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Oct 14, 2016 - Department of Chemistry, Texas A&M University Commerce, Commerce, ... Cu(I) or Cu(II) ions, the deprotonation sites, and likely Cu(I/II...
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Applying Ion Mobility−Mass Spectrometry Techniques for Explicitly Identifying the Products of Cu(II) Reactions of 2His-2Cys Motif Peptides Yashodharani Vytla and Laurence A. Angel* Department of Chemistry, Texas A&M UniversityCommerce, Commerce, Texas 75428, United States S Supporting Information *

ABSTRACT: The Cu(II) and pH titrations of four structurally similar 2His-2Cys motif peptides were investigated by electrospray ionization− ion mobility−mass spectrometry. The results provided insight into the pH dependent redox processes that took place in solution and identified the number of inter- or intramolecular disulfide bridges, the number of Cu(I) or Cu(II) ions, the deprotonation sites, and likely Cu(I/II) coordination of the various products. Competitive Cu(II) titrations of binary peptide mixtures at pH 5 indicated which species would preferably bind Cu(I) ions over forming the intramolecular disulfide bridge. Moreover, these reactions were pH dependent and included the formation of various multimers and multiple Cu(I/II) binding. For example, for the mildly acidic solution (pH ∼ 3−6) each monomer (whether it was free or in a multimer) primarily bound up to 3 Cu(I) ions, whereas at pH ∼ 8−11 the fully oxidized monomer or multimer (where all Cys formed a disulfide bond) primarily bound up to 2 Cu(II) ions. This behavior was indicative of linear bridging of Cu(I) by Cys thiolate and His imidazole groups, whereas the coordination of Cu(II) involved His and the nitrogens of deprotonated backbone amide groups, resulting in either distorted T-shaped or square planar geometries.

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only just starting to be recognized and understood. Disruption in copper metabolism or homeostasis is directly implicated in Menkes and Wilson’s disease9 as well as in cancers10 and neurological diseases such as Alzheimer’s11 and prion disease.12 Our previous work on two alternative metal binding (amb) peptides amb113 and amb214 (Figure 1) showed the formation of multiply Cu(I/II)-bound complexes, and the goal of this research is to investigate their formation further by studying how the amb structure and pH influence their formation. Here we report on the redox and Cu(I/II)-binding activity resulting from Cu(II) and pH titrations of four amb peptides which all contain the 2His-2Cys motif. These results elucidate the fundamental Cu(I/II)-binding and redox-activity mechanisms exhibited by copper-binding peptides containing the 2His-2Cys motif. For this study, four amb peptides were designed (Figure 1) which in addition to the 2His-2Cys binding motif contain a proline residue which acts as a hinge for the N- and C-terminal arms to locate around the Cu(I/II) ions,14 and an acetylated Nterminal to block its Cu(I/II)-binding capacity. The Tyr residue which can stabilize Cu(II) coordination via a second solvation shell π−cation interaction15 was also included in the sequences of amb2, amb3, and amb4.

he redox activity of copper ions is indispensable for a wide variety of life for regulating energy production in chloroplasts1 and mitochondria.2 The redox potential of the Cu(I/II) couple varies depending on the type of ligands and the coordination geometry.3 Common ligands include Cys, His, and Met with coordination geometries of linear, trigonal planar, square planar, and tetrahedral.4 However, copper ions can also react with oxygen derivatives to produce reactive oxygen species that can produce oxidative stress. Glutathione is a redox active tripeptide with a sequence of γ-Glu-Cys-Gly where the γlinkage is between the carboxyl side chain of Glu and the amine of Cys. Glutathione acts as an antioxidant in a wide range of organisms5 and will also bind with Cu(I) ions.6 The activity of glutathione is due to the Cys thiol group which can form a disulfide bridge with another glutathione to form a dimer and in the process deliver two reducing equivalents. A recent investigation7 of the mechanisms and rate of Cys disulfide formation (oxidation) in the presence of Cu(II) showed that the overall oxidation process consists of several steps with the early stages dominated by the formation of Cu(I) complexes and competition from disulfide bond formation. Copper trafficking proteins are also important for avoiding the detrimental effects of free copper ions and copper chaperones safely transport copper to locations in the organism where copper ions are needed as cofactors for metalloenzymes.3 The role copper chaperones have in the complex nature of copper regulation and metabolism is an active area of research8 and is © XXXX American Chemical Society

Received: June 14, 2016 Accepted: October 14, 2016

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Figure 1. Schematic structures of the alternative metal binding (amb) peptides, representing the neutral charged state of each molecule (a) acetylHis1-Cys2-Gly3-Pro4-His5-Cys6 (MW = 694.81 Da, amb1), (b) acetyl-His1-Cys2-Tyr3-Pro4-His5-Cys6 (MW = 800.93 Da, amb2), (c) acetyl-His1-Cys2Gly3-Tyr4-Pro5-His6-Cys7 (MW = 857.99 Da, amb3), and (d) acetyl-His1-Cys2-Gly3-Ser4-Pro5-His6-Cys7-Ser8 (MW = 1032.15 Da, amb4). The shading shows the substituent groups of histidine and cysteine (shaded in brown), which are potential binding sites for Cu(I/II); the proline (shaded in blue), which acts as the hinge of the C- and N-terminal arms of the peptide; the acetyl group (shaded in green) used to block metal binding ability of the N-terminal; and the substituent group of tyrosine (shaded in yellow), which can stabilize Cu(I/II) coordination via a second solvation shell π− cation interaction.

contain the Tyr phenol group (pKa ∼ 11.0).20 These six protic groups result in the metal-free amb peptides predominantly forming positively charged cations below pH 6 and negatively charged anions above pH 6. By using positive ion analyses to monitor the reactions resulting from the competitive Cu(II) titration (pH 5) and positive and negative ion analyses for monitoring the pH titration, the individual products produced by the redox activity, oligomerization, and Cu(I/II) binding can all be determined.

Ion mobility−mass spectrometry (IM-MS) is developing into a powerful technique for studying protein and peptide complexes resulting from redox and Cu(I/II)-binding reactions.13,14,16,17 The strength of IM-MS is that it simultaneously measures the mass-to-charge ratios (m/z) and arrival times that relate to the identity, interactions, and conformation of the peptide complex. Our IM-MS technique utilizes electrospray ionization (ESI) which is a method capable of transferring a peptide from the solution phase to the gas phase while conserving its mass, charge state, binding interactions, and conformational structure.18 Precisely how much solution-phase information is retained in the gas phase is an active area of research.19 Two important parameters that need to be addressed are sample preparation and instrumental conditions. In our research our peptides are prepared with only aqueous solvent, CuCl2, and either dilute acetic acid or dilute ammonium hydroxide to regulate the pH. The overall charge of the peptide and its complexes is an important criterion for mass spectrometry analyses and must be considered for unbiased analyses. For instance moderate voltages (20−30 V) must be used on the sampling cone of the ESI source to eliminate dissociation of the 2+ and 3+ cations while still effectively sampling the 1+ cations into the instrument. A similar approach must be used on selecting the appropriate injection voltages into the IM traveling wave cell. The overall charge of the complex is determined by the number and charge of the copper ions in the complex and the protonation state of the acidic and basic sites of the peptide. The four amb peptides studied here all contain the carboxyl group of the C-terminus (pKa ∼ 3.0), two His imidazoles (pKa ∼ 6.0), and two Cys thiols (pKa ∼ 8.4), and three of them also

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EXPERIMENTAL SECTION Reagents and Stock Solutions. The four amb peptides (Figure 1) were purchased from Neo BioLab (https:// neobiolab.com/). All stock solutions were prepared with Milli-Q-deionized water (>17.8 MΩ cm, Milli-Q, Millipore (http://www.millipore.com/)) and CuCl2 (99.9% purity) purchased from Sigma-Aldrich (http://www.sigmaaldrich. com/). Ammonium hydroxide (trace metal grade) and glacial acetic acid (Optima grade) were purchased from Fisher Scientific (http://www.Fishersci.com) to adjust the pH for the pH titration studies. Cu(II) Titrations of ambs. The competitive Cu(II) titrations of binary amb mixtures were prepared using only >17.8 MΩ cm water with final concentrations of 25.0 μM of each amb and adding 0.25, 0.50, 1.0, and 2.0 mol equiv of CuCl2 as expressed for the total amb concentration. Preliminary experiments showed that the same results were exhibited whether using anaerobic or aerobic conditions, so the experiments were carried out in aerobic conditions. The pH of these solutions started at pH 5.0 and dropped to about pH B

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Synapt G1 and G2 (which yields improved mobility resolution relative to the G1) with comparison to absolute Ω He measurements made using a RF confining drift tube concluded accurate ΩHe measurements could be made using both G1 and G2 instruments. The method provided ΩHe for the amb4 species with estimated 2% relative uncertainties and 4% absolute errors based on previous analyses of the calibration procedure with the Synapt instrument.22 Details of the procedure are given in the Supporting Information. Computational Methods. The B3LYP/LanL2DZ level of theory containing the Becke three parameter hybrid functionals26 with the Dunning basis set27 and electron core potentials28−30 from Gaussian 0931 was used to locate the lowest energy, geometry optimized, gas-phase structures of the [amb4 + 3Cu(I)]+ and [amb4(ox) + 2Cu(II)]2− complexes. Their theoretical ΩHe were calculated using the ion size scaled Lennard-Jones (LJ) method from the Sigma program which was developed and written by the Bowers group from the Department of Chemistry and Biochemistry at the University of CaliforniaSanta Barbara.32 The LJ values are derived from the mean and standard deviation from 10 repeated calculations.

4.5 after the addition of 2.0 mol equiv of CuCl2. Our previous work on amb214 showed the multiply Cu(I)-bound complexes decreased over time, so in this study a short 5 min incubation was used. All combinations of the four ambs were tried, but only the results that were reproduced by three replicate titrations are shown. Equimolar Cu(II):amb pH Titrations. The metal-binding pH titrations were conducted using solutions prepared by adding 20.0 μL of 1.25 mM amb stock solution to 100.0 μL of deionized water with preadjusted pH using diluted glacial acetic acid or diluted ammonium hydroxide. To this solution was added 20.0 μL of 1.25 mM stock solutions of CuCl2. The solution was mixed and incubated at room temperature for 5 min followed by further dilution to the final concentration of 25.0 μM amb with the acetic acid or ammonium hydroxide pH adjusted deionized water. Samples were then incubated for an additional 5 min, and the final pH was recorded using a micro pH electrode (Orion EA940, Thermo Fisher Scientific Inc.). A pH study was also undertaken where all the pH adjusted equimolar Cu(II):amb samples were made in an N 2 atmosphere to check whether O2 was contributing to the redox activity. The anaerobic results reproduced the pH titration made in air and confirmed Cu(II) was the oxidizing agent of the amb4 peptide. Waters Synapt High-Definition Mass Spectrometer. The samples were analyzed using the Waters Synapt highdefinition mass spectrometer (G1) equipped with an ESI source and a quadrupole−ion mobility−orthogonal time-offlight configuration 21 as described in the Supporting Information. Prior to sample preparation, the entrance tubing and needle capillary of the ESI were cleaned with 1.0 mM aqueous solution of Cu-free methanobactin from Methylosinus trichosporium-OB3b to remove any trace amounts of Cu(II). Three trials of the pH titration were conducted using different settings in the source and interaction region to test the effect of these on the reproducibility of the results (Table S1). All three trials produced the same product channels but showed the relative intensities of the singly and multiply charged ions of the same molecular species changed due to the relative efficiencies of focusing these ions through the source and IM region. Analyses of the Cu(II) and pH Titrations. The sequential IM-MS analyses allow for effective separation and identification of all the various amb species including coincidental m/z monomers and their various oligomers, i.e., [amb4 + 3Cu(I)]+, [diamb4 + 6Cu(I)]2+, [triamb4 + 9Cu(I)]3+ and [tetraamb4 + 12Cu(I)]4+ (Supporting Information Figures S1 and S2). For both positive and negative ion analyses, the arrival-time distribution (ATD) for each of the species was extracted and the integrated area used as the measure of its population (Figures S2 and S3). To calculate the percent relative intensities, the summation of the integrated ATD for all extracted species at the titration point was used to normalize to the percent scale. Some of the species had unresolved ATDs, and their population was determined by reproducing the isotope pattern associated with the ATD with an overlapped isotope pattern made from percent contributions of the two or three unresolved species as shown in Figures S4 and S5. Collision Cross-Sections. A calibration method was used to determine the collision cross-sections of the various amb4 species using previously published collision cross-sections in helium (ΩHe) for positive and negative poly-DL-alanine ions measured in a radio frequency (RF) confining drift cell.22−24 A study25 of ΩHe measurements of human insulin using the

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RESULTS AND DISCUSSION Competitive Cu(II) Titrations of ambs: Redox Activity and Cu(I) Binding. Our previous research13,14 on amb1 and amb2 (Figure 1a,b) showed that Cu(II) reactions produced positively charged Cu(I) complexes below pH 6, and both positively and negatively charged Cu(II) complexes of oxidized amb(ox) above pH 6. The study of the Cu(II) titration of amb2 showed the oxidation of amb2 was via the formation of intraand intermolecular Cys-Cys disulfide bridges and the multiple Cu(I) coordination complexes were by unoxidized amb2 or the partially oxidized dimer and trimer of amb2. The major product of these reactions was [amb2 + 3Cu(I)]+ which coordinated three Cu(I) ions via two bridging thiolate groups of Cys2 and Cys6 and the δN of the imidazole groups of His6 and His1 (or the O of the C-terminus) as supported by geometry optimized structures at the B3LYP/LanL2DZ level of theory and their theoretical ΩHe calculated by the LJ method.14 Here we report the results of the competitive Cu(II) titrations of a mixture of two of the amb peptides (Figure 1) and compare their individual Cu(I)-binding and oxidation reactions. The results indicate which amb preferentially binds Cu(I) ions or which amb prefers to form an intramolecular disulfide bridge. The free Cys thiolate and His imidazole groups are the ligands for Cu(I) ions, and the formation of a disulfide bridge limits the binding ability. Panels a and b of Figure 2 show the results of the competitive Cu(II) titration of a binary mixture of amb1 and amb2. The comparison shows amb1 formed an intramolecular disulfide bridge to a greater extent (36−41%) than amb2 (23−28%), over the range of 0.5−1.5 Cu(II) molar equivalents (Meq). This indicates that Tyr3 of amb2, which replaced Gly3 of amb1, has either imparted a steric hindrance toward the formation of the Cys2-Cys6 disulfide bridge or has formed a hydrogen bond to either the free Cys or C-terminus, making amb2 less reactive to oxidation. Another possibility is the Tyr deprotonated33 and formed a salt bridge to one of the positively charged histidine side groups, resulting in a conformational change. Based on the pKas of 3.0, 8.4, and 11.0 for the C-terminus, Cys and Tyr, respectively,20 the hydrogen bond to the deprotonated C-terminus is more favorable at pH 5 and would produce a single positively charged ion [amb4]+ as observed if both His imidazolium groups C

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dimers and trimers than the other amb peptides; product channels that are not included in Figure 2. Overall, these results indicated that amb1 and amb3 were more conducive to forming intramolecular disulfide bridges, whereas amb2 and amb4 favored coordinating 3 Cu(I) ions. However, the analyses did not include the multiply charged and multimeric products that amb peptides can also produce. Previously, we reported on the Cu(II) and pH titration of amb2 which produced the dimer complex with 1−4 Cu(I) ions and the trimer complex with 1 or 3 Cu(I) ions.14 The Cu(II) and pH titration of amb4 exhibited a greater range of multiply charged and oligomerization products, and the results of the pH titration of an equimolar Cu(II):amb4 mixture will be discussed next. Cu(I/II) Binding of Monomers of amb4 Resulting in Positive Ions. The pH range of 3−11 was chosen because proton-induced chemistry, including electron-transfer reactions, can occur over an extended pH range because many small enzymes, e.g., lysozyme, have optimal activity in a range of biological fluids, e.g., tears, saliva, and blood.34 Figure 3 shows

Figure 2. Percent relative intensities of singly positive ion products from the competitive Cu(II) titration of a binary mixture of amb species. Results of the Cu(II) titration of 1:1 mol equiv of amb1:amb2 showing (a) the reaction products of amb1 and (b) the products of amb2. Results of the Cu(II) titration of amb2:amb3 showing (c) the reaction products of amb2 and (d) the products of amb3. Results of the Cu(II) titration of amb3:amb4 showing (e) the reaction products of amb3 and (f) the products of amb4. In all titrations the unoxidized ambbound Cu(I) ions apart from [amb + Cu(I)]+ which was oxidized amb(ox) at 1.00 and 1.50 Cu(II) molar equivalents.

remained protonated. A final possibility was Tyr formed a π− cation interaction with Cu(II) that lowered the redox activity of Cu(II) with the thiolate groups. The amb2 + Cu(II) complex, however, was not observed during the Cu(II) titration which undermines this possibility. The efficiency of coordinating 3 Cu(I) ions may also have been lowered by Tyr3 because during 0.5−1.0 Meq Cu(II), the formation of [amb2 + 3Cu(I)]+ was only 23−26%, whereas [amb1 + 3Cu(I)]+ was slightly higher at 26−29%, even though there was more disulf ide-f ree amb2 available for coordinating Cu(I) ions than amb1. However, at the point of 1.5 Meq Cu(II) the amb2 did bind 3 Cu(I) (33%) more efficiently than amb1 (28%) and the extent of binding 1 or 2 Cu(I) ions by amb2 (16−21%) was similar to that of amb1 (11−22%) indicating Tyr3 did not significantly affect the binding of 8.0. The tetraamb4 which contained no disulfide bridges could coordinate 12 Cu(I) ions via the 8 Cys and 8 His groups, and [tetraamb4 + 12Cu(I)]4+ existed over the extended range of pH 3−10. Cross-Sections and Coordination Conformers of Positively Charged amb4 Species. The collision crosssections (ΩHe) of the major amb4 species are shown in Table 1. Comparison of the positively charged Cu(I)-bound and their analogous Cu(II)-bound species showed they were of similar size (e.g., row 2 [amb4 + Cu(I)]+ and [amb4(ox) + Cu(II)]+, 230 and 228 Å2, respectively) and the small differences were within the 2% relative uncertainty of the ΩHe measurements. The diamb4 with 6 Cu(I) ions, [diamb4 + 6Cu(I)]2+, also exhibited the same ΩHe as the fully oxidized diamb(ox) with 4

Figure 4. Changing relative intensities of identities of the Cu(I/II) complexes of the monomer, dimer, and trimer of amb4 over the pH range of 3−11: (a) monomer with one Cu(I/II) ion, (b) dimer with two Cu(I/II) ions, and (c) trimer with three Cu(I/II) ions. The notations indicate how many disulfide bonds were present in the complex.

species was [amb4 + 2Cu(I)]+ below pH 5.5, which changed to [amb4(ox) + 2Cu(I)]+ between pH 5.5 and pH 8.0, and finally to [amb4(ox) + 2Cu(II)]+ above pH 8. However, the singly and doubly charged complexes with 3 Cu(I) ions solely exhibited the isotope patterns for [amb4 + 3Cu(I)]+ over the range of pH 3−9.6 and [amb4 + 3Cu(I)]2+ over the range of pH 3−6.5, indicating the 3 Cu(I) ions restricted the formation of the disulfide bridge in amb4(ox), and the disulfide bridge also prevented the coordination of the third Cu(I/II) ion. Our group previously determined that amb2 coordinated 3 Cu(I) ions via linear bridging by the thiolate groups of Cys2 and Cys8 and the imidazole nitrogens of His1 (or the C-terminus) and His7.14 This linear bridging can also explain the amb4-binding behavior and will be discussed again later. Formation of Positively Charged amb4 Multimers. Dimers, trimers, and tetramers of amb4 were also observed and their complexes coordinated with Cu(I) or Cu(II) ions in a pH trend similar to that observed for the amb4 monomer. Figure 5 shows the general pH behavior by comparing the summed populations and general identities of the monomers, dimers, trimers, or tetramers over the titration. For example, diamb4 coordinated 2−6 Cu(I) ions over the range of pH 3−6 and the f ully oxidized diamb(ox), which contained 2 disulfide bridges, coordinated 1−4 Cu(I/II) ions between pH 6 and pH 11 (see E

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Table 1. Collision Cross-Sections As Measured in He (ΩHe)1 of the Major Species Detected from the Ion Mobility−Mass Spectrometric Analysis of 1:1 mol equiv Solutions of amb4:CuCl2 at Various pH positive ionsa [amb4(ox)]+ [amb4 + Cu(I)]+ [amb4 + 2Cu(I)]+ [amb4 + 3Cu(I)]+ [amb4(ox)]2+ [amb4 + Cu(I)]2+ [amb4 + 2Cu(I)]2+ [amb4 + 3Cu(I)]2+ [diamb4(ox)]2+ [diamb4 + 2Cu(I)]2+ [diamb4 + 4Cu(I)]2+ [diamb4 + 6Cu(I)]2+ [triamb4(ox)]3+ [triamb4 + 3Cu(I)]3+ [triamb4 + 6Cu(I)]3+ [triamb4 + 9Cu(I)]3+ [tetraamb4 + 12Cu(I)]4+

ΩHe (Å2)b (no. of disulfide bondsc) 228 230 232 237 228 230 235 235 354 365 373 375 474 483 490 503 496

(1) (0) (0) (0) (1) (0) (0) (0) (2) (1) (0) (0) (3) (2) (0) (0) (0)

positive ionsd

ΩHe (Å2)b,c

negative ionse

ΩHe (Å2)b,c

[amb4(ox)]+ [amb4(ox) + Cu(II)]+ [amb4(ox) + 2Cu(II)]+

227 228 232

[amb4(ox)]− [amb4(ox) + Cu(II)]− [amb4(ox) + 2Cu(II)]−

214 219 220

[amb4(ox)]2+ [amb4(ox) + Cu(II)]2+ [amb4(ox) + 2Cu(II)]2+

228 230 230

[diamb4(ox)]2+ [diamb4(ox) + 2Cu(II)]2+ [diamb4(ox) + 4Cu(II)]2+

350 367 375

[amb4(ox)]2− [amb4(ox) + Cu(II)]2− [amb4(ox) + 2Cu(II)]2− [amb4(ox) + 3Cu(I)]2− [diamb4(ox)]2− [diamb4(ox) + 2Cu(II)]2− [diamb4(ox) + 4Cu(II)]2−

213 208 213 218 322 343 336

[triamb4(ox)]3+ [triamb4(ox) + 3Cu(II)]3+ [triamb4(ox) + 6Cu(II)]3+

474 487 500

[triamb4(ox)]3− [triamb4(ox) + 3Cu(II)]3− [triamb4(ox) + 6Cu(II)]3−

424 434 436

a Measured at pH 4.0. bΩHe have estimated relative errors of 2% for measurements made using the same calibration curve or 4% based on comparison of data from separate calibration curves.22,25 cShown in parentheses unless the species is designated with ox: i.e., amb4(ox) = 1 disulfide bond, diamb4(ox) = 2 disulfide bonds, and triamb4(ox) = 3 disulfide bonds. dMeasured at pH 10.6. e[amb4(ox)]− measured at pH 4.0, [diamb4(ox)]2−, [triamb4(ox)]3−, and [amb4(ox) + 3Cu(I)]2− measured at pH 9.3, and [amb4(ox)]2− and all Cu(II)-bound species measured at pH 10.5.

Figure 6. Lowest energy, geometry optimized complexes located using the B3LYP/LanL2DZ level of theory (hydrogens not shown). (a) [amb4 + 3Cu(I)]+ showing the linear bridging of 3Cu(I) ions via the δN of His1, S of Cys2, δN of His7, and S of Cys8. The complex has ΩHe = 256 ± 5 Å2 as calculated by the ion size scaled Lennard-Jones (LJ) method. (b) [amb4(ox) + 2Cu(II)]2− showing the square planar coordination of one Cu(II) via the δN of His1 and the backbone amide nitrogens of Cys2, Gly3, and Ser4 while the other Cu(II) ion is coordinated by the δN of His7, the backbone amide nitrogens of Cys8 and Ser9, and the C-terminal carboxylate oxygen. The complex has ΩHe = 243 ± 5 Å2 as calculated by the LJ method.

Cu(II) ions, [diamb4(ox) + 4Cu(II)]2+, indicating a similar coordination structure existed between these two complexes. Using the B3LYP/LanL2DZ level of theory we located six minima structures of amb4 binding 3 Cu(I) ions, and the lowest energy complex is shown in Figure 6a. The 3 Cu(I) ions are coordinated via linear bridging by the 2His-2Cys substituent groups, and the complex has ΩHe of 256 ± 5 Å2 as calculated by the LJ method32 which compares to the 237 ± 9 Å2 measured by IM-MS (Table 1). The Cu(I) linear bridging by Cys thiolate and His imidazole groups can be extrapolated to diamb4, which between pH 3 and pH 5.5 coordinates up to 6 Cu(I) ions, as illustrated in Figure 7a. This type of coordination also accounts for [diamb4 + 5Cu(I)]2+ which maintains the 2+ overall charge because the charge of Cu(I) is replaced by a proton on the former ligating His imidazole. Likewise the replacement of Cu(I) by a proton on all four His imidazoles will account for

the 4, 3, and 2 Cu(I) diamb4 complexes with the overall 2+ charge being conserved (Figure S8a). The 5 and 6 Cu(I) complexes do not convert to 5 and 6 Cu(I/II) diamb4(ox) complexes because the formation of two disulfide bonds prevents the bridged Cys-Cu(I)-Cys coordination. Therefore, during the pH range of 6−7.5, the oxidized dimer [diamb4(ox) + 4Cu(I)]2+ can only coordinate up to 4 Cu(I) ions via the bridging between the four imidazole groups of His and four sulfurs of the disulfide bonds (Figure 7b).35 The sulfurs of a disulfide group are softer ligands than the thiolate sulfur anions and would also explain the decrease in the formation of Cu(I) complexes as observed in Figure 5 over pH 6−7.5. At pH 8−11, the oxidized dimer binds 2−4 Cu(II) ions and the m/z of the species are consistent with Cu(II) coordination via His and the deprotonated nitrogen from the backbone amide group between the His and Cys (Figure 7c) as supported by our F

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Figure 7. Proposed coordination structures of (a) [diamb4 + 6Cu(I)]2+, (b) [diamb4(ox) + 4Cu(I)]2+, and (c) [diamb4(ox) + 4Cu(II)]2+.

resulted in negative ions with amb4(ox) observed as singly and doubly charged ions over pH 3−11(Figure S11) and Cu(I/II)bound amb4(ox) observed above pH 5.5 with the principle species being the [amb4(ox) + Cu(II)]−, [amb4(ox) + Cu(II)]2−, and [amb4(ox) + 2Cu(II)]2− complexes. The ΩHe of the negatively charged amb4 and Cu(II)-bound complexes (Table 1) are 4−13% smaller than their counterpart positively charged amb4 and Cu(I/II)-bound complexes. The difference between the singly negatively and singly positively charged amb 4 (ox) monomers could be accounted for by the deprotonation of the two imidazolium groups and a decrease in electrostatic charge repulsion between two neutral imidazole groups, with only a single negative charge on the C-terminus, resulting in a more compact negative ion complex. The m/z of the negatively charged Cu(II)-bound complexes suggests that each Cu(II) is coordinating via the His imidazole group and two or three sequential deprotonated nitrogens from the backbone amide groups. The B3LYP/LanL2DZ method located geometry optimized structures for [amb4(ox) + 2Cu(II)]2− and the lowest energy structure is shown in Figure 6b. In this complex one of Cu(II) ions is coordinated via the His1 imidazole group and the backbone amide nitrogens of Cys2, Gly3, and Ser4 in a square planar geometry and the other Cu(II) ion is coordinated via the imidazole of His7 and backbone amide nitrogens of Cys8 and Ser9 with the C-terminus completing a second square planar geometry. This dual square planar coordination involves seven residues of amb4(ox) solvating the 2 Cu(II) ions and is predicted by the LJ method to have ΩHe of 243 ± 5 Å2 which is about 5% smaller than the ΩHe for [amb4 + 3Cu(I)]+ (Figure 6a,b), which is consistent with the decrease in size of the negatively charged complexes as compared to the positively charged complexes as shown in Table 1.

previous work on amb1.13 This coordination could either be distorted trigonal planar which is a favorable Cu(II) coordination geometry36 or a distorted T-shaped geometry that was previously located as the lowest energy Cu(II) coordination by amb1 via the imidazole group of His1 and the deprotonated amide groups of Cys2 and Gly3.13 This type of coordination seems likely as Cu(II) has a preference for S or N ligands4 over O ligands such as Tyr and will preferably deprotonate and coordinate to amide nitrogens at a pH well below the pKa of the amide group.36 For the [diamb4(ox) + 4Cu(II)]2+ to cancel out 6 of the 8+ charges from the 4 Cu(II) ions, diamb4(ox) must have six negative charge sites and these can be accounted for by the two C-termini and four deprotonated backbone amide groups. Alternatively, binding via four doubly deprotonated His groups would also give the correct m/z, but this would suggest linear coordination and Cu(II) prefers higher coordination numbers.4,36 The same coordination trends can account for the trimer [triamb4 + 9Cu(I)]3+ linearly coordinating 9 Cu(I) ions via 6 Cys and 6 His side groups (Figure S10a), the fully oxidized trimer [triamb4(ox) + 6Cu(I)]3+ coordinating 6 Cu(I) ions via 6 His and 6 sulfurs of 3 disulfide bonds (Figure S10b), and [triamb4(ox) + 6Cu(II)]3+ coordinating 6 Cu(II) ions via 6 His, 6 sulfurs of disulfides, and 6 deprotonated nitrogens from amide groups between His and Cys (Figure S10c). A recent study of biologically relevant Cu(II) thiolate complexes reported a similar conversion from a Cu(II) complex to a Cu(I) disulfide complex driven by protonation.35 Cross-Sections and Coordination Conformers of Negatively Charged amb4 Species. The titration also

CONCLUSIONS ESI-IM-MS is an effective technique for determining the various molecular identities and relative populations of products that result from the solution-phase redox chemistry of Cu(II) and small 2His-2Cys motif peptides. For the competitive titrations various species containing Cu(I) ions or disulfide bridges were identified and quantified from chemically similar products, a task that no other analytical technique can achieve in such detail. The pH titration of Cu(II):amb4 showed that in moderately acidic solutions amb4 would bind 1−3 Cu(I) ions, whereas in moderately basic solutions the oxidized amb4(ox) coordinated 1−2 Cu(II) ions. The oligomers of amb4 exhibited a similar pH dependence for Cu(I/II) binding. For instance, the diamb4 (dimer with 0 or 1 disulfide bridge) coordinated 1−6 Cu(I) ions in moderately acidic solutions, while diamb4(ox) (2 disulfide bridges) coordinated 2−4 Cu(II) ions in moderately basic solutions. Likewise the trimer or tetramer coordinated a maximum of 9 Cu(I) or 12 Cu(I) ions at low pH, respectively, and in their fully oxidized form coordinated a maximum of 6 Cu(II) or 8 Cu(II) ions, respectively, at higher pH. The overall m/z states and ΩHe of amb4 complexes are consistent with the findings of our previous research on amb113 and amb214 and indicate Cu(I) coordination is via linear bridging by the Cys and His substituent groups and Cu(II) coordination is via His and the nitrogens of backbone amide groups, resulting in distorted Tshaped and square planar geometries. The results of this study showed the combined resolving power of ion mobility and time-of-flight mass spectrometry allowed for explicitly identifying this large set of reaction products. Moreover, IM-MS

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DOI: 10.1021/acs.analchem.6b02313 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

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validated the identity of the starting molecular state of the peptide ensuring no preliminary redox reactions had taken place prior to the start of the reaction study.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b02313. Additional results and instrumental and experimental details including tune settings used, collision crosssection calculations, mass spectra of positive ion products, IM-MS analyses of products, changing isotope patterns for Cu(I/II)-bound amb4, arrival-time distributions and percent relative intensities, and proposed coordination structures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank NSF for instrument support from Grant MRI0821247 and Welch Foundation Grant T-0014 for scholarship and financial support. We also thank Manogna Deeconda, Jacob W. McCabe, and Hind R. Alshehri for conducting preliminary experiments and Swetha Chintala and Rajpal Vangala for repeating the experiments in the nitrogen atmosphere.

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REFERENCES

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DOI: 10.1021/acs.analchem.6b02313 Anal. Chem. XXXX, XXX, XXX−XXX