pubs.acs.org/IC Article Histidine-Rich C‑Terminal Tail of Mycobacterial GroEL1 and Its Copper Complex�The Impact of Point Mutations Anna Rola,* Oscar Palacios, Merce Capdevila, Daniela Valensin, Elżbieta Gumienna-Kontecka, and Sławomir Potocki* Cite This: Inorg. Chem. 2023, 62, 6893−6908 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: The mycobacterial histidine-rich GroEL1 protein differs signifi- cantly compared to the well-known methionine/glycine-rich GroEL chaperonin. It was predicted that mycobacterial GroEL1 can play a significant role in the metal homeostasis of Mycobacteria but not, as its analogue, in protein folding. In this paper, we present the properties of the GroEL1 His-rich C-terminus as a ligand for Cu(II) ions. We studied the stoichiometry, stability, and spectroscopic features of copper complexes of the eight model peptides: L1�Ac-DHDHHHGHAH, L2� Ac-DKPAKAEDHDHHHGHAH, and six mutants of L2 in the pH range of 2−11. We revealed the impact of adjacent residues to the His-rich fragment on the complex stability: the presence of Lys and Asp residues significantly increases the stability of the system. The impact of His mutations was also examined: surprisingly, the exchange of each single His to the Gln residue did not disrupt the ability of the ligand to provide three binding sites for Cu(II) ions. Despite the most possible preference of the Cu(II) ion for the His9−His13 residues (Ac-DKPAKAEDHDHHH-) of the model peptide, especially the His11 residue, the study shows that there is not only one possible binding mode for Cu(II). The significance of this phenomenon is very important for the GroEL1 function�if the single mutation occurs naturally, the protein would be still able to interact with the metal ion. ■ INTRODUCTION Infections caused by Mycobacterium species remain a huge folding supported by the co-chaperonin GroES, in a manner dependent on ATP.8 The mycobacterial GroEL1 histidine-rich problem for the modern world with the rapidly growing protein exists only as a monomer, even in the presence of number of multidrug-resistant (MDR) bacteria.1−4 The WHO stabilizers of oligomeric forms.8,9 It has been discovered that report reveals that the infection caused by Mycobacterium Cu(II) induces a response of the mycobacterial GroEL1 (M. tuberculosis is the most difficult to cure.5 In 2020, an estimated tuberculosis) and its histidine-rich C-terminal region plays an 1.3 million patients died due to tuberculosis and approximately essential role in this process.10 The histidine-rich motif is 9.9 million people fell ill with the disease worldwide. M. responsible for metal ion trafficking in a variety of organisms, including bacteria.11−13 It was predicted that proteins tuberculosis survives in the host organism by penetrating the containing such specific sequences may be used as a molecular macrophages and manipulating their metal cation trafficking. target for a new generation of medicines.14 The GroEL1 The host organism defends itself from M. tuberculosis, e.g., by protein containing the HRCT tail cannot exert the same increasing copper and zinc concentrations in phagosomes.6 In biological function as well-studied, typical GroEL proteins order to develop a new method of treatment, it is necessary to containing the methionine- and glycine-rich C-terminus understand the mechanisms of metal homeostasis in M. (Figure 1).14 Therefore, a unique role played by the HRCT tuberculosis. More detailed studies on the role of important tail of mycobacterial GroEL1 in maintaining metal homeostasis mycobacterial virulence factors are required to achieve this is very probable. With the use of the UniProtBlast database, we goal. have identified a large number of metal-transporting/binding Chaperonins containing a histidine-rich C-terminal (HRCT) tail are potentially significant factors of maintaining metal homeostasis in Mycobacteria. In this paper, we investigate a Received: December 22, 2022 particular type of mycobacterial histidine-rich chaperonins� Published: April 24, 2023 GroEL1. The GroEL1 participates in metabolic and energetic adaptation under stress.7 The well-known Escherichia coli GroEL has the ability to oligomerize in two heptameric structures stacked back to back, in order to promote protein © 2023 The Authors. Published by American Chemical Society https://doi.org/10.1021/acs.inorgchem.2c04486 6893 Inorg. Chem. 2023, 62, 6893−6908 Inorganic Chemistry pubs.acs.org/IC Article Figure 1. C-terminal sequence comparison of mycobacterial GroELs with the E. coli homologue. It suggests the existence of two protein families. The first shares a glycine−rich domain with the E. coli GroEL. The second possesses a distinct histidine-rich sequence.14 proteins derived from different bacterial and fungal organisms, containing histidine-rich motifs, of which sequences are 100% ■ EXPERIMENTAL SECTION Materials. All peptide ligands were purchased from KareBay identical with the motifs occurring in different GroEL1 Biochem, Inc. (certified purity: L1 = 98.27%, L2 = 98.08%, L3 = proteins. The HDHHHGHAH motif from M. tuberculosis 98.52%, L4 = 99.12%, L5 = 98.96%, L6 = 98.19%, L7 = 98.32%, L8 = GroEL1 (UniProt Code: P9WPE9)15 is also present, e.g., in 98.12%). The identity of the peptides was evaluated based on mass copper-transporting ATPase protein (UniProt Code: Q2K000, spectrometry. The purity was checked based on potentiometric Rhizobium etli),16 metal tolerance protein 12 (UniProt Code: titrations using the Gran method.19 The solutions of metal ions were W9RBJ6, Morus notabilis),17 and nickel/cobalt efflux system prepared using Cu(ClO4)2·6H2O (Merck, HPLC grade) and filtered, using double-distilled water. The concentration of the stock solution (UniProt Code: A4Z171, Bradyrhizobium sp.).18 was periodically checked via ICP. All solvents were prepared with The results of this bioinformatic research confirm that freshly doubly distilled water. For the preparation of peptide and peptides containing histidine-rich motifs may indeed be complex solutions, 4 × 10−3 M HClO4 (Merck) acid was used. The involved in maintaining metal homeostasis. In this paper, we ionic strength was adjusted to 0.1 M by adding NaClO4 (Merck). analyze the properties of the Cu(II)−HRCT systems from the Mass Spectrometry Measurements. The high-resolution mass coordination chemistry point of view. With a variety of spectra of all samples were obtained by means of electrospray methods, we examined the stability and stoichiometry of the ionization time-of-flight mass spectrometry (ESI-TOF MS) using a formed complexes as well as their geometry and number or Micro TOF-Q instrument (Bruker Daltonics, Bremen, Germany) interfaced to a Series 1200 HPLC Agilent pump and controlled using characteristic of metal-binding sites. We have chosen two Compass software. ESI-L low-concentration tuning mix (Agilent model peptides as ligands for metal ions: L1: Ac- Technologies, Santa Clara, CA) was used as a calibrator. A 5:95 DHDHHHGHAH and L2: Ac-DKPAKAEDHDHHHGHAH. mixture of acetonitrile:ammonium acetate (15 mM) was used as a L1 is a HRCT fragment of M. tuberculosis GroEL1 (UniProt running buffer for neutral (pH 7.5) conditions. Instrument conditions kode: P9WPE9), and L2 is a longer fragment of the same were as follows: 10−45 μL of sample solution was injected through a protein. This approach allowed us to analyze the impact polyether heteroketone (PEEK) tube (0.5−1.5 m, 0.18 mm i.d.) at adjacent to histidine-rich fragment amino acids on the 25−50 μL·min−1, applying a capillary counter-electrode voltage of properties of complexes formed by these ligands with divalent 3.5−5. 5 kV, a dry temperature of 90−110 °C, dry gas at 6 L min−1, and a spectral collection range of 300−2000 m/z. All spectra were metal ions. Studies on the longer sequence have one more processed using Bruker Data Analysis software. The metal-to-ligand advantage: because of the high flexibility of the histidine-rich molar ratio was 1:1. domain of GroEL1, three-dimensional (3D) structures of Potentiometric Measurements. Stability constants for the mycobacterial GroEL1 obtained so far by X-ray diffraction proton and Cu(II) complexes were calculated from the pH-metric (XRD) crystallography research lack approximately 20 amino titration curves, in the pH range of 2.5−11 at 298 K and an ionic acids on the C-terminal tail.14 We also studied six mutants of strength of 0.1 M NaClO4. All the experiments were performed under L2, in which one His residue is replaced with a Gln residue an argon atmosphere, to protect the sample from carbonate (L3: Ac-DKPAKAEDQDHHHGHAH, L4: Ac-DKPA- appearance. The measurements were carried out using a Dosimat 665 Methrom titrator connected to a Methrom 691 pH meter KAEDHDQHHGHAH, L5: Ac-DKPAKAEDHDHQH- equipped with a pH electrode InLab Semi-Micro (Mettler Toledo). GHAH, L6: Ac-DKPAKAEDHDHHQGHAH, L7: Ac-DKPA- The thermostabilized glass cell was equipped with a microburette KAEDHDHHHGQAH, L8: Ac-DKPAKAEDHDHHH- delivery tube, a magnetic stirring system, and an inlet−outlet tube for GHAQ) to investigate the impact of each of the His argon. 0.1 M carbonate-free NaOH was used as a titrant. The residues on the Cu(II) complex properties. electrodes were calibrated daily for hydrogen ion concentration by 6894 https://doi.org/10.1021/acs.inorgchem.2c04486 Inorg. Chem. 2023, 62, 6893−6908 Inorganic Chemistry pubs.acs.org/IC Article Table 1. Protonation Constants of L1−L8 Peptides at T = 298 K and I = 0.1 M (NaClO4) and Potential Assignments to Appropriate Side Chains/Chemical Groupsa,b species log βjkc pKad residue species log βjkc pKad residue L1: Ac-DHDHHHGHAH L2: Ac-DKPAKAEDHDHHHGHAH [HL]3− 7.83 (1) 7.83 His [HL]5− 10.48 (1) 10.48 Lys [H2L]2− 15.10 (1) 7.28 His [H2L]4− 20.24 (1) 9.76 Lys [H3L]− 21.96 (1) 6.86 His [H3L]3− 28.35 (1) 8.11 His [H4L] 28.38 (1) 6.42 His [H4L]2− 35.68 (1) 7.33 His [H5L]+ 34.44 (1) 6.06 His [H5L]− 42.75 (1) 7.07 His [H6L]2+ 39.88 (1) 5.44 His [H6L] 49.25 (1) 6.50 His [H7L]3+ 43.69 (2) 3.81 Asp [H7L]+ 55.46 (1) 6.21 His [H8L]4+ 46.77 (2) 3.08 Asp [H8L]2+ 60.95 (1) 5.49 His [H9L]5+ 49.27 (2) 2.49 C-terminus [H9L]3+ 65.22 (1) 4.27 Glu [H10L]4+ 69.11 (2) 3.89 Asp [H11L]5+ 72.40 (2) 3.29 Asp [H12L]6+ 75.30 (2) 2.90 Asp [H13L]7+ 77.83 (2) 2.53 C-terminus L3: Ac-DKPAKAEDQDHHHGHAH L4: Ac-DKPAKAEDHDQHHGHAH [HL]5− 10.82 (1) 10.82 Lys [HL]5− 11.01 (1) 11.01 Lys [H2L]4− 20.74 (1) 9.92 Lys [H2L]4− 20.96 (1) 9.95 Lys [H3L]3− 28.59 (2) 7.84 His [H3L]3− 28.74 (1) 7.78 His [H4L]2− 35.81 (1) 7.23 His [H4L]2− 35.98 (1) 7.24 His [H5L]− 42.68 (1) 6.86 His [H5L]− 42.83 (1) 6.85 His [H6L] 48.98 (1) 6.30 His [H6L] 49.17 (1) 6.35 His [H7L]+ 55.96 (1) 5.98 His [H7L]+ 55.17 (1) 6.00 His [H8L]2+ 59.71 (2) 4.74 Glu [H8L]2+ 59.88 (2) 4.71 Glu [H9L]3+ 63.78 (2) 4.07 Asp [H9L]3+ 63.94 (3) 4.05 Asp [H10L]4+ 67.34 (3) 3.56 Asp [H10L]4+ 67.55 (3) 3.61 Asp [H11L]5+ 70.79 (2) 3.45 Asp [H11L]5+ 70.97 (2) 3.42 Asp L5: Ac-DKPAKAEDHDHQHGHAH L6: Ac-DKPAKAEDHDHHQGHAH [HL]5− 11.11 (1) 11.11 Lys [HL]5− 11.02 (1) 11.02 Lys [H2L]4− 21.26 (1) 10.16 Lys [H2L]4− 21.01 (1) 9.98 Lys [H3L]3− 29.13 (1) 7.87 His [H3L]3− 28.87 (2) 7.86 His [H4L]2− 36.29 (1) 7.16 His [H4L]2− 36.10 (2) 7.23 His [H5L]− 43.07 (1) 6.78 His [H5L]− 43.07 (2) 6.97 His [H6L] 49.36 (1) 6.30 His [H6L] 49.42 (2) 6.35 His [H7L]+ 55.10 (1) 5.74 His [H7L]+ 55.59 (2) 6.17 His [H8L]2+ 59.46 (1) 4.36 Glu [H8L]2+ 60.54 (2) 4.96 Glu [H9L]3+ 63.36 (2) 3.90 Asp [H9L]3+ 64.63 (3) 4.08 Asp [H10L]4+ 66.73 (2) 3.37 Asp [H10L]4+ 68.27 (4) 3.65 Asp [H11L]5+ 69.78 (2) 3.06 Asp [H11L]5+ 71.82 (2) 3.55 Asp L7: Ac-DKPAKAEDHDHHHGQAH L8: Ac-DKPAKAEDHDHHHGHAQ [HL]5− 11.10 (1) 11.10 Lys [HL]5− 11.06 (2) 11.06 Lys [H2L]4− 21.11 (1) 10.01 Lys [H2L]4− 21.45 (1) 10.39 Lys [H3L]3− 28.95 (1) 7.84 His [H3L]3− 29.54 (3) 8.09 His [H4L]2− 36.15 (1) 7.20 His [H4L]2− 36.49 (3) 6.95 His [H5L]− 42.98 (1) 6.83 His [H5L]− 43.39 (3) 6.89 His [H6L] 49.27 (1) 6.29 His [H6L] 49.51 (3) 6.12 His [H7L]+ 55.06 (1) 5.79 His [H7L]+ 55.31 (3) 5.80 His [H8L]2+ 59.51 (1) 4.45 Glu [H8L]2+ 59.75 (3) 4.44 Glu [H9L]3+ 63.44 (2) 3.93 Asp [H9L]3+ 63.73 (3) 3.98 Asp [H10L]4+ 66.77 (3) 3.33 Asp [H10L]4+ 67.04 (4) 3.31 Asp [H11L]5+ 70.02 (2) 3.25 Asp [H11L]5+ 70.34 (3) 3.30 Asp a The ligand concentration was 0.0005 M. The Cu(II)-to-ligand molar ratio was 0.9:1. I = 0.1 M NaClO4 and T = 298 K. bThe standard deviations are reported in parentheses as uncertainties on the last significant figure. cProtonation constants are presented as cumulative log βjk values. Standard deviations of the last digits are given in parentheses, at the values obtained directly from the experiment. L stands for a peptide with acid−base active groups. β(HjLk) = [HjLk]/([H]j[L]k), in which [L] is the concentration of the fully deprotonated peptide. dpKa = log β(HjLk) − log β(Hj−1Lk). titrating HClO4 with NaOH under the same experimental conditions as mentioned above. The Gran method allowed us to determine the peptide concentration was 0.5 mM, and the Cu(II)-to-ligand molar purity and the exact concentration of the ligand solutions.19 The ratio was 0.9:1. 6895 https://doi.org/10.1021/acs.inorgchem.2c04486 Inorg. Chem. 2023, 62, 6893−6908 Inorganic Chemistry pubs.acs.org/IC Article Table 2. Potentiometric Data for Cu(II)-L1−Cu(II)-L8 Complexes in an Aqueous Solution of HClO4 at I = 0.1 M (NaClO4), [Cu(II)] = 0.00045 M, T = 298 K (of Potentiometric Titration); Molar Ratio M/L�0.9:1 species log βijka pKab donor set species log βijka pKab donor set Cu(II)-Ac-DHDHHHGHAH (L1) Cu(II)-Ac-DKPAKAEDHDHHHGHAH (L2) [CuH−3L]4− −13.47 (4) 8.76 {1Nim. 3N−} [CuH−3L]6− −13.26 (3) 10.20 {1Nim. 3N−} [CuH−2L]3− −4.71 (2) 8.35 {2Nim. 2N−} [CuH−2L]5− −3.06 (3) 10.18 {2Nim. 2N−} [CuH−1L]2− 3.64 (2) 8.17 {2Nim. 1N−} [CuH−1L]4− 7.12 (3) 8.99 {3Nim. 1N−} [CuL]− 11.81 (2) 6.81 {2Nim} [CuL]3− 16.10 (4) 8.46 {3Nim} [CuHL] 18.62 (2) 6.16 {2Nim} [CuHL]2− 24.57 (2) 8.36 {3Nim} [CuH2L]+ 24.78 (2) 5.47 {2Nim} [CuH2L]− 32.93 (2) 6.84 {3Nim} [CuH3L]2+ 30.25 (4) 4.75 {2Nim} [CuH3L] 39.77 (3) 6.14 {3Nim} [CuH4L]3+ 34.99 (3) 3.97 {2Nim} [CuH4L]+ 45.91 (3) 5.37 {3Nim} [CuH5L]4+ 38.96 (3) {1Nim} [CuH5L]2+ 51.28 (3) 4.85 {2Nim} [CuH6L]3+ 56.13 (4) 4.23 {2Nim} [CuH7L]4+ 60.35 (7) 4.03 {1Nim} [CuH8L]5+ 64.38 (8) {1Nim} Cu(II)-Ac-DKPAKAEDQDHHHGHAH (L3) Cu(II)-Ac-DKPAKAEDHDQHHGHAH (L4) [CuH−3L]6− −14.05 (10) 10.65 {1Nim. 3N−} [CuH−3L]6− −14.49 (5) 10.43 {1Nim. 3N−} [CuH−2L]5− −3.40 (10) 10.20 {1Nim. 3N−} [CuH−2L]5− −4.06 (5) 9.58 {1Nim. 3N−} [CuH−1L]4− 6.80 (10) 9.45 {2Nim. 2N−} [CuH−1L]4− 5.52 (5) 9.24 {2Nim. 2N−} [CuL]3− 16.25 (9) {3Nim. 1N−} [CuL]3− 14.76 (5) {2Nim. 2N−} [CuH2L]− 32.30 (8) 6.36 {3Nim} [CuH2L] − 32.30 (4) 6.63 {3Nim} [CuH3L] 38.66 (10) 6.28 {2Nim} [CuH3L] 38.94 (3) 5.65 {2Nim} [CuH4L]+ 44.94 (8) 4.93 {2Nim} [CuH4L]+ 44.59 (5) 5.18 {2Nim} [CuH5L]2+ 49.87 (10) 4.67 {2Nim} [CuH5L]2+ 49.77 (4) 4.67 {2Nim} [CuH6L]3+ 54.77 (10) {1Nim} [CuH6L]3+ 54.44 (6) {1Nim} Cu(II)-Ac-DKPAKAEDHDHQHGHAH (L5) Cu(II)-Ac-DKPAKAEDHDHHQGHAH (L6) [CuH−3L]6− −14.10 (3) 11.25 {1Nim. 3N−} [CuH−3L]6− −16.68 (3) 11.65 {1Nim. 3N−} [CuH−2L]5− −2.85 (3) 10.30 {1Nim. 3N−} [CuH−2L] 5− −5.02 (3) 10.36 {1Nim. 3N−} [CuH−1L]4− 7.45 (3) 9.43 {2Nim. 2N−} [CuH−1L]4− 5.34 (3) 9.80 {2Nim. 2N−} [CuL]3− 16.88 (2) {3Nim. 1N−} [CuL]3− 15.13 (3) {3Nim. 1N−} [CuH2L]− 33.64 (2) 6.15 {3Nim} [CuH2L] − 32.53 (3) 7.31 {3Nim} [CuH3L] 39.79 (2) 5.44 {2Nim} [CuH3L] 39.84 (2) 5.72 {3Nim} [CuH4L]+ 45.23 (2) 4.81 {2Nim} [CuH4L]+ 45.57 (2) 5.17 {2Nim} [CuH5L]2+ 50.04 (3) 4.52 {2Nim} [CuH5L]2+ 50.73 (2) 4.71 {2Nim} [CuH6L]3+ 54.55 (3) {1Nim} [CuH6L]3+ 55.45 (2) {1Nim} Cu(II)-Ac-DKPAKAEDHDHHHGQAH (L7) Cu(II)-Ac-DKPAKAEDHDHHHGHAQ (L8) [CuH−3L]6− −14.25 (4) 10.92 {1Nim. 3N−} [CuH−3L]6− −13.99 (4) {1Nim. 3N−} [CuH−2L]5− −3.33 (3) 10.46 {1Nim. 3N−} [CuH−1L]4− −6.73 (4) 9.57 {1Nim. 3N−} [CuH−1L]4− 7.14 (4) 9.51 {2Nim. 2N−} [CuL]3− 16.30 (4) 8.51 {2Nim. 2N−} [CuL]3− 16.65 (3) {3Nim. 1N−} [CuHL] 2− 24.80 (3) {3Nim. 1N−} [CuH2L]− 32.32 (2) 6.60 {3Nim} [CuH3L] 39.46 (2) 5.68 {3Nim} [CuH3L] 38.92 (2) 5.83 {2Nim} [CuH4L]+ 45.14 (3) 5.10 {2Nim} [CuH4L]+ 44.75 (3) 5.10 {2Nim} [CuH5L]2+ 50.24 (3) 4.68 {2Nim} [CuH5L]2+ 49.85 (3) 4.58 {2Nim} [CuH6L]3+ 54.92 (3) {1Nim} [CuH6L]3+ 54.43 (3) {1Nim} a Cu(II) stability constants are presented as cumulative log βijk values. L stands for a fully deprotonated peptide ligand that binds Cu(II). Standard deviations of the last digits are given in parentheses at the values obtained directly from the experiment. β(MiHjLk) = [MiHjLk]/([M]i[H]j[L]k), where [L] is the concentration of the fully deprotonated peptide. bpKa = log β(MiHjLk) − log β(MiHj−1Lk). Stability constant calculations were performed using HYPER- (charges omitted; p might also be 0). Standard deviations were QUAD 2006 software.20 The reported log β values refer to the overall calculated using HYPERQUAD 2006 and refer to random errors only. equilibria The speciation, Kd values, and competition diagrams were obtained with the HYSS program.21 pM + qH + rL = M pHqLr (1) CD, UV−vis, and EPR Spectroscopy Measurements. The absorption spectra were recorded on a Jasco V-750 (Jasco) [MpHqLr ] spectrophotometer, and the circular dichroism (CD) spectra were = obtained on a Jasco J-1500 spectrometer at 298 K in the 750−250 nm [M ] p [H ]q [L]r (2) range. For the experiments, the total sample volume of 2.5 mL was where charges are omitted for clarity; log Kstep values refer to the used. The parameters of the instruments were as followed: scanning protonation process speed: 200 nm/min, data pitch: 0.5 nm, and number of accumulations: 3. The concentration of the peptides was 0.5 mM, MpHq 1Lr + H = MpHqLr (3) and the complex samples were prepared by adding metal ions to the 6896 https://doi.org/10.1021/acs.inorgchem.2c04486 Inorg. Chem. 2023, 62, 6893−6908 Inorganic Chemistry pubs.acs.org/IC Article Figure 2. continued 6897 https://doi.org/10.1021/acs.inorgchem.2c04486 Inorg. Chem. 2023, 62, 6893−6908 Inorganic Chemistry pubs.acs.org/IC Article Figure 2. Distribution diagram of complex forms in the Cu(II)-L systems. L�Ac-DHDHHHGHAH (A), Ac-DKPAKAEDHDHHHGHAH (B), Ac-DKPAKAEDQDHHHGHAH (C), Ac-DKPAKAEDHDQHHGHAH (D), Ac-DKPAKAEDHDHQHGHAH (E), Ac-DKPAKAEDHDHHQ- GHAH (F), Ac-DKPAKAEDHDHHHGQAH (G), and Ac-DKPAKAEDHDHHHGHAQ (H). M/L = 1:1. pH range: 2−11. peptide solution. The molar ratio of metal to ligand were 0.9:1. Data and the last two pKa values arise from the deprotonation of two were processed using Origin 2016. lysine residues. For the secondary structure studies, the parameters of the CD The L3−L8 behave like H11L ligands. The first three pKa spectrometer were changed as follows: scanning speed: 100 nm/min, values arise from the deprotonation of the three carboxylic side data pitch: 0.5 nm, number of accumulations: 6, and range: 280−180 nm. The optical pathway for these experiments was 0.1 cm. chain groups of aspartic acid residues, the following one Electron paramagnetic resonance (EPR) spectra were recorded at corresponds to the deprotonation of the glutamic acid side an X-band frequency (9.5 GHz) at 77 K on a Bruker ELEXSYS E500 chain group, and the next five correspond to the deprotonation CW−EPR spectrometer equipped with an ER 036TM NMR of histidine residues. The last two pKa values arise from the teslameter and E41 FC frequency counter. The complex solutions deprotonation of two lysine residues. were prepared in an aqueous solution containing 4 mM HClO4 and The exact pKa values of all ligands are listed in Table 1. pKa 0.1 M NaClO4, with 25% ethylene glycol as a cryoprotectant. The constant values of His residue deprotonation are similar to concentration of Cu(II) was 1 mM with 0.9:1 metal-to-peptide molar ratio. The pH was adjusted with HClO4 and NaOH solutions. The those found in other similar His-rich systems.22−24 In the case EPR parameters were obtained from computer simulations of the of all ligands, at acidic pH, the formation of highly positively experimental spectra using Bruker’s WIN-EPR SIMFONIA software, charged species occurs, e.g., [H10L]4+, [H9L]5+, [H12L]6+, and version 1.2 (Billerica). All spectra were drawn in Origin 2016. [H13L]7+. It is caused by the presence of 5−6 His and 0−2 Lys residues. These species correspond to the deprotonation of ■ RESULTS Protonation Equilibria of Ac-DHDHHHGHAH (L1), Ac- Asp residues or the C-terminus. pKa values of these steps (2.90−3.65) are lower than the pKa value of the free Asp DKPAKAEDHDHHHGHAH (L2), and Mutants of the residue (3.86), and the peptide charge can be an explanation Latter. Protonation constants of the examined peptides and for it. Highly positively charged species tend to favor the probable assignments to the particular chemical groups are deprotonation, resulting in the charge decrease, which presented in Table 1. The peptides were protected at the N- contributes to the system stability.25,26 In the case of terminus. Ac-DHDHHHGHAH (L1) behaves like H9L acid, [H9L]3+, the pKa constant values (3.89−4.08) also correspond- Ac-DKPAKAEDHDHHHGHAH (L2) behaves like H13L acid, ing to the Asp residue deprotonation are more like the pKa and L2 mutants (L3−L8) exhibit 11 protonation constants in value of the free Asp residue because the charge of this species the pH range of 2.5−11. is not highly positive anymore. Potentiometric measurements detected nine protonation Cu(II) Complexes. The presence of Cu(II) complexes with constants for the L1 peptide (H9L). The first pKa value the model peptides was determined by a variety of methods. corresponds to the deprotonation of the C-terminus. The next Signals in the mass spectra have been assigned to ions of two pKa values arise from the deprotonation of the carboxyl ligands or Cu(II)−L complexes. ESI-MS peak assignments groups of the two aspartic acids. Another six pKa values can be were based on the comparison between the precisely calculated assigned to the deprotonations of the imidazole groups of the and experimental m/z values and their isotopic patterns. To six histidine residues. establish the number and chemical characteristic of metal The L2 can be considered the H13L ligand. The first pKa binding sites as well as the geometry and stability of the value arises from the deprotonation of the C-terminus. The complexes, potentiometric titrations, EPR, CD, and UV−Vis next three pKa values correspond to the deprotonation of the spectroscopy have been used. Stability constants for the three carboxylic side chain groups of aspartic acid residues, studied complexes are collected in Table 2; the distribution while the following one corresponds to the deprotonation of diagrams are presented in Figure 2, and MS/UV−Vis/EPR/ the glutamic acid side chain group. The next six pKa values can CD spectra are presented in the Supporting Information. Table be assigned to the deprotonation of histidine imidazole groups, S1 shows the major coordination modes of the Cu(II)-L 6898 https://doi.org/10.1021/acs.inorgchem.2c04486 Inorg. Chem. 2023, 62, 6893−6908 Inorganic Chemistry pubs.acs.org/IC Article system at a pH range of 4.0−11.0, where L means the In the case of Cu(II)-L1, His binding starts when the charge appropriate model peptide. is +4. The deprotonation of a second, metal binding His Electrospray ionization mass spectrometry revealed the residue is followed by the change of charge to +3. The pKa of stoichiometry of the studied complexes. In the spectrum of [CuH4L]3+ is low compared to the pKa values of the free ligand the Cu(II)-L1 system (equimolar mixture), two main types of related to the deprotonation of the His residue: 5.44, 6.06, ions were detected, [L]+ (m/z = 1241.52, z = +1) and [CuL]+ 6.42, 6.86, 7.28, and 7.83 (Table 2). The low value of (m/z = 1302.44, z = +1), corresponding to the single-charged [CuH4L]3+ pKa is a result of a relatively high positive ion of the ligand and its Cu(II) complex, respectively (Figure charge.25,26 With the decrease of the charge, the pKa values S1). In the spectra of Cu(II) and L2 equimolar mixture of particular His residues increase. Amide nitrogen deproto- (Figure S2A) also, two main types of ions were observed: [L]2+ nation starts when the complex is relatively highly negatively (z = 2+, m/z = 991.44) and [CuL]2+ (z = 2+, m/z = 1022.40). charged (−3) and is forced by the basic pH. They correspond to the double-charged ion of the ligand and In the case of Cu(II)-L2, the first His residue deprotonation its Cu(II) complex, respectively. In addition, we observed a starts when the molecule is more positively charged than weak signal corresponding to [Cu2L]2+. However, the intensity Cu(II)-L1 (+5), due to the presence of two Lys residues. of this signal is not significant compared to that of [CuL]2+. In Another three deprotonations followed by the change of the case of the equimolar mixtures of Cu(II) and mutants charge to +2 and most probably are related to the (L3−L8), the same types of ions as for Cu(II)-L2 were deprotonation of one Glu and two His residues. pKa values detected. The spectra of all mutants were identical, and an of [CuH7L]4+, [CuH6L]3+, and [CuH5L]2+ are decreased example of a spectrum is presented in Figure S3A. The 1: 1 compared to the pKa of Glu (4.27) and His residues in the free metal-to-ligand interaction in equimolar mixture is confirmed ligand: 5.49, 6.21, 6.50, 7.07, and 7.33 (Table 2). This is due to by potentiometric calculations. The comparison of the the increase of the charge in the presence of the Cu(II) simulated and experimental isotopic distribution of [CuL]+ ion.25,26 pKa values of three next species, [CuH4L]+, [CuH3L], and [CuL]2+ confirmed the identity of signals at m/z = and [CuH2L]−, related to the deprotonation of another three 1302.44 for Cu(II)-L1, 1022.40 for Cu(II)-L2, and 1017.43 for His residues, are higher due to the decrease of the charge. Cu(II)-L3−Cu(II)-L8. Amide nitrogen deprotonations are forced by the basic pH and In the presence of an excess of metal ions, the intensity of cause the further decrease of the charge to a highly negative [Cu2L]2+ ions increases in the case of the studied systems value: −6. Cu(II)-L2−Cu(II)-L8 (Figures S2B, S3B, S4), but the Cu(II)-L3−Cu(II)-L8 systems are of lower charge at acidic pH compared to Cu(II)-L2, due to the lack of one His residue. abundance ratio of [CuL]2+/[Cu2L]2+ differs between the The general correlation between the decreased pKa values particular systems. It confirms that the studied peptides could related to His residue deprotonation and highly positive charge bind more than one copper ion per molecule when an excess of of species is preserved. The details of the Cu(II)-L1−Cu(II)- copper is applied. Unfortunately, due to the problems with L8 systems are described in further sections. complex peptide solubility that commonly occur in the high Cu(II)-L1. The potentiometric titrations of the Cu(II)-L1 concentration of metal ions, we could not obtain reliable system revealed the existence of nine complex forms at the pH results of potentiometric and spectroscopic (UV−Vis, CD) range of 2.5−11: [CuH 5L] 4+ , [CuH 4L] 3+ , [CuH 3L] 2+ , titrations. [CuH 2 L] + , [CuHL], and [CuL], and [CuH −1 L] 2− , MS studies provide interesting results concerning the [CuH−2L]3−, and [CuH−3L]4−. L1 peptide starts to bind potential role of particular His residues in Cu(II) binding Cu(II) ions at pH above 3 (Figure 2A). The first complex and the role of the HRCT itself. The intensity of the MS species is [CuH5L]4+ with a maximum concentration at pH signals does not always correlate with the concentration, as the 4.0, and most probably, it comes from the deprotonation of the ability of the molecule to be ionized is also important in terms first His residue. Asp residues are also deprotonated in this of intensity. Here, we compare the signals of two very similar form. The presence of a band with a maximum absorption at species with the same charge�[CuL]2+ and [Cu2L]2+�so we 720 nm in the UV−vis spectra suggests one nitrogen atom in can assume that their ionization should be very similar, and the Cu(II) ion coordination sphere (Figure S5A).22,27 EPR consequently, the relative intensity could give information parameters for the complex at pH 4.05 (A = 160.5 G, g∥ = 2.31, about the abundance of these particular forms. The intensity Figure S5B) also confirm the interactions of Cu(II) with one ratio of [CuL]2+/[Cu2L]2+ = 2:1, approximately, in the case of nitrogen atom and suggest a {1Nim} coordination mode for Cu(II)-Ac-DKPAKAEDHDHHHGHAH (L2) (Figure S2B). [CuH5L]4+.22 The next species, [CuH4L]3+ (maximum In the case of Cu(II)-Ac-DKPAKAEDHDQHHGHAH (L4), concentration at pH 4.5), is formed as a result of the the intensity ratio [CuL]2+/[Cu2L]2+ is the biggest and equal deprotonation of the second His residue. The pKa value of 3.97 to approximately 6:1 (Figure S4A). In the case of Cu(II)-Ac- for this step probably corresponds to pKa = 7.83 for one His DKPAKAEDQDHHHGHAH (L3) and Cu(II)-Ac-DKPA- residue in the free ligand and is significantly reduced, KAEDHDHQHGHAH (L5) systems, the intensity ratio suggesting the presence of other His side chains in the [CuL] 2+/[Cu2L]2+ is close to 1:1 (Figures S3B and S4B). coordination sphere of Cu(II). Above pH 4.0, a significant blue The reverse correlation is observed in the case of Cu(II)-Ac- shift (720−600 nm) is observed in the UV−vis spectra, DKPAKAEDHDHHQGHAH (L6), Cu(II)-Ac-DKPA- suggesting the formation of a complex with a second or third KAEDHDHHHGQAH (L7), and Cu(II)-Ac-DKPA- nitrogen atom bond (Figure S5A).22,27 EPR parameters for the KAEDHDHHHGHAQ (L8) (Figure S4C−E); the intensity complex at pH 5.07 (A = 176.5 G, g∥ = 2.27, Figure S5B) ratio is reversed in favor of binuclear species and equal to 1:6, confirm the interaction of Cu(II) with two nitrogen atoms.22 1:4, and 1:7, respectively. The results obtained suggest that the The other four species, [CuH3L]2+, [CuH2L]+, [CuHL], and ability to form binuclear species depends on the rearrangement [CuL]−, with maximum concentrations at pH 5.0, 6.0, 6.5, and of His residues. 7.5, respectively, arise from the deprotonation of four, 6899 https://doi.org/10.1021/acs.inorgchem.2c04486 Inorg. Chem. 2023, 62, 6893−6908 Inorganic Chemistry pubs.acs.org/IC Article probably, non-metal-binding His residues. pKa values of 4.75, presence of Cu(II), suggesting that a second His imidazole ring 5.47, 6.16, and 6.81 for these steps could probably correspond is in the coordination sphere of the metal ion. At pH 5.08, a to pKa = 5.44, 6.06, 6.42, and 7.28, respectively, for His significant blue shift (720−600 nm) is observed in the UV−vis residues in the free ligand. However, this assignment is only spectra, confirming the involvement of an additional nitrogen approximate�we cannot precisely indicate which pK a atom in Cu(II) binding (Figure S6A).22,27 EPR parameters for corresponds to the particular His residue deprotonation. It is the complex at pH 5.11 (A = 177.1 G, g∥ = 2.27, Figure S6B) obvious that the adjacent residues and their acceptor/donor also support the interaction of Cu(II) with two nitrogen abilities as well as the overall complex charges impact the atoms.22 A {2Nim} coordination mode is therefore assigned to scheme of deprotonation and pKa values. They are not reduced the [CuH6L]3+ and [CuH5L]2+ species. [CuH4L]+, with a in the presence of Cu(II), suggesting that four His residues do maximum concentration at pH 6.0, could arise from the not bind Cu(II). Under the conditions of the UV−vis/CD deprotonation of a fourth His residue. The pKa value of 5.37 experiment, the complex at pH 6.00−8.01 was not soluble. for this step could probably correspond to pKa = 8.11 for His However, the EPR parameters for the complex (A = 180.0 G, residue in the free ligand and is significantly reduced. EPR g∥ = 2.26 at pH 6.12 and A = 175.3 G, g∥ = 2.25 at pH 7.03) parameters for the complex at pH 6.12 (A = 179.1 G. g∥ = 2.26. allowed us to establish the {2Nim} coordination mode for Figure S6B) confirm the interaction of Cu(II) with three [CuH3L]2+, [CuH2L], [CuHL]−, and [CuL]2− (Figure S5B).22 nitrogen atoms, suggesting a {3Nim} coordination mode for No significant changes in the UV−vis spectrum were observed [CuH4L]+.22,23,34,35 Another two species, [CuH3L] and at pH 8.01, what supports the above-mentioned result. [CuH2L]− (maximum at pH 6.5 and 7.5, respectively), most [CuH−1L]2−, [CuH−2L]3−, and [CuH−3L]4− forms most likely come from the deprotonation of two non-metal-binding probably arise from the deprotonation of three amide nitrogen His residues. The pKa values of 6.14 and 6.84 for these steps atoms (maximum concentration at around pH 8.0, 9.0, and could probably correspond to pKa = 6.50 and 7.07, 10.0, respectively). The blue shift (600 to 540 nm) at pH 9.11 respectively, for the His residue in the free ligand�they are observed in the UV−vis and the changes in the EPR spectra at not significantly reduced. No significant changes in the UV− pH 9.07 (A = 196.0 and g∥ = 2.21) indicate that the third vis, EPR, and CD spectra were observed at pH 6.07−9.09, nitrogen atom, arising from the deprotonation of the first confirming the above-mentioned result. [CuHL]2−, [CuL]3−, amide bond, coordinates with Cu(II) (Figures 2A and and [CuH−3L]6− presumably come from the deprotonation of S5A,B).22,27,28 This finding is supported by the presence of three amide nitrogen atoms (maximum concentration at the d−d band at 500 and 620 nm (negative and positive around pH 8.0, 9.0, and 10.0, respectively), and [CuH−1L]4− Cotton effect), corresponding to the copper(II)−amide and [CuH−2L]5− (maximum concentrations at around pH 9.5 nitrogen interactions in peptide complexes (Figure and 10.2, respectively) could arise from the deprotonation of S5C).29−32 A further increase of pH causes the increase of two Lys residues that are not involved in Cu(II) binding. The the d−d band in the CD spectrum, a slight blue shift in the EPR parameters at pH 9.01 (A = 193.2 and g∥ = 2.21) suggest UV−vis spectrum, and the change of EPR spectral parameters that the formation of a complex with a fourth nitrogen atom (A = 194.0 and g∥ = 2.19).22,33 It confirms the involvement of a bond started (Figure S6B).22,33 Additionally, this finding is second and third amide nitrogen atom in Cu(II) binding, supported by the presence of the d−d band at 500 and 620 nm replacement of two Nim with the N− atom in the Cu(II) (negative and positive Cotton effect) at pH 9.09, correspond- coordination sphere, and formation of a complex with a square ing to the copper(II)−amide nitrogen interactions in peptide planar geometry. The coordination mode of [CuH−1L]2−, complexes (Figure S6C).29−32 A further increase of pH to 10.0 [CuH−2L]3−, and [CuH−3L]4− is {2Nim. 1N−}, {2Nim. 2N−}, and 11.0 causes the increase of the d−d band in the CD and {1Nim. 3N−}, respectively. spectra and the shift of a band in the UV−vis spectra (Figure Cu(II)-L2. The potentiometric titrations of the Cu(II)-L2 S6A,C).22,27,29−32 It confirms the involvement of a second and system revealed the existence of 12 complex forms at the pH third amide nitrogen atom in Cu(II) binding, replacement of range of 2−11: [CuH 8 L] 5+ , [CuH 7 L] 4+ , [CuH 6 L] 3+ , two Nim with a N− atom in the Cu(II) coordination sphere, [CuH5L]2+, [CuH4L]+, [CuH3L], [CuH2L]−, [CuHL]2−, and and formation of a complex with a square planar geometry. [CuL]3− and [CuH−1L]4−, [CuH−2L]5−, and [CuH−3L]6−. The final UV−vis (λmax = 525 nm) and EPR (A = 196.9, g∥ = Similar to L1, L2 starts to bind Cu(II) ions at pH above 3.0 2.19) set of parameters at pH 11.04 (λmax = 530 nm) strongly (Figure 2B). [CuH8L]5+ and [CuH7L]4+, with a maximum confirms the interaction of Cu(II) with four nitrogen atoms concentration at around pH 4.0, most probably come from the (Figure S6A,B).22,27,33 The coordination modes of [CuHL]2−, deprotonation of a His and Glu residue, respectively. Asp [CuL]3−, and [CuH−1L]4− are {3Nim. 1N−}. For [CuH−2L]5− residues are also deprotonated in this form. In the UV−vis and [CuH−3L]6−, the coordination mode would be {2Nim. spectra at pH 3.99, we can observe a band with a maximum 2N−} and {1Nim, 3N−}, respectively. absorption at 720 nm, suggesting that one nitrogen atom is in Cu(II)-L3. The potentiometric titrations of the Cu(II)-L3 the metal ion coordination sphere (Figure S6A).22,27 EPR system revealed the existence of nine complex forms at the pH parameters for the complex at pH 4.02 (A = 163.3 G, g∥ = 2.31, range of 2−11: [CuH6L]3+, [CuH5L]2+, [CuH4L]+, [CuH3L], Figure S6B) indicate the interactions of Cu(II) with one [CuH2L]−, and [CuL]3− and [CuH−1L]4−, [CuH−2L]5−, and nitrogen atom, suggesting a {1Nim} coordination mode for [CuH−3L]6−. Similar to L1 and L2, L3 peptide starts to bind [CuH 7 L] 4+ and [CuH 8 L] 5+ . 22 The next two species, Cu(II) ions at pH above 3.0 (Figure 2C). [CuH6L]3+ with a [CuH6L]3+ and [CuH5L]2+, (maximum concentrations at pH maximum concentration at pH 4.5 most probably comes from 4.5 and 5.0, respectively) could be formed as a result of the the deprotonation of the first His side chain. Asp and Glu deprotonation of a second and third His residue. The pKa residues are also deprotonated in this form. The arise of a band values of 4.23 and 4.85 most probably correspond to pKa = with a maximum absorption at 690 nm in the UV−vis 7.34 and 5.49, respectively, for His residues in the free ligand. spectrum (pH 4.16) indicates that one nitrogen atom is Only one of these values is significantly reduced in the involved in Cu(II) binding (Figure S7A).22,27 Additionally, the 6900 https://doi.org/10.1021/acs.inorgchem.2c04486 Inorg. Chem. 2023, 62, 6893−6908 Inorganic Chemistry pubs.acs.org/IC Article EPR parameters (A = 163.0, g∥ = 2.30) of the Cu(II)-L3 [CuH −3 L] 6− (Figure 2D). The first form [CuH 6 L] 3+ system at pH around 4.0 support this observation, confirming a (maximum concentration at pH 4.0) most probably appears {1Nim} coordination mode for [CuH6L]3+ (Figure S7B).22 The as a result of the first His deprotonation. In the UV−vis next species [CuH5L]2+ with a maximum at a pH of around 5.0 spectrum at pH 4.12, we can observe a band with a maximum presumably appears in the solution as a result of the second at around 700 nm, and the parameters of the EPR spectrum at His residue deprotonation. The pKa value of 4.67 could pH 4.10 are as follows: A = 161.0 and g∥ = 2.31 (Figure correspond to pKa = 7.23 for one of the His residues in the free S8A,B). Both phenomena indicate that one nitrogen atom is in ligand and is significantly reduced, suggesting that another the coordination sphere of Cu(II). The coordination mode for imidazole nitrogen atom is involved in Cu(II) binding. The [CuH6L]3+ is therefore {1Nim}.22,27 The next species shift of a band in the UV−vis spectrum at pH 5.15 (690 to 630 [CuH5L]2+ (maximum concentration at pH 5.0) most likely nm) and EPR parameters (A = 173.4, g∥ = 2.27) suggest the comes from the second His residue deprotonation. The pKa interaction of two nitrogen atoms with Cu(II) (Figure value of 4.67 for this step could probably correspond to pKa = S7A,B).22,27 It indicates a {2Nim} coordination mode for 7.24 for one of the His residues in the free ligand and is [CuH5L]2+. The species [CuH4L]+ and [CuH3L] with a significantly reduced, suggesting that another imidazole maximum concentration at pH 5.5 and 6.5, respectively, could nitrogen atom is involved in Cu(II) binding. This result is come from the deprotonation of the third and fourth His confirmed by the presence of a blue shift in the UV−vis residues. The pKa values of 4.93 and 6.28 probably correspond spectrum (700 to 630 nm) and the change of EPR parameters to pKa = 5.98 and 6.83, respectively, for two of the His residues (A = 171.9, g∥ = 2.27) (Figure S8A,B).22,27 {2Nim} can be in the free ligand and are not significantly reduced, suggesting therefore assigned to [CuH5L]2+. Another two species, the non-metal binding characteristic of these residues. The [CuH4L]+ and [CuH3L] (maximum concentration at pH 5.5 shift of a band in the UV−vis spectrum (630 to 600 nm) along and 6.0, respectively), are probably formed as a result of the with the EPR spectral parameters (A = 180.5, g∥ = 2.25) at pH third and fourth His imidazole group deprotonation that most around 6.0 and 7.0 suggests that there is an equilibrium probably are not involved in Cu(II) binding. The pKa values of between forms in which two or three nitrogen atoms bind 5.18 and 5.65 for these steps could be related to pKa = 6.00 Cu(II) (Figure S7A,B).22,23,27 It is related to the formation of and 6.35, respectively, for two of the His residues in the free another species, [CuH2L]−�it presumably comes from the ligand and are not significantly reduced. The small blue shift in deprotonation of the fifth His imidazole ring. The pKa value of the UV−vis (630 to 600 nm) and EPR parameters (A = 181.3, 6.36 could probably correspond to pKa = 7.84 for one of the g∥ = 2.25) at pH around 6.0 and 7.0 (Figure S8A,B) suggest His residues in the free ligand and is reduced, supporting the that there could be an equilibrium of species in which two or metal binding characteristic of this residue. The shift of a band three nitrogen atoms are present in the coordination sphere of in the UV−vis spectrum (600 to 580 nm) and the change of the metal ion.22,27 A similar situation was observed in the case EPR parameters (A = 181.3, g∥ = 2.24) at pH around 8.0 finally of the copper complex of the first mutant�L3. This is in indicate that three nitrogen atoms bind Cu(II) (Figure agreement with the potentiometric results (Figure 2D)�at pH S7A,B).22,23,27,35 The coordination mode for [CuH2L]− is around 6.0 and 7.0, another species starts to form, [CuH2L]− {3Nim}. [CuL]3− species with a maximum concentration at pH (with a maximum concentration at pH 8.0). It most likely 8.5 is most likely related to the deprotonation of one amide comes from the deprotonation of the fifth His imidazole group. bond and one non-metal binding under the conditions of the The pKa value of 6.63 for this step could probably correspond experimental Lys residue. Arising of a d−d band at pH 8.11− to pKa = 7.78 for one of the His residues in the free ligand and 9.27 in the CD spectrum and a shift in the UV−vis spectrum is reduced, suggesting that another imidazole nitrogen atom is (580 to 550 nm) confirm the interactions between Cu(II) and involved in Cu(II) binding. This is confirmed by the presence amide nitrogen (Figure S7C).29−32 The coordination mode for of a blue shift in the UV−vis spectrum (600 to 580 nm) and [CuL] 3− is therefore {3N im ,N − }. [CuH −1 L] 4− and the change of the EPR parameters (A = 183.6, g∥ = 2.24, Figure [CuH−2L]5−, with a maximum concentration at pH 10.0 and S8A,B).22,27,34,35 The coordination mode for [CuH4L]+ and 10.5, respectively, appear in the solution most probably as a [CuH3L] is {2Nim}, and for [CuH2L]−, it is {3Nim}. The next result of the second and third amide bond deprotonation. The species [CuL]3− (maximum concentration at pH 9.0) comes changes of EPR parameters (A = 189.9, g∥ = 2.20) and from the deprotonation of two amide nitrogen atoms. The appearance of a blue shift in the UV−vis spectrum (550 to 530 appearance of a blue shift at pH 9.10 along with the EPR nm) at pH around 10 confirm the existence of forms in which parameter set (A = 195.3, g∥ = 2.21) indicates that another only four and not three nitrogen atoms bind Cu(II) (Figure nitrogen atom is involved in metal binding (Figure S7A,B).22,27,33 In the CD spectrum, a d−d band at 500 nm S8A,B).22,27,33 Additionally, the d−d band at 500 and 600 (negative Cotton effect) and at 620 nm (positive Cotton nm (negative and positive Cotton effect), characteristic for effect) increases, which confirms the interaction of amide Cu(II) complexes of amide nitrogen, supports this finding nitrogens with Cu(II) and formation of a complex with a (Figure S8C).29−32 It indicates a {2Nim, 2N−} coordination square planar geometry (Figure S7C).29−32 No further changes mode for [CuL]3−. [CuH−1L]4− (maximum concentration at were observed when the pH increases. It indicates that pH 10.0) most probably arises from the deprotonation of a [CuH−3L]6− comes from the deprotonation of the second non- non-metal-binding Lys residue, while [CuH−2L]5− (maximum metal-binding Lys residue and that the coordination mode for concentration at pH 10.0) is formed as a result of the third [CuH −1 L] 4− is {2N im , 2N − } and for [CuH −2 L] 5− / amide nitrogen deprotonation. The increase of pH to 10.11 [CuH−3L]6−, it is {Nim, 3N−}. causes the increase of the CD band at 500 and 600 nm and a Cu(II)-L4. The potentiometric titrations of the Cu(II)-L4 further blue shift in the UV−Vis spectrum (580 to 540 nm), system indicate that nine complex forms exist at the pH range which supports this finding (Figure S8A,C).22,27,29−32 The of 2−11: [CuH6L]3+, [CuH5L]2+, [CuH4L]+, [CuH3L], further increase of pH does not significantly affect the spectra. [CuH2L]−, and [CuL]3− and [CuH−1L]4−, [CuH−2L]5−, and This is in agreement with the potentiometric data, since 6901 https://doi.org/10.1021/acs.inorgchem.2c04486 Inorg. Chem. 2023, 62, 6893−6908 Inorganic Chemistry pubs.acs.org/IC Article [CuH−3L]6− that dominates at pH 11.0 is most probably at 500 and 620 nm indicates that Cu(II) starts to form a bond related to the second non-metal-binding Lys residue with amide nitrogen (Figure S9A−C).22,27,29−32 A coordina- deprotonation. The coordination mode for [CuH−3L]6− tion mode for [CuL]3− is {3Nim, 1N−}. Two other species, should be {1Nim, 3N−}. [CuH−1L]4− and [CuH−2L]5− (maximum concentration at pH Cu(II)-L5. The potentiometric titrations of the Cu(II)-L5 10.0 and 11.0, respectively), presumably arise from the system revealed that nine exact complex forms exist at the pH deprotonation of two other amide bonds that bind Cu(II). range of 2−11: [CuH6L]3+, [CuH5L]2+, [CuH4L]+, [CuH3L], This finding is supported by the presence of a blue shift in the [CuH2L]−, and [CuL]3− and [CuH−1L]4−, [CuH−2L]5−, and UV−vis spectra at pH 10.12 (570 to 540 nm), changes in EPR [CuH−3L]6− (Figure 2E). The first species [CuH6L]3+ with a parameters (A = 194.5, g∥ = 2.20), and the increase of a d−d maximum concentration at pH 4.0 most probably arises from band at 500 and 620 nm in the CD spectrum (related to the the deprotonation of the first His imidazole group. In the UV− the formation of the N−−Cu(II) bond) (Figure S9A− vis spectrum at pH 4.09, we can observe a band with a C).22,27,29−33 No significant changes were then observed with maximum at around 690 nm that corresponds to the d−d band a further increase of the pH, which supports the idea that all characteristic for a copper complex with one nitrogen atom amide bonds deprotonated before pH around 11.0 and that the (Figure S9A).22,27 This observation is supported by the EPR last species [CuH−3L]6− most likely comes from the spectrum parameters for the Cu(II)-L5 system at pH 4.19 (A = deprotonation of a non-metal-binding Lys residue. Therefore, 161.0, g∥ = 2.31) (Figure S9B), suggesting a {1Nim} the coordination mode for [CuH−1L]4−, [CuH−2L]5−, and coordination mode for [CuH6L]3+.22 Another species, [CuH−3L]6− is, respectively, {2Nim, 2N−}, {1Nim, 3N−}, and [CuH5L]2+ (maximum concentration at pH 4.8), most likely {1Nim, 3N−}. comes from the deprotonation of another His residue. The pKa Cu(II)-L6. The potentiometric titrations of the Cu(II)-L6 value of 4.52 for this step could correspond to pKa = 7.16 for system indicated that nine exact complex forms exist at the pH one of the His residues in the free ligand and is significantly range of 2−11: [CuH6L]3+, [CuH5L]2+, [CuH4L]+, [CuH3L], reduced, suggesting that a second imidazole nitrogen atom is in [CuH2L]−, and [CuL]3− and [CuH−1L]4−, [CuH−2L]5−, and the Cu(II) coordination sphere. This is confirmed by the [CuH−3L]6−. [CuH6L]3+ with a maximum at pH 4.3 most presence of a blue shift in the UV−vis spectra (690 to 640 nm) probably arises from the deprotonation of the first His and the change of EPR parameters (A = 171.9, g∥ = 2.27) at imidazole group (Figure 2F). The involvement of this residue pH around 5.0 (Figure S9A,B).22,27 Therefore, the {2Nim} in metal binding is confirmed using two techniques: in the coordination mode can be assigned to [CuH5L]2+. [CuH4L]+ UV−vis spectrum at pH 4.09 (Figure S10A), the presence of a and [CuH3L] (maximum concentration at pH 5.3 and 6.0, band with a maximum at 680 nm indicates that one imidazole respectively) species presumably arise from the deprotonation nitrogen is in the coordination sphere of Cu(II) and the EPR of another two His imidazole groups. The pKa values of 4.81 parameters at pH 4.15 are in agreement with the UV−vis and 5.44 for these steps could probably correspond to pKa = results (A = 161.4, g∥ = 2.30, Figure S10A,B).22,27 It suggests a 5.74 and 6.30 for two of the His residues in the free ligand and {1Nim} coordination mode for [CuH6L]3+. Another species, are not significantly reduced, suggesting that these His [CuH5L]2+ (maximum concentration at pH 5.0), most likely imidazole nitrogen atoms do not bind Cu(II). The increase comes from the deprotonation of the second His residue. The of pH to 6.13 causes a small shift of a band in the UV−vis pKa value of 4.71 for this could correspond to pKa = 6.97 for spectra (640 to 600 nm) and the change of EPR parameters (A one of the His residues in the free ligand and is significantly = 181.3, g∥ = 2.25) (Figure S9A,B).22,23,27,35 It suggests the reduced�it indicates that a second imidazole nitrogen atom existence of an equilibrium of species in which two and three binds Cu(II). The changes in the UV−vis and EPR spectra at nitrogen atoms interact with Cu(II)�this is in agreement with pH around 5 (the presence of a blue shift: 680−615 nm, the potentiometric results and related to the arise of another Figure S10A; A = 171.1, g∥ = 2.27, Figure S10B) confirm that species�[CuH2L]− (Figure 2E). [CuH2L]−, the most two nitrogen atoms bind Cu(II) and that the {2Nim} abundant form with a maximum concentration at pH 7.4, coordination mode can be assigned to [CuH5L]2+.22,27 most probably comes from the deprotonation of the fifth His Another species, [CuH4L]+ (maximum concentration at pH residue. The pKa value of 6.15 for this step could correspond to 5.5), most probably arises from the deprotonation of a non- pKa = 7.87 for one of the His residues in the free ligand and is binding His residue. The pKa value of 5.17 for this step could significantly reduced, suggesting that a third imidazole nitrogen correspond to pKa = 6.17 for one of the His residues in the free atom binds Cu(II). In the UV−vis spectrum at pH 7.13 (and ligand and is not significantly reduced, suggesting that this 8.17), we can observe a blue shift (600 to 580 nm), and in the residue is not involved in Cu(II) binding. However, the EPR spectrum, a shift of parameters was detected (A = 183.6, changes in the UV−vis and EPR spectra at pH around 6.0 (the g∥ = 2.25 at pH 7.07 and g∥ = 2.24 at pH 8.06), confirming that presence of a slight blue shift: 615−600 nm, Figure S10A; A = a third His residue is present in the Cu(II) coordination sphere 174.4, g∥ = 2.26, Figure S10B) suggest the coexistence of two and that a coordination mode for this species should be {3Nim} species in which two and three nitrogen atoms are in the (Figure S9A,B).22,23,27 Another species, [CuL]3− (maximum coordination sphere, which is in agreement with the concentration at pH 9.0), most probably arises from the potentiometric data (Figure 2F).22,23,27,34,35 This is due to deprotonation of one amide nitrogen and one Lys residue. the formation of [CuH3L] (maximum concentration at pH Three phenomena could support this suggestion: (1) the 6.5)�a species that is most probably related to the presence of a slight blue shift of a band in the UV−vis deprotonation of the fourth His residue. The pKa value of spectrum (580 to 570 nm) at pH 9.09 suggests that there is an 5.72 for this step could correspond to pKa = 7.23 for one of the equilibrium between species in which three and four nitrogen His residues in the free ligand and is significantly reduced, atoms are involved in Cu(II) binding; (2) EPR parameters at suggesting that this residue strongly interacts with Cu(II). The pH 9.12 change to A = 195.3 and g∥ = 2.21; and (3) in the CD increase of pH to 7.12 causes the appearance of a blue shift in spectrum at pH 9.09, the appearance of an abundant d−d band the UV−vis spectrum (600 to 585 nm), and the EPR 6902 https://doi.org/10.1021/acs.inorgchem.2c04486 Inorg. Chem. 2023, 62, 6893−6908 Inorganic Chemistry pubs.acs.org/IC Article parameters at pH 7.07 change to A = 177.3 and g∥ = 2.25 and 6.5, respectively) are most likely related to the (Figure S10A,B). It confirms that at pH around 9.0, the species deprotonation of two non-metal-binding His residues. The in which three nitrogen atoms bind Cu(II) dominates.22,23,27,34 pKa values of 5.10 and 5.83 for these steps could correspond to A coordination mode for [CuH4L]+ and [CuH3L] is {2Nim} pKa = 5.79 and 6.29 for two of the His residues in the free and {3Nim}, respectively. [CuH2L]− (maximum concentration ligand, respectively. They are not significantly reduced, at pH 8.0) most probably arises from the deprotonation of the suggesting that these residues do not bind Cu(II). The fifth His residue. The pKa value of 7.31 for this could be related coordination mode for [CuH4L]+ and [CuH3L] is still {2Nim}. to pKa = 7.86 for one of the His residues in the free ligand and However, in the UV−vis spectrum at pH 6.14−7.11, we can is not significantly reduced. It suggests that this residue does observe the blue shift (630−600 nm) (Figure S11A), not bind Cu(II). At pH around 8.0, no significant changes in suggesting the presence of an equilibrium between species in the UV−vis and EPR spectra were observed, which supports which two and three nitrogen atoms are involved in Cu(II) this assumption (Figure S10A,B).22,27,33 The next species binding.22,27 This finding is supported by the EPR parameters [CuL]3− (maximum concentration at pH 9.0) most probably (A = 172.1, g∥ = 2.26 at pH 6.07; A = 182.0, g∥ = 2.25 at pH corresponds to the deprotonation of one non-binding Lys 7.10) (Figure S11B)22,23,34,35 and is in agreement with residue and amide nitrogen. The slight changes in the UV−vis potentiometric titrations�at the pH range of 5.5−8.5, another and EPR spectra at pH around 9.0 (the presence of a slight species occurs, [CuH2L]− (maximum concentration at pH blue shift: 585−575 nm, Figure S10A; A = 195.4, g∥ = 2.21, 7.5). Most probably, it comes from the deprotonation of an Figure S10B) suggest the coexistence of a species in which imidazole group of the fifth, metal-binding His residue. The three and four nitrogen atoms are in the coordination sphere. pKa value of 6.60 for this step could correspond to pKa = 7.84 This is in agreement with the potentiometric data (Figure for one of the His residues in the free ligand and is significantly 2F).22,23,27,33 Additionally, in the CD spectrum at pH 9.09, we reduced. The coordination mode for [CuH2L]− is therefore can observe the arising of a d−d band at 500 and 600 nm, {3Nim}. [CuL]3− (maximum concentration at pH 8.5) most commonly found in the spectra of Cu(II) complexes with an probably is related to the deprotonation of one Lys residue and amide nitrogen atom (Figure S10C).29−32 The coordination one amide bond. This finding is supported by the arise of a d− mode for [CuL]3− is {3Nim, N−}. [CuH−1L]4− reaches its d band at 540 and 620 nm in the CD spectrum at pH 8.12, maximum concentration at pH 10.0. The increase of pH to commonly found in Cu(II)−N−-type complexes (Figure 10.10 causes a significant blue shift in the UV−vis spectrum S11C).29−32 Interestingly, this band is present at lower pH (575−540 nm, Figure S10A), the change of EPR parameters compared to CD spectra of Cu(II)-L of previous mutants (A = 198.9, g∥ = 2.21, Figure S10B), and the increase of a d−d (Figures S7C−S9C). This is however in agreement with the band at 500 and 600 nm in the CD spectrum (Figure S10C), potentiometric data: in Cu(II)-L systems of L4-L6, [CuL]3− confirming the increasing abundance of a form in which four species is less abundant than its co-species�in the case of nitrogen atoms are present in the Cu(II) coordination sphere Cu(II)-L7, [CuL]3− species is slightly more abundant than its and another nitrogen atom deprotonates.22,23,27,29−33 The co-partner [CuH2L]−. Additionally, in the UV−vis spectrum at coordination mode for [CuH−1L]4− is therefore {2Nim, 2N−}. pH 8.12, the presence of a blue shift is observed (600−560 [CuH−2L]5− and [CuH−3L]6− (maximum concentration at pH nm), suggesting that there are two species in which three and 11.0 and above 11.0, respectively) species are most probably four nitrogen atoms are present in the Cu(II) coordination related to the deprotonation of the second Lys residue and sphere (Figure S11A).22,27 This is supported by the EPR third amide bond. The increase of pH to 11.10 causes the shift parameters at pH 8.03: A = 192.1, g∥ = 2.22 (Figure of a band in the UV−vis spectrum (540−520 nm, Figure S11B).22,23,32,33 The increase of pH to 9.20 (in which S10A) and the change of EPR parameters (A = 191.4, g∥ = [CuL]3− dominates) causes a blue shift (560 to 540 nm), 2.20,Figure S10B), confirming that another amide nitrogen is which strongly supports the assumption that four nitrogen in the Cu(II) coordination sphere.22,27 The [CuH−2L]5− and atoms bind Cu(II). The coordination mode for [CuL]3− is [CuH−3L]6− coordination mode is {Nim, 3N−}.33 {3Nim, 1N−}. [CuH−1L]4− and [CuH−2L]5− (maximum Cu(II)-L7. The potentiometric titrations of the Cu(II)-L7 concentration at pH 10.0 and 10.5, respectively) most system indicated that similar to Cu(II)-L4, Cu(II)-L5, and probably come from the deprotonation of the second and Cu(II)-L6, nine exact complex forms exist at the pH range of third amide bonds. In the CD spectra at pH 10.03 and 11.02, 2−11: [CuH 6 L] 3+ , [CuH 5 L] 2+ , [CuH 4 L] + , [CuH 3 L], we can observe that the intensity of the d−d band elevates with [CuH2L]−, and [CuL]3− and [CuH−1L]4−, [CuH−2L]5−, and increasing pH, suggesting that the formation of a square planar [CuH−3L]6− (Figure 2G). [CuH6L]3+ and [CuH5L]2+ most complex and a second and third Cu(II)−N− bond is achieved probably arise from the deprotonation of the first and second (Figure S11C).29−32 Additionally, in the UV−vis spectrum, we His residues (maximum concentration at pH 4.0 and 5.0, can observe a blue shift (540−525 nm), confirming that at pH respectively). Both of the histidine imidazole groups are 11.02, only 4N-type complex species occur (Figure S11A).22,27 involved in Cu(II). This is confirmed by the arise of a band at The last species, [CuH−3L]6−, most likely arises from the around 700 nm in the UV−vis spectrum at pH 4.01 deprotonation of a second Lys residue. The coordination mode (corresponding to a bond of Cu(II) with one nitrogen for [CuH−1L]4−, [CuH−2L]5−, and [CuH−3L]6− is as follows: atom) and a shift of this band to 630 nm when the pH {2Nim, 2N−}, {1Nim, 3N−}, and {1Nim, 3N−}. increases to 5.21 (corresponding to a bond of Cu(II) with two Cu(II)-L8. The potentiometric titrations of the Cu(II)-L8 nitrogen atoms) (Figure S11A).22,27 Additionally, this finding system revealed that eight complex forms exist at the pH range is supported by the EPR parameters: A = 161.3, g∥ = 2.31 at of 2−11: [CuH6L]3+, [CuH5L]2+, [CuH4L]+, [CuH3L], pH 4.12 and A = 167.2, g∥ = 2.28 at pH 5.08 (Figure S11B).22 [CuHL]2− , [CuL] 3− , and [CuH ‑1L]4− and [CuH −3L] 6− The coordination modes for [CuH6L]3+ and [CuH5L]2+ are (Figure 2G). [CuH6L]3+ and [CuH5L]2+ most probably {1Nim} and {2Nim}, respectively. The next two species come from the deprotonation of the first and second His [CuH4L]+ and [CuH3L] (maximum concentration at pH 5.5 imidazole groups (maximum concentration at pH 4.5 and 5.0, 6903 https://doi.org/10.1021/acs.inorgchem.2c04486 Inorg. Chem. 2023, 62, 6893−6908 Inorganic Chemistry pubs.acs.org/IC Article respectively). Both of the residues are involved in Cu(II) does not significantly impact this correlation (Figure S13B). binding. This assumption is supported by the arise of a band at Peptides are not likely to form secondary and tertiary around 700 nm in the UV−vis spectrum at pH 4.11�it is structures, compared to native proteins. Some amino acid related to the formation of a bond between Cu(II) and one residues, such as Gly/Pro (common “helix breaker”) or with nitrogen atom�and a shift of this band to 640 nm when the amphiphilic side chains having both, a polar group and a pH increases to 5.18 (Figure S12A).22,27 This phenomenon is flexible hydrocarbon such as His, Lys, Glu, Arg, and Gln supported by the EPR parameters: A = 161.0, g∥ = 2.31 at pH additionally decrease the chances of peptides to form 4.12 and A = 169.5, g∥ = 2.28 at pH 5.08 (Figure S12B).22 The secondary structures.36 This is in agreement with the results coordination modes for [CuH6L]3+ and [CuH5L]2+ should be of XRD study of GroEL1 (M. tuberculosis)�the solved therefore {1Nim} and {2Nim}, respectively. The next species structure of this protein lacks approximately 20 amino acids [CuH4L]+ (maximum concentration at pH 5.5) most likely on the C-terminus, due to the high flexibility of this arises from the deprotonation of the third His imidazole ring. fragment.14 The pKa value of 5.10 for this step could be related to pKa = 5.80 for one of the His residues in the free ligand. It is only slightly reduced, suggesting that this residue is not involved in ■ DISCUSSION Stability of Cu(II) Complexes. To compare the stability of Cu(II) binding. In the UV−vis spectrum at pH 6.10−7.12, we the studied copper complexes and the affinity of different can observe a blue shift (630 to 620 and then 610 nm) (Figure ligands for the Cu(II) ion, the competition plots were drawn S10A), suggesting the presence of an equilibrium of species in (Figures 3 and 4). These graphs are based on the protonation which two and three nitrogen atoms are involved in Cu(II) binding.22,27 This conclusion is supported by the EPR parameters (A = 178.1, g∥ = 2.26 at pH 6.10; A = 179.7, g∥ = 2.25 at pH 7.12) (Figure S10B).22,23,35 It is also in agreement with the potentiometric data�at the pH range of 5.0−8.0, another species occurs, [CuH 3L] (maximum concentration at pH 6.5) (Figure 2H). Most probably, it comes from the deprotonation of an imidazole group of the fourth His residue. The pKa value of 5.68 for this step could correspond to pKa = 8.09 for one of the His residues in the free ligand and is significantly reduced�which means that this residue is involved in Cu(II) binding. Coordination modes for [CuH4L]+ and [CuH3L] are {2Nim} and {3Nim}, respectively. The next species [CuHL]2− with a maximum concentration at pH 8.0 is most probably related to the deprotonation of one non-metal binding His residue and one amide bond. This is confirmed by the presence of a d−d band in the CD spectrum at pH 8.12, corresponding to the formation of the Cu(II)−N− bond and the change of geometry to square planar (Figure S10C).29−32 It suggests that the formation of a complex in Figure 3. Competition plot between L1 and L2 ligand complexes with which four nitrogen atoms bind Cu(II) starts. In addition, in the Cu(II) ion, showing complex formation in a hypothetical solution the UV−vis spectrum at pH 8.12, a slight blue shift was in which the metal ion and two peptides are present. The calculation detected (610−585 nm, Figure S10A) and the EPR parameters based on the potentiometric data for the studied systems is given in changed significantly (A = 195.3, g∥ = 2.21, Figure S10B), the table. suggesting that the abundance of a form containing more Cu(II)−N-type bonds increased.23,33 The coordination mode and stability constants and show a hypothetical situation in for [CuHL]2− is {3Nim, 1N−}.22 Two other species [CuL]3− which equimolar concentrations of all reagents are present. and [CuH‑1L]4− (maximum concentration at pH 9.0 and 10.0, They are theoretical, based on the potentiometric data. Figure respectively) most likely arise from the deprotonation of 3 reveals that the longer counterpart (L2) forms a more stable another two amide bonds. We can observe two effects that complex with Cu(II) than the shorter one (L1). This can be confirm this result: (1) the presence of a blue shift at pH 9.01 easily explained: the potentiometric and spectroscopic studies (585−560 nm) and then at pH 10.10 (560−540 nm) suggests (Figures S5 and S6) revealed that in L1, two His residues are the increase of the species in which four nitrogen atoms bind most likely to bind Cu(II) with three His residues in the case Cu(II) (Figure S12A) and (2) the d−d band in the CD of L2. Theoretical explanation can also be given: L2 is more spectrum becomes more abundant (Figure S12C). An increase hydrophilic, so in aqueous solutions, it will show better of the pH to 11.06 does not significantly impact the parameters properties as a ligand because of its significant amount of of CD and UV−vis spectra. This is in agreement with the charged amino acid residues (e.g., Lys and Asp). These potentiometric data�the most abundant species at pH around residues should stabilize the complex through the formation of 11.0 is [CuH−3L]6− that presumably is related to the ion pairs, hydrogen bonds, and other less specific electrostatic deprotonation of two non-metal-binding Lys residue. interactions.37,38 The formation of Lys−Asp interactions, so Secondary Structure Studies. Under the conditions of the called “salt bridges”, is possible. Nevertheless, we do not CD experiment at pH 2−11 that were adjusted to observe the α-helix formation (Figure S13A,B), perhaps due to potentiometric titrations, all of the studied ligands exhibit a the conditions of the CD experiments and the presence of the random-coil structure. An example of the CD spectrum of Pro residue and many His residues.36 Another explanation can ligand L2 is presented in Figure S13A. The presence of Cu(II) be given by discussing the charge of the peptides: in the initial 6904 https://doi.org/10.1021/acs.inorgchem.2c04486 Inorg. Chem. 2023, 62, 6893−6908 Inorganic Chemistry pubs.acs.org/IC Article still able to bind Cu(II). We can see it from the results of the potentiometric, MS, and spectroscopy experiments (Figures 2 and S1−S14). However, we can also observe that the original L2 is more likely to interact with the Cu(II) ion: in the previous sections, we discussed that L2 reaches the {3Nim} coordination mode at pH 6.07 (Figure S6). All of the mutations “disrupt” the system: we can see that all of the mutants can finally bind three His residues (Figures S7−S12), but these systems struggle to find this third binding site for the metal ion: in the 6.0−8.0 pH range, we can observe an equilibrium of species in which two or three nitrogen atoms bind Cu(II). This indicates that the deletion of one His residue weakens the coordination properties of L3−L8 ligands. We can observe this correlation in Figure 4. It also shows that in the most biologically important physiological pH around 7.4, the stability of the Cu(II) complex with all mutants except L5 is very similar but lower compared to that of Cu(II)-L2. The reason for it could be that every positively charged His residue Figure 4. Competition plot between L2−L8 complexes with the forms strong electrostatic interactions with negatively charged Cu(II) ion, showing complex formation in a hypothetical solution in amino acid residues such as Glu and Asp and C-terminus that which the metal ion and seven peptides are present. The calculation stabilize the structure of peptides, proteins, and their metal based on the potentiometric data for the studied systems is given in complexes.39 On the other hand, even in the presence of five the table. His residues, not six (like in L2), the system manages to finally reach the {3Nim} coordination mode. What could this mean phase, i.e., low pH, acidic residues deprotonate. Then, the for the biological role of GroEL1? If an undesirable, accidental positive sum charge of L2 is higher than that of L1; therefore, mutation occurs and the HRCT will end with less than six L1 forms slightly more stable complexes with Cu(II) at the low potential binding sites, the GroEL1 could still function as a pH of 3.0−4.0. With the deprotonation of successive His metal transporter. This is very important for the whole residues (above pH around 4.3), this correlation is reversed biological system. It also indicates one more thing: most and Cu(II)-L2 is more stable than Cu(II)-L1 because it is less probably, there is not only one proper binding mode for positively charged. As a result of the deprotonation of Lys GroEL1 HRCT but a few possible binding modes. residues in L2, the electrostatic interactions of Asp−Lys However, is it possible to point out which “side” of the significantly weaken, and the stabilities of Cu(II)-L2 and GroEL1 HRCT has greater affinity for Cu(II)? The most Cu(II)-L1 at pH above 9.5 are again very similar. accurate methods for these studies could be NMR spectros- Which His mutations significantly impact the stability of the copy or XRD crystallography. In our systems, the Cu(II) Cu(II) complex? The answer to this question is intriguing. It is paramagnetic properties and the very close neighborhood of clear that even in the absence of one His residue, the ligand is each His residue do not make it straightforward to obtain Figure 5. ESI-MS/MS spectrum of the Cu(II)-L2 sample at (A) 1325−1575 m/z range and (B) 1550−1775 m/z range. Parent ion m/z = 1021.93, z = 2+; collision energy 40 eV. The fragmentation pattern was assigned.41 6905 https://doi.org/10.1021/acs.inorgchem.2c04486 Inorg. Chem. 2023, 62, 6893−6908 Inorganic Chemistry pubs.acs.org/IC Article informative NMR spectra. The flexibility and inability to form on His11 significantly disrupts the stability of Cu2L species secondary structures of our ligands�a property mentioned under the ESI conditions or relatively prevents from binding before in the Introduction section and confirmed by CD the second Cu(II) ion. Mutation on His9 and His12 causes the secondary structure studies�revealed that XRD is also not a increase of Cu2L species abundance (Figures S3B and S4B). suitable analytical method in this case. Therefore, we tried Interestingly, His13, His15, and His17 mutations cause the another approach: tandem mass spectrometry studies. We huge increase of Cu2L signal intensity (Figure S4C−E). isolated and fragmented the parent ions of [L]2+ (m/z = Opposite to the other mutations, these also caused the 991.44, z = +1) and [CuL]2+ (m/z = 1021.93, z = +2) of L2, significant decrease of mononuclear species. We could assume and the results of these experiments are presented in Figures that the His11 is the most important residue for the binuclear S14 and 5. The comparison of the two spectra revealed in species stability in the presence of Cu(II) ion excess and is an Figure 5 additional signals corresponding to ions of Cu(II)- important Cu(II)-binding site, which is in agreement with the containing fragments: Cua12, Cua13, Cub12, Cuc14, and Cuz15� MS/MS result and also supports the competition plot of the they correspond to a Cu(II) complex of appropriate fragments. equimolar M/L mixture (Figures S4A, 4, and 5). On the other The presence of the above-mentioned signals in the MS/MS hand, His13, His15, and His17 residues seem to prevent the spectrum strongly suggests that three of the His9−His13 binuclear species formation under the ESI experiment residues, Ac-DKPAKAEDHDHHH, preferably bind Cu(II). conditions and may not be so important in Cu(II) binding. This is in agreement with the potentiometric results�the L4 His11−12 most probably are also important for Cu2L species mutant lacking the His11 residue forms the least stable stability and could provide binding sites for Cu(II). complex with Cu(II) in the widest pH range. The reason for this preference might be that the close neighborhood of two Asp residues in this region additionally supports Cu(II) ■ CONCLUSIONS In this work, we fully characterized the properties of the binding�the Asp residue itself is known for being a binding mycobacterial GroEL1 C-terminus and its mutants. We site for Cu(II), although Cu(II) prefers the His residue.40 If established that in the equimolar mixture of reagents, all of His11 was the main binding site for Cu(II) in L2−L3 and L5− the ligands form equimolar complexes with Cu(II). In the case L8, the three amide bonds involved in metal binding at high of L2−L8, the coordination sphere of the metal ion most pH above 9 would preferably belong to the two next amino probably is occupied by three His residues, and in the case of acid residues on the left: Ac-DKPAKAEDHDHHHGHAH (in L1, it is occupied by two His residues. The amino acid residues the N-terminus direction)�the presence of negatively charged adjacent to the HRCT fragment significantly increase the Asp residues could stabilize the metal ion on this site. stability of the complexes. The mutations on all six His Another interesting result is that Cu(II)-L5 has a very residues do not impact the number of the binding sites for similar stability as that of Cu(II)-L2 in the wide pH range. Cu(II) but affect the stability of the complexes�it is lower for What can be the reason for it? The sequence of Ac- the peptides containing five His residues instead of six. DKPAKAEDHDHQHGHAH (L5) has a very special pattern: Surprisingly, L5 appeared to be the most stable of all all His residues are equally remote from each other, by one mutants�this is probably due to its unique His arrangement, amino acid residue. It suggests that the Cu(II) ion has a similar equally remote from each other, by one amino acid residue. distance to all of its potential binding sites, and this simple This could increase the chances of reaching the Cu(II) regularity could be a reason for additional stability to the coordination sphere equally for all His residues of L5 and complex system.42 GroEL1 proteins of different Mycobacteria elevate the stability of this system. Most probably, in the case species possess a repeatable pattern of His residue arrangement of L2, the metal-binding sites are His residues arranged closer at the C-terminus: -DKPAEEADDHGHGHHHH (Mycobacte- to the “left side” of the peptides�the stability of Cu(II)-L4, in rium avium (strain 104), UniProt code: CH601_MYCA1),43 which the His11 residue is replaced with the Gln residue, is the -EKPAEAEDDGHGHGHGHHHH (Mycolicibacterium gilvum lowest in the widest pH range. This is supported by the MS/ (strain PYR-GCK), UniProt code: CH601_MYCGI),44 MS fragmentation spectral analysis. -DEPANAHEHDHGHGHGHHHHH (Mycobacterium kansa- sii, UniProt code: A0A653F4Z6_MYCKA), 45 -DKPA- KAEDHDHHHGHAH (Mycobacterium bovis (strain ATCC ■ * ASSOCIATED CONTENT sı Supporting Information BAA-935/AF2122/97), UniProt code: A0A679LKC3_MYC- The Supporting Information is available free of charge at BO),46 and -DKPAEEADDHGHGHHHH (Mycolicibacterium https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c04486. paratuberculosis (strain ATCC BAA-968/K-10) (Mycobacterium paratuberculosis), UniProt code: CH601_MYCPA).47 It Complementary mass spectra, UV−vis/CD/EPR spec- supports the suspicion that the probable binding sites are tra, and MS/MS spectrum (PDF) arranged similarly like in the case of L5�at the same distance from each other. This would be in agreement with our findings and could support the explanation of the competition plot ■ AUTHOR INFORMATION Corresponding Authors (Figure 4). Anna Rola − Faculty of Chemistry, University of Wroclaw, 50- We do not exclude the possibility to form binuclear 383 Wroclaw, Poland; Email: anna.rola@chem.uni.wroc.pl complexes in the presence of an excess of metal ions. MS Sławomir Potocki − Faculty of Chemistry, University of studies at pH 7.5 revealed that all L2−L8 ligands tend to form Wroclaw, 50-383 Wroclaw, Poland; stable binuclear complexes with Cu(II), when the mixture of Email: slawomir.potocki@chem.uni.wroc.pl M/L = 2:1 is applied (Figures S2B, S3B, S4A−E). However, we can see that in the case of particular mutants, this tendency Authors along with the ability to be ionized decreases or increases, Oscar Palacios − Departament de Química, Universitat compared to the original sequence. It seems like the mutation Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain 6906 https://doi.org/10.1021/acs.inorgchem.2c04486 Inorg. Chem. 2023, 62, 6893−6908 Inorganic Chemistry pubs.acs.org/IC Article Merce Capdevila − Departament de Química, Universitat (10) Ansari, M. Y.; Batra, S. D.; Ojha, H.; Dhiman, K.; Ganguly, A.; Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Tyagi, J. S.; Mande, S. C. A Novel Function of Mycobacterium Spain; orcid.org/0000-0002-2246-0994 Tuberculosis Chaperonin Paralog GroEL1 in Copper Homeostasis. Daniela Valensin − Department of Biotechnology, Chemistry FEBS Letters 2020, 594 (20), 3305−3323. and Pharmacy, University of Siena, 53100 Siena, Italy (11) Battistoni, A.; Pacello, F.; Mazzetti, A. P.; Capo, C.; Kroll, J. S.; Langford, P. R.; Sansone, A.; Donnarumma, G.; Valenti, P.; Rotilio, G. Elżbieta Gumienna-Kontecka − Faculty of Chemistry, A Histidine-Rich Metal Binding Domain at the N Terminus of Cu,Zn- University of Wroclaw, 50-383 Wroclaw, Poland; Superoxide Dismutases from Pathogenic Bacteria: A NOVEL orcid.org/0000-0002-9556-6378 STRATEGY FOR METAL CHAPERONING *. J. Biol. Chem. Complete contact information is available at: 2001, 276, 30315−30325. https://pubs.acs.org/10.1021/acs.inorgchem.2c04486 (12) Cheng, T.; Xia, W.; Wang, P.; Huang, F.; Wang, J.; Sun, H. Histidine-Rich Proteins in Prokaryotes: Metal Homeostasis and Notes Environmental Habitat-Related Occurrence. Metallomics 2013, 5, The authors declare no competing financial interest. 1423−1429. (13) Rowinska-Zyrek, M.; Witkowska, D.; Potocki, S.; Remelli, M.; ■ ACKNOWLEDGMENTS Financial support by the National Science Centre (UMO- Kozlowski, H. His-Rich Sequences − Is Plagiarism from Nature a Good Idea? New J. Chem. 2013, 37, 58−70. (14) Colaco, C. A.; MacDougall, A. 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