Erik Sedlák
email: erik.sedlak@upjs.sku

Kinetics of cyanide binding as a probe of local stability/flexibility of cytochrome c

Keywords: cyt c, ferricytochrome c, MCD, magnetic circular dichroism flexibility, protein dynamics, ligand binding, Hofmeister series

Effect of anions of Hofmeister series (thiocyanate, perchlorate, iodide, bromide, nitrate, chloride, sulfate, and phosphate) on local and global stability and flexibility of horse heart ferricytochrome c (cyt c) has been studied. Global stability of cyt c was determined by iso/thermal denaturations monitored by change in ellipticity in the far-UV region and its local stability was determined from absorbance changes in the Soret region. Particularly, relative stability/flexibility of the Met80heme iron bond has been assessed by analysis of binding of cyanide into the heme iron. Both global and local stabilities of cyt c exhibited monotonous increase induced by a change of anion from chaotropic to kosmotropic species. However, this monotonous dependence was not observed for the rate constants of cyanide association with cyt c. As expected more chaotropic ions induced lower stability of protein and faster binding of cyanide but this correlation was reversed for kosmotropic anions. We propose that the unusual bell-shaped dependence of rate constant of cyanide association is a result of modulation of Met80-heme iron bond strength and/or flexibility of heme region by Hofmeister anions independently on global stability of cyt c. Further, our results demonstrate sensitivity of cyanide binding to local change in stability/flexibility in heme region of cyt c.

Introduction

It is generally accepted that stability and conformational dynamics/flexibility are inversely related to each other [START_REF] Vihinen | Relationship of protein flexibility to thermostability[END_REF][START_REF] Tang | Native protein fluctuations: the conformational-motion temperature and the inverse correlation of protein flexibility with protein stability[END_REF]. Although the terms dynamics and flexibility in complex systems are usually interchangeable, they, in general, describe different properties. The dynamics describes motion(s) with a certain time scale, while the flexibility has no particular time reference but implies multiple structures that are of comparable energy and can be interconverted with a series of small changes [START_REF] Richards | Protein Folding[END_REF].

Apparently, their interchangeable use follows from the fact that decreasing a barrier between two states increases rate of interconversion (dynamics) as well as accessibility of these two states (flexibility).

Stability and dynamics/flexibility of protein structure are intimately coupled to the properties (namely dynamics) of the solvent [START_REF] Ansari | The role of solvent viscosity in the dynamics of protein conformational changes[END_REF][START_REF] Fenimore | Slaving: solvent fluctuations dominate protein dynamics and functions[END_REF][START_REF] Prabhu | Protein-solvent interactions[END_REF][START_REF] Finkelstein | Viscosity-dependent protein dynamics[END_REF][START_REF] Samuni | Ligand recombination and hierarchy of solvent slaved dynamics: The origin of kinetic phases in hemoproteins[END_REF]. Structural fluctuations that occur on the fastest (~picoseconds) timescales permit the protein to sample a rugged energy landscape and ultimately facilitate slower, larger scale protein rearrangements [START_REF] Frauenfelder | The role of structure, energy landscape, dynamics, and allostery in the enzymatic function of myoglobin[END_REF]. Water interactions cause a softening of many of the free energy barriers between conformational sub/states [START_REF] Prabhu | Protein-solvent interactions[END_REF][START_REF] Chaplin | Do we underestimate the importance of water in cell biology?[END_REF]. The closest hydration shell surrounding proteins responsible for slaving of protein dynamics/flexibility to solvent motions can be efficiently affected by Hofmeister effect of ions [START_REF] Omta | Negligible effect of ions on the hydrogen-bond structure in liquid water[END_REF][START_REF] Batchelor | Impact of protein denaturants and stabilizers on water structure[END_REF][START_REF] Näslund | X-ray absorption spectroscopy study of the hydrogen bond network in the bulk water of aqueous solutions[END_REF][START_REF] Sedlák | Effect of Hofmeister ions on protein thermal stability: roles of ion hydration and peptide groups?[END_REF][START_REF] Roche | Molecular level probing of preferential hydration and its modulation by osmolytes through the use of pyranine complexed to hemoglobin[END_REF]. The effect of anions on interfacial water structure next to proteins apparently may change the free energy barriers between the conformational sub/states [START_REF] Neagu | Fluctuations and the Hofmeister effect[END_REF].

While it is generally accepted that effect of ions on stability of proteins depends on the position of the ion in Hofmeister series [START_REF] Sedlák | Effect of Hofmeister ions on protein thermal stability: roles of ion hydration and peptide groups?[END_REF][START_REF] Hippel | Neutral salts: the generality of their effects on the stability of macromolecular conformations[END_REF][START_REF] Hippel | Ion effects on the solution structure of biological macromolecules[END_REF][START_REF] Baldwin | How Hofmeister ion interactions affect protein stability[END_REF], an effect of anions on dynamical properties of protein structure is not well understood. In fact, we have only recently showed that ions (both anions and cations) of Hofmeister series significantly affect dynamics/flexibility of the active site of NADH oxidase from Thermus

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thermophilus resulting in unusual bell-shaped dependence of the enzyme activity on position of ions in Hofmeister series [START_REF] Žoldák | Modulation of activity of NADH oxidase from Thermus thermophilus through change in flexibility in the enzyme active site induced by Hofmeister series anions[END_REF][START_REF] Tóth | Flexibility and enzyme activity of NADH oxidase from Thermus thermophilus in the presence of monovalent cations of Hofmeister series[END_REF]. Moreover, Hofmeister effect of anions can affect not only transitions among substates (changing thus local dynamics/flexibility of the protein structure) but also "macroscopic" conformational transitions [START_REF] Vogel | Conformation and stability of alpha-helical membrane proteins. 1. Influence of salts on conformational equilibria between active and inactive states of rhodopsin[END_REF][START_REF] Tomášková | Conformational stability and dynamics of cytochrome c affect its alkaline isomerization[END_REF].

Recently, it was shown that binding of phosphate anions to cyt c destabilizes/increases flexibility of its heme region, specifically the Met80-heme iron bond [START_REF] Shah | Structural changes of horse heart ferricytochrome c induced by changes of ionic strength and anion binding[END_REF]. This is surprising finding as there are well-known general stabilization properties of phosphate anions on protein structures and particularly on heme region of cyt c [START_REF] Osheroff | Mapping of anion binding sites on cytochrome c by differential chemical modification of lysine residues[END_REF][START_REF] Osheroff | Electrostatic interactions in cytochrome c. The role of interactions between residues 13 and 90 and residues 79 and 47 in stabilizing the heme crevice structure[END_REF].

In the effort to investigate this finding in more details, we have systematically

Materials and Methods

B Materials

Horse heart cytochrome c, type VI, was obtained from Sigma and used without purification. All other chemicals were purchased from Fluka and Sigma. Stock solutions of the salts were filtered before use.

Absorption measurements

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Absorption experiments were made with a Varian Cary Bio 100 spectrophotometer equipped with a Peltier element. Thermally induced unfolding of cyt c was monitored at 396 nm. Data were fitted by using the equation:

( ) ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ - Δ + ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ - Δ + + + = trs trs trs trs U U N N obs T T R H T T R H T m A T m A A 1 1 exp 1 1 1 exp (1)
where A obs is observed absorbance at temperature T, A N and A U , and m N and m U , represent intercepts and slopes of the native (predenaturation) and unfolded (postdenaturation) baselines, respectively, and ΔH trs is the enthalpy at the transition temperature T trs .

Circular dichroism measurements

The isothermal denaturation of cyt c was followed by measurement of the ellipticity using a Jasco J-810 spectropolarimeter. The circular dichroism (CD) measurements were done in a solution of 7-9 μM cyt c, 5 mM sodium cacodylate (pH 6.0), 1 μM potassium ferricyanide, and the salt. For measurements in the 250-450 nm region a 1 cm pathlength cuvette was used, and for the 190-250 nm region a 0.1 cm pathlength cuvette was used. The urea-induced spectral changes were detected at 222 nm (no salt, NaH 2 PO 4 , Na 2 SO 4 , KCl), 225 nm (NaClO 4 ), 235 nm (KBr) and 417 nm (NaNO 3 , NaI, KSCN). The different wavelengths were used to minimize the contribution from the salt in the CD signal. Measurements were performed at 24 °C.

The Gibbs free energy of unfolding, ΔG°, that expresses the protein stability and the m value, relating to the amount of protein surface exposed to solvent upon unfolding [START_REF] Myers | Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding[END_REF], were fitted using following equation:

[ ] [ ] ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ + °Δ - + ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ + °Δ - + Δ + + Δ = RT D m G RT D m G D m D m U U N N exp 1 exp ]) [ ( ] [ θ θ θ (2)
where θ is observed ellipticity at the denaturant concentration [D], Δθ N and Δθ U represent the intercepts of the pre-and postdenaturation baselines, respectively, and m N and m U represent the slopes of the pre-and postdenaturation baselines, respectively. This equation takes into account the linear dependence of the pre-and postdenaturation region of the unfolding curve [START_REF] Santoro | Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl αchymotrypsin using different denaturants[END_REF].

The pH values were measured directly in the cuvettes using a Sensorex glass microelectrode, before and after measurements.

Near-infrared magnetic circular dichroism measurements

Data were collected in JASCO J-730W spectropolarimeter at room temperature. All samples of 1 mM oxidized cytochrome c were in D 2 O buffers.

Buffers were prepared by freeze and dry water solutions and solubilization of solid residues with the adequate amount of D 2 O.

Kinetic studies

The kinetic measurements were performed with a Specord S 300 UV-VIS diode-array spectrophotometer equipped with a Peltier element. The protein was fully oxidized prior to the kinetic measurements by the addition of 1 μl of 10 mM potassium ferricyanide per 1.5 ml of sample volume. The final protein concentration was ~ 9 μM. The samples were in 50 mM sodium cacodylate buffer, pH 6.0, in the presence or absence of 0.5, 1.0, 1.5 and 2.0 M salts (NaH 2 PO 4 , Na 2 SO 4 , NaCl, NaBr, NaNO 3 , NaI, NaClO 4 and KSCN) at 24.0 ± 0.1 °C.

A freshly prepared 0.5 M sodium cyanide stock solution was adjusted to pH 6 with concentrated HCl and used within four hours. In a typical experiment the

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reaction was initiated by quickly mixing a given volume of 0.5 M HCN with the solution of cyt c. The final concentration of HCN in the cuvette ranged from 10 to 90 mM. The formation of the complex was followed at 418 nm, which is the maximum in the difference spectrum between the cyt c-CN -complex and the free (unliganded) cyt c. The absorbance data as a function of time were fitted to a single exponential function of the form:

A t = A 0 + A 1 exp(-k obs t) (3)
where A t is the absorbance at time t, A 0 is the final absorbance at time t = ∞ and A 1 is the total change in absorbance between t = 0 and t = ∞, and k obs is the observed pseudo first-order rate constant. The association rate constant, k a, was estimated from the slope of the best-fit plot of the pseudo first-order rate constants versus the concentration of HCN according to equation [START_REF] Sutin | Mechanisms of the reactions of cytochrome c: rate and equilibrium constants for ligand binding to horse heart ferricytochrome c[END_REF]:

k obs = k d + k a [HCN] (4)
where k d represents the dissociation rate constant of the cyt c-CN -complex.

The pH of the solution was determined after each kinetic experiment.

Results

To study the effect of Hofmeister anions on stability of cyt c, and to avoid complications due to specific anion binding to cyt c, we used high (≥0.5M) concentrations of anions when Hofmeister effect prevails [START_REF] Sedlák | Effect of Hofmeister ions on protein thermal stability: roles of ion hydration and peptide groups?[END_REF][START_REF] Ramos | Sulfate anion stabilization of native ribonuclease A both by anion binding and by the Hofmeister effect[END_REF][START_REF] Sedlák | Role of cations in stability of acidic protein Desulfovibrio desulfuricans apoflavodoxin[END_REF]. The native conformation of cyt c is unaffected in high ionic strength [START_REF] Sanishvili | The low ionic strength crystal structure of horse cytochrome c at 2.1 Å resolution and comparison with its high ionic strength counterpart[END_REF]. This is evidenced by an absence of significant spectral change in the Soret region that monitors close environment of sensitive probe -heme, as well as the band 695 nm which monitors integrity of charge-transfer bond between sulfur of Met80 and heme iron (Fig. 1).

The stability of cyt c, expressed in Gibbs free energy (ΔG°), in the presence of

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different salts and their concentrations (0.5 and 1.0 M) was determined by isothermal urea denaturation monitored by ellipticity changes in the far-UV or the Soret region of CD spectra (Table S1). A dependence of ΔG° values on the position of the anions in Hofmeister series shows a monotone increasing trend from chaotropic to kosmotropic anions (Fig. 2A) in accordance with generally accepted effect of Hofmeister anions on protein stability [START_REF] Hippel | Ion effects on the solution structure of biological macromolecules[END_REF].

To To examine our hypothesis that modulated stability of heme region, particularly the Met80-heme iron bond, is reflected in kinetics of cyanide binding to cyt c, we studied effect of Hofmeister anions on association rate of interaction between cyanide and the protein. In fact, the association rate constant of cyanide binding with cyt c strongly depends both on a position of anion in Hofmeister series (Fig. 3) and anion concentration (Fig. 3B, Table S3). The progress of the reaction of cyanide binding into the protein was fitted with monophasic exponentials at all

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studied conditions (Fig. 3A). Observed pseudo first-order rate constants, k obs , obtained from these fits, were plotted vs concentration of added HCN. From the linear dependences (inset Fig. 3A) association, k a , and dissociation, k d , rate constants of cyanide binding were determined in accordance with the equation ( 4). Because the precision of k d values is negatively affected by an extrapolation to zero concentration of the ligand (Table S3), analysis of the k d values was omitted in the further work.

The plot of k a vs type of anion (Fig. 3B) shows the strong dependence of this parameter on the position of the anion in Hofmeister series: while neutral (from Hofmeister effect standpoint) anions has only minimal effect on the k a values, chaotropic and in less extent also kosmotropic anions led to an increase in the association rate constant of cyanide binding with cyt c. The effect of chaotropic anions on the association rate constant is even more pronounced at 2 M concentration when the rate constant in the case of KSCN increases ~ 40 times in comparison with that in the absence of salt (Table S3). The effect of kosmotropic anions on the association rate was not possible to analyze at such high salt concentrations due to their limited solubility at given conditions. It seems that penetration of cyanide through the protein matrix and/or cyanide binding to heme iron are alleviated in the presence of chaotropic and kosmotropic anions in comparison with the neutral anions. This is demonstrated as a minimum of k a value in the presence of chloride anions in the plot shown in Figure 3B.

Unexpected enhancement of the observed rate of cyanide binding in both kosmotropic (phosphate) and chaotropic (thiocyanate) solutions relative to the plain buffer and neutral Hofmeister salt (sodium chloride) prompted us to investigate the stability of axial coordination of cyt c under these conditions. Near-infrared magnetic circular dichrosim (MCD) spectroscopy is one of the tools for an identification of the

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ACCEPTED MANUSCRIPT 10 axial ligands in the low spin heme proteins [START_REF] Gadsby | Assignment of the axial ligands of ferric ion in lowspin hemoproteins by near-infrared magnetic circular dichroism and electron paramagnetic resonance spectroscopy[END_REF][START_REF] Cheesman | Magnetic circular dichroism of hemoproteins[END_REF]. As Figure 4 illustrates MCD spectra of cyt c in the buffer and in the presence of 1M chloride are identical. On the other hand, the presence of either 1 M phosphate or 1M thiocyanate results in the decrease of the band intensity at ~1730 nm. This decrease indicates that the native axial coordination of His-Fe-Met in cyt c is partially disrupted by both anions.

Discussion

B Factors affecting cyanide binding to hemoproteins

The major determinants that govern the affinity of the cyanide for hemoproteins such as metmyoglobins and methemoglobins that have heme iron connected with bulk solvent through channel are: (i) the ease of displacement of the sixth ligands (in the cases of these proteins: water molecule), (ii) the acid dissociation constant of HCN inside the protein, and (iii) steric hindrance and electrostatic interactions at the sixth coordination position [START_REF] Brancaccio | Structural factors governing azide and cyanide binding to mammalian metmyoglobins[END_REF][START_REF] Dou | Mechanism of hydrogen cyanide binding to myoglobin[END_REF]. The observed association rate constant k a is proportional to the major parameters that can be expressed by the simplified equation:

k a ~ k diff Q CN Q 5c,
where k diff is the rate of diffusion of HCN through the protein matrix, Q CN is the equilibrium distribution of the protein molecules that have a cyanide anion in the heme pocket poised to bind to the heme iron once the sixth ligand dissociates, and Q 5c is the equilibrium distribution of pentacoordinate heme.

In the hemoproteins such as cyt c, where there is no apparent channel connecting bulk solvent with heme iron, there might be important one more determinant: a barrier caused by protein environment surrounding the heme. However, Lakowicz and Weber showed that small molecules such as oxygen and even soft iodide anion diffusion rate through proteins, k diff , is very fast, diminished only by two

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11 orders of magnitude in comparison with diffusion rate in water [START_REF] Lakowicz | Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale[END_REF]. On the other hand, example supporting an existence of the barrier induced by protein environment is ferricytochrome c' from Chromatium vinosum. In this case, kinetics of cyanide binding to the heme iron is despite the fact that it contains penta-coordinated heme, i.e. Q 5c ~1, 10 2 slower than to horse heart cyt c (hexa-coordinated heme) and more than 10 5 slower than that to metmyoglobin and methemoglobin [START_REF] Motie | Kinetics of cyanide binding to Chromatium vinosum ferricytochrome c[END_REF]. Assuming similar value of pK a constant of HCN in heme region on cyanide binding in the proteins, the association rate has to be affected mainly by k diff . Taking into account results of Lakowicz and Weber, the value of k diff is proportional to accessibility of heme iron. This is arguably modulated by flexibility of the crowded heme region of ferricytochrome c' from Ch. vinosum or stability/flexibility of heme region, particularly of bond between heme iron and the sixth ligand in the case of horse heart cyt c.

B Effect of (Hofmeister) salts on properties of cyt c

Advantage of the modification of solvent properties by Hofmeister salts is that solvent modification affects only stability/flexibility of the sixth ligand without an effect on the other determinants of the affinity of cyanide for heme. The unaffected spectral properties of cyt c even at high concentrations of Hofmeister anions imply an absence of any major conformational change (Fig. 1). Structural studies performed by NMR [START_REF] Feng | Mechanism of hydrogen cyanide binding to myoglobin[END_REF][START_REF] Moench | Proton-NMR studies of the effects of ionic strength and pH on the hyperfine-shifted resonances and phenylalanine-82 environment of three species of mitochondrial ferricytochrome c[END_REF], X-ray crystallography [START_REF] Sanishvili | The low ionic strength crystal structure of horse cytochrome c at 2.1 Å resolution and comparison with its high ionic strength counterpart[END_REF], and ultraviolet resonance Raman spectroscopy [START_REF] Liu | Ionic strength dependence of cytochrome c structure and Trp-59 H/D exchange from ultraviolet resonance Raman spectroscopy[END_REF] also indicate only minor and locally distributed changes on Met80 side of the heme in the structure of cyt c due to high ionic strength. On the other hand, small-angle X-ray scattering measurements indicate decrease of the radius of gyration of cyt c by ~8% due to neutralization of high positive charge of cyt c surface by high ionic strength of solvent [START_REF] Trewhella | Differences in the solution structures of oxidized and reduced cytochrome c measured by small-angle X-ray scattering[END_REF]. One might expect that the neutralization of the

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repulsive positive charges in the protein structure will stabilize the structure either locally and/or globally. However, the effect of high ionic strength on cyt c at neutral pH region strongly depends on the nature of ions in accordance with the Hofmeister effect. While it is generally accepted that chaotropic, soft anions efficiently screen charge interactions in proteins due to their tendency to bind to hydrophobic patches on protein surface [START_REF] Perez-Jimenez | The efficiency of different salts to screen charge interactions in proteins: a Hofmeister effect?[END_REF][START_REF] Lund | Specific ion binding to nonpolar surface patches of proteins[END_REF] and to induce a salt in effect on the peptide group [START_REF] Baldwin | How Hofmeister ion interactions affect protein stability[END_REF], the situation is more complex for kosmotropic anions. Kosmotropic anions are strongly hydrated and therefore, considered to be excluded from protein surface [START_REF] Arakawa | Preferential interactions of proteins with salts in concentrated solutions[END_REF][START_REF] Timasheff | Control of protein stability and reactions by weakly interacting cosolvents: the simplicity of the complicated[END_REF][START_REF] Moelbert | Kosmotropes and chaotropes: modelling preferential exclusion, binding and aggregate stability[END_REF].

Moreover, according Colins "law of matching water affinities" kosmotropic anions should have low affinities for chaotropic ammonium groups on protein surface [START_REF] Collins | Charge density-dependent strength of hydration and biological structure[END_REF][START_REF] Collins | Ions in water: characterizing the forces that control chemical processes and biological structure[END_REF]. This hypothesis was supported experimentally by investigation of ion effects on interfacial water structure next to macromolecules with vibrational sum frequency spectroscopy [START_REF] Chen | Differences in the solution structures of oxidized and reduced cytochrome c measured by small-angle X-ray scattering[END_REF]. In accordance, it was shown that Hofmeister effect of anions on stability of proteins is nearly independent on charge of protein surface implicating an absence of specific electrostatic interactions [START_REF] Perez-Jimenez | The efficiency of different salts to screen charge interactions in proteins: a Hofmeister effect?[END_REF]. On the other hand, it was experimentally shown the stabilization effect of kosmotropic anions through their specific interactions with protein surface [START_REF] Ramos | Sulfate anion stabilization of native ribonuclease A both by anion binding and by the Hofmeister effect[END_REF][START_REF] Makhatadze | Anion binding to the ubiquitin molecule[END_REF][START_REF] Waldron | The salt-dependence of a protein-ligand interaction: ion-protein binding energetics[END_REF], and recent molecular dynamic simulations show tendency of sulfate anions to be strongly attached to the proteins and generally, polycationic surfaces [START_REF] Vrbka | Specific ion effects at protein surfaces: a molecular dynamics study of bovine pancreatic trypsin inhibitor and horseradish peroxidase in selected salt solutions[END_REF][START_REF] Mason | Specificity of ion-protein interactions: complementary and competitive effects of tetrapropylammonium, guanidinium, sulfate, and chloride ions[END_REF][START_REF] Vlachy | Hofmeister series and specific interactions of charged headgroups with aqueous ions[END_REF]. Cyt c contains high affinity binding site for sulfate and phosphate anions localized at the heme crevice [START_REF] Osheroff | Mapping of anion binding sites on cytochrome c by differential chemical modification of lysine residues[END_REF]. Therefore, it is likely that the effect of kosmotropic anions on cyt c might be combined effect of specific (high affinity site) and nonspecific (Hofmeister effect) interactions.

Chaotropic and kosmotropic anions modulate stability/flexibility of heme region of cyt c.

In accordance with our findings in this work, a plausible explanation for an show that axial coordination of His-Fe-Met is partially disrupted not only by chaotropic thiocyanate but also by phosphate anions. Thus it appears that despite kosmotropic effect of phosphate anions on global stability of protein at the same time these ions impose a local destabilization of the iron axial coordination either through nonspecific Hofmeister effect or through specific interaction with high affinity site at the heme crevice [START_REF] Osheroff | Mapping of anion binding sites on cytochrome c by differential chemical modification of lysine residues[END_REF][START_REF] Osheroff | Electrostatic interactions in cytochrome c. The role of interactions between residues 13 and 90 and residues 79 and 47 in stabilizing the heme crevice structure[END_REF]. To reconcile observed increased global and local stabilities with MCD spectrum in the presence of phosphate anions, we suggest that the decreased MCD band is an expression of an increased flexibility in the heme region.

The MCD spectrum is then average spectrum of several substates of heme region of cyt c in solutions containing phosphate anions.

In fact, our MCD data are consistent with the results of the work of Shah and Sweitzer-Stenner [START_REF] Shah | Structural changes of horse heart ferricytochrome c induced by changes of ionic strength and anion binding[END_REF]. The authors studied structural changes of horse heart cyt c induced by ionic strength and anion binding. They concluded that binding of phosphate anion (kosmotrope) to cyt c stabilizes the protein, however, at the same time, increases heterogeneity of the 695-nm band that indicates a higher flexibility of the protein segment that contains Met80 as a result of entropic stabilization [START_REF] Dumortier | Protein dynamics in the region of the sixth ligand methionine revealed by studies of imidazole binding to Rhodobacter capsulatus cytochrome c 2 hinge mutants[END_REF]. In fact, it was shown that charge-transfer band at 695 nm in ferricytochrome c spectrum is composed of at least three bands, assignable to different substates [START_REF] Dragomir | Optical band splitting and electronic perturbations of the heme chromophore in cytochrome c at room temperature probed by visible electronic circular dichroism spectroscopy[END_REF] with different thermal stabilities [START_REF] Schweitzer-Stenner | Conformational substates of horse heart cytochrome c exhibit different thermal unfolding of the heme cavity[END_REF]. It should be pointed out that iso/thermal denaturation experiments showed that both local and global stabilities of cyt c increased in the presence of kosmotropic anions. On the other hand, only cyanide binding "feels" increased flexibility of Met80-heme iron linkage in the presence of kosmotropic anions in accordance with recent findings of Shah and Sweitzer-Stenner

A C C E P T E D M A N U S C R I P T [START_REF] Shah | Structural changes of horse heart ferricytochrome c induced by changes of ionic strength and anion binding[END_REF]. The increased flexibility is therefore paradoxical consequence of a strengthening of Met80-heme iron bond that is in all substates likely accompanied by changed equilibrium distribution between penta-and hexa-coordinated hemes in favor of the former state as is indicated by MCD spectra. On the other hand, the effect of chaotropic anions on the heme region of cyt c can be looked at as solvent-protein interaction induced dynamic rearrangement which weakens the strength of methionine sixth axial ligation and permits its replacement by exogenous strong field ligands.

3 B Physiological implications of limited flexibility of cyt c.

Apparently, anions can modulate stability/flexibility of heme region of cyt c with a consequence of different accessibility of heme iron from the bulk solvent with likely of functional significance [START_REF] Shah | Structural changes of horse heart ferricytochrome c induced by changes of ionic strength and anion binding[END_REF]. The minimum value of k a , i.e. the smallest accessibility of heme region to solvent, at low (physiological) ionic strength in vitro might therefore have physiological role because an exposure of heme as well as modified mobility in heme region affect both redox properties [START_REF] Kassner | Effects of nonpolar environments on the redox potentials of heme complexes[END_REF][START_REF] Michel | Heme attachment motif mobility tunes cytochrome c redox potential[END_REF] and stability [START_REF] Wen | Effects of heme pocket structure and mobility on cytochrome c stability[END_REF] of cyt c. In fact, the dynamics of dissociation/association of Met80-heme iron bond (~30 s -1 ) appears to be conserved throughout evolution in class I of c-type cytochromes [START_REF] Cheng | Local stability of Rhodobacter capsulatus cytochrome c 2 probed by solution phase hydrogen/deuterium exchange and mass spectrometry[END_REF]. Interestingly, it was shown in bacterial cyt c that dynamics of the hinge region (in horse cyt c includes residues 70-85), responsible for dissociation/association of Met80-heme iron, facilitate its dissociation from its physiological partner suggesting thus a role of this movement in turnover under physiological conditions [START_REF] Devanathan | Binding of oxidized and reduced cytochrome c 2 to photosynthetic reaction centers: plasmon-waveguide resonance spectroscopy[END_REF].

Conclusions.

In conclusion, the unusual dependence of cyanide association rate constant on the position of anions in Hofmeister series demonstrates sensitivity of cyanide

  explored the Hofmeister effect of anions (0.5-2.0 M range) on local and global stability of cyt c examined by iso/thermal denaturation experiments and specifically on the Met80-heme iron bond examined by kinetics of cyanide binding to cyt c. Our findings show that cyanide binding to cyt c is useful probe for monitoring local stability/flexibility of heme region of cyt c. In the present work, we use the term flexibility in relation of cyanide binding to the protein as our results do not address fluctuations of polypeptide structure in specific timeframe.

  investigate a local stability of cyt c on Hofmeister anions, we determined thermal stability of the heme region monitored by absorbance of cyt c in the Soret region. The dependence of transition temperatures on the position of the anions in Hofmeister series is shown in Figure 2B. The thermal denaturation of cyt c was also determined by monitoring an ellipticity change in the far-UV region of CD spectra. This provided us with information about stability of the secondary structure of protein reflecting the global stability of cyt c (Fig. 2B). Both local (absorbance in the Soret region) and global (ellipticity in the far-UV region) stabilities of cyt c, expressed by transition temperatures, increase in a monotone way from chaotropic to kosmotropic anions. Resemblance of thermal transition temperatures measured by absorbance and ellipticity points to two-state character of the thermal transition of cyt c at given conditions.

  shaped dependence of k a vs anions of Hofmeister series, particularly the increase of k a values in the presence of kosmotropic anions provides MCD data. Data

Figure 1 .

 1 Figure legends:

Figure 2 .

 2 Figure 2. A: Dependence of stability of cyt c expressed in free Gibbs energy on the

Figure 3 .Figure 4 .

 34 Figure 3. A: Time dependence of the absorbance change monitored at 418 nm dur-

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Acknowledgement

The authors thank the Slovak Grant Agency for support through grants 1/4319/07 and 1/3252/06 and the P.J. Šafárik University VVGS Grant No 29/07-08.