Investigation of the metal-binding site 2 of Y. pestis YfeA protein: solution studies of Fe(II), Mn(II), and Zn(II) complexes

Abstract

YfeABCD is a metal-ion transport system found in Yersinia pestis, responsible for Fe(II) and Mn(II) uptake. YfeA is a periplasmic substrate-binding protein (SBP) that binds the metal ion in the periplasm and facilitates its transfer to the transmembrane YfeC and YfeD proteins. Interestingly, ITC and crystallographic data indicate that YfeA can also bind Zn(II) ions as a preferred substrate, even though the YfeABCD system most likely does not transport zinc. YfeA possesses two metal-binding sites. Site 2 was shown to bind Zn(II) and Mn(II), but not Fe(II) ions. In this work, we have investigated the metal-binding properties of a peptide model of YfeA Site 2. We have utilized a variety of physicochemical methods to characterize the stoichiometry, stability constants, and metal-binding residues in Fe(II), Mn(II), and Zn(II) complexes.

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Introduction

The ability to sequester essential elements in sufficient amounts to support growth and development is often a decisive factor in bacterial survival.^1,2 The importance of metal uptake is underscored by a great variety of metal transporters expressed by bacteria.^3,4 In the case of iron acquisition, bacteria have developed a variety of strategies for the uptake of both Fe(II) and Fe(III), an evolutionary adaptation to thrive in both anaerobic and aerobic niches (in which ferrous and ferric iron, respectively, are prevalent). Fe(II) exhibits sufficient solubility to be transported across the inner bacterial membrane in the form of a free metal ion.⁵ This process is carried out by a variety of transport systems, usually consisting of a transmembrane protein acting as a direct metal transporter, which can be supported by accessory proteins located either in the bacterial cytoplasm, membrane, or periplasm.

Only a few Fe(II) transport systems are widely distributed across Gram-positive and Gram-negative bacteria. An example of such a system would be Feo, regarded as the most widespread and important bacterial ferrous iron uptake system.⁶ Other systems are rather specific to narrow pathogen groups, or even certain pathogens, like IroT,⁷ FbpABC,⁸ SitABCD,⁹ characteristic of Legionella, Neisseria, and Salmonella genera of bacteria, respectively. Another example can be the YfeABCD system, found in Yersinia pestis, a causative agent of the plague in its three forms: bubonic, septicemic, and pneumonic.¹⁰

YfeABCD or Yfe system was discovered in the late 1990s and classified as a member of the ABC-transporter family.^11–13 The system comprises four proteins: periplasmic metal-binding YfeA, ATP-binding YfeB, and transmembrane YfeC and YfeD proteins (Fig. 1).

Fig. 1 Scheme of Fe(II) and Mn(II) transport by Yfe system. The system is comprised of periplasmic substrate-binding protein YfeA, transmembrane proteins YfeC and YfeD, an ATP-binding protein YfeB, and inner membrane protein of unknown function, YfeE. Fe(II) and Mn(II) are transported across the outer membrane most probably by porins, and chelated in the periplasm by YfeA. Next, the metal ions are trafficked through the inner membrane by YfeC and YfeD proteins, with the use of the energy released from the ATP hydrolysis. UniProt entries: Q56952 (YfeA), Q56953 (YfeB), Q56954 (YfeC), Q56955 (YfeD), Q56956 (YfeE). Proteins visualized with UCSF Chimera.¹⁴

Yfe system has been shown to transport both Fe(II) and Mn(II), and therefore is not metal-specific, similarly to many other divalent metal ion transporters, such as MntH or ZupT.⁵ It is regulated by Fur protein¹⁵ and is TonB-independent, with M(II) metal ions probably being transported through the outer membrane by general or metal-specific porins.¹⁶ As iron assimilation is crucial during the infection of the host, unsurprisingly, Yfe plays an important role in the pathogenicity of Y. pestis. It has been shown that while a siderophore, yersiniabactin,¹⁷ is essential for iron uptake during the initial stages of the plague, the Yfe system could play a greater role in iron assimilation during the disease progression from the lymphatic nodes to other organs.¹³ On the contrary, neither Feo nor Yfe systems are significant for the development of the pneumonic plague, however, they seem to play a role in its bubonic form.^18,19 Mutations in Yfe system genes generated growth defects in Y. pestis exhibited only in limited oxygen conditions, consistent with the system being a transporter of Fe(II), stable in anaerobic conditions.¹⁹ However, both Feo and Yfe systems have been shown to transport iron under reducing and non-reducing conditions. Moreover, they have shown the capacity to use both Fe(II) and Fe(III), albeit ferric iron is most likely reduced to ferrous form before the transport.²⁰

As a periplasmic substrate-binding protein (SBP), YfeA plays a key role in determining the metal ion specificity of the Yfe transport system. While Yfe is regarded as an Fe(II) and Mn(II) transporter, it has been shown by ITC experiments that Zn(II) can also bind to YfeA with affinity similar to that of Mn(II). The determined dissociation constant values are in the nanomolar range, suggesting a very tight binding of both metal ions.²¹ The possibility of Zn(II) binding to YfeA was further corroborated by the crystal structures of the protein, which have shown zinc as the preferred metal incorporated into two metal-binding sites, however, it is not clear whether this behavior is an example of mismetalation of the protein or an indication of Yfe being also a zinc transporter under certain conditions.²² As many other SBPs, YfeA adopts a c-clamp structure. Deeply buried canonical metal-binding site consists of two histidine residues, one glutamic acid, and one aspartic acid residue (H76, H141, E207, D282). X-ray fluorescence data have shown that this site binds Fe(II), Mn(II), and Zn(II) in order of increasing preference. Interestingly, a second metal-binding site has been discovered on the surface of the protein. It consists of glutamic acid and histidine residues, with two water molecules completing the coordination sphere of Zn(II) and Mn(II). Curiously, no crystals of YfeA with iron-bound site 2 have been obtained. The exposure of site 2 on the surface of the protein, as well as the presence of water molecules in the metal coordination sphere, suggests a labile metal binding and possibly a regulatory or communication role with other parts of YfeA. Furthermore, it has been shown that site 2 can participate in Zn(II) and Mn(II) coordination when the metal is bound by more than one YfeA molecule.²³ When site 2 is metal-bound, four other residues can bind zinc, with three of them (D164, H167, E169) being in the vicinity of the site (E162, H163).²²

While X-ray data provided an excellent overview of the location of the metal-binding sites and identity of the bound metal ions, we are very much interested in additional insight into the metal-binding peculiarities, such as the affinity toward the discussed metal ions. The solution studies could aid in characterizing the protein's coordination chemistry and identifying its metal-binding sites under conditions that more closely reflect its native environment. However, due to the size of the protein and the large number of deprotonating residues it contains, studying the metal-ion binding preferences in solution is rather challenging. In such cases, carefully designed model peptides mimicking binding sites of various proteins can be used as simplified protein models to better understand the factors influencing the complexation process and the coordination chemistry of the studied metal ions. We acknowledge that peptide models cannot fully reproduce long-range electrostatic interactions or the rigid scaffold of a folded protein, both of which may influence the entropy of binding. Nevertheless, studies employing peptide models and full-length proteins provide different yet complementary insights. Investigations on intact proteins primarily address biological function and the role of the overall fold, whereas peptide models enable the isolation and precise characterization of the fundamental coordination chemistry of the binding site itself, including thermodynamics, stoichiometry, and geometry. These intrinsic properties are often obscured within the complex structural environment of the whole protein. This reductionist approach is particularly valuable for intrinsically disordered proteins or well-defined local binding motifs, where metal coordination is governed mainly by local chemical affinity rather than by tertiary structure.^24,25 Moreover, peptide models are highly cost-effective and allow rapid screening without the need for protein overexpression. They also facilitate systematic modifications, such as alanine scanning or the incorporation of non-natural residues, providing detailed insight into the specific contribution of individual amino acids to metal binding.^26,27 Finally, the use of peptide models reduces spectral complexity, enabling high-resolution characterization by techniques such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) (vide infra).

The Y. pestis YfeA site 2 seems very suited for such detailed studies with the use of a peptide model, as all of the metal-binding residues are located close to each other in the protein sequence, in contrast to site 1. We have decided to select YfeA fragment containing site 2 (E162, H163) and three ancillary zinc-binding residues (D164, H167, E169), Ac-V₁₆₁EHDPAHAET₁₇₀-NH₂ (Fig. 2). The peptide has been acetylated and amidated at the N-terminus and C-terminus, respectively, to resemble protein conditions. While site 2 has been shown to bind only Zn(II) and Mn(II), we have decided to investigate also Fe(II) complexes, in the hope of finding differences in the metal binding, which could explain the metal preferences of site 2 in the native YfeA protein.

Fig. 2 YfeA structure with the selected peptide sequence visualized with stick representation. The sequence contains metal-binding site 2 of YfeA, consisting of E162 and H163 (colored red in the peptide sequence), as well as ancillary zinc-binding residues, D164, H167, and E169 (colored blue). PDB entry: 6Q1C. Visualized with UCSF Chimera.

Considering all the challenges when working with Fe(II) and Mn(II) complexes, such as high susceptibility of Fe(II) ions to oxidation, or the limitations in the use of spectroscopic methods in identifying Mn(II), or Fe(II) binding residues, to thoroughly characterize the complexes formed between the designed peptide model and Fe(II), Mn(II), and Zn(II), we have utilized a variety of available physicochemical methods, such as (MS), potentiometric titrations, NMR, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies. As there is undoubtedly an urgent need to deepen the knowledge about factors influencing the thermodynamic stability of complexes with those metal ions, we believe that these studies enrich the literature of peptide interactions with Fe(II) and Mn(II) ions, that is restricted to several examples only.^28–31 This knowledge may be of further help in elucidating the metal binding-structure–function relationships of naturally occurring proteins.

Experimental section

Materials

The peptide ligand L1, Ac-VEHDPAHAET-CONH₂, was ordered from KareBay® Biochem. Its identity was confirmed by mass spectrometry experiments, while its purity of >98% was confirmed using the Gran method on potentiometric data. Titripur® 0.1 M carbonate-free sodium hydroxide (Sigma-Aldrich) was standardized with potassium hydrogen-phthalate (Sigma-Aldrich) and used as a titrant in potentiometric titrations. Concentrated 70% perchloric acid (Sigma-Aldrich) and solid sodium hydroxide (Sigma-Aldrich) were used to prepare solutions of various concentrations for manual pH changes in NMR, EPR, and CD experiments. Sodium perchlorate (Sigma-Aldrich) was used to adjust the ionic strength in potentiometric titrations, CD, and EPR experiments. Deuterium oxide (Sigma-Aldrich) was used for sample preparation in NMR experiments. Solutions of the Mn(II) and Zn(II) were prepared from corresponding perchlorates (Sigma-Aldrich), while Mohr's salt (ammonium iron(II) sulfate, Sigma-Aldrich) was used to prepare Fe(II) solution in anaerobic conditions. Mn(II) and Zn(II) solutions were standardized using ICP-OES and complexometric titration with murexide and standardized ethylenediaminetetraacetic acid disodium salt (Na₂H₂EDTA). Fe(II) solution concentration was determined by the colorimetric method, for which 1,10-phenanthroline (Sigma-Aldrich) was used. Due to the oxidation-prone nature of Fe(II), all samples containing Fe(II) were prepared in an argon atmosphere inside the glove box, using deoxygenated solvents, immediately before the experiments. Before taking the samples for the experiments outside of the glove box, laboratory glassware containing the samples (potentiometric vessel, CD cuvette, NMR tube, vial for MS experiments) was carefully sealed. Throughout the experiments, the samples remained colorless, and we did not observe oxidation to Fe(III). Only after exposing the samples to air after finished experiments, a yellow color could be observed, indicating the presence of Fe(III). No signals related to Fe(III)-complexes could be identified in the mass spectra. No formation of red-colored Fe(III) complex has been observed after the addition of thiocyanate anions in anaerobic conditions to the sample after the NMR experiments. Therefore, we believe that our procedure of sample preparation for Fe(II) experiments is reliable, preventing oxidation to Fe(III) during the experiments.

Electrospray-ionization mass spectrometry (ESI-MS)

Mass spectra for Mn(II) and Zn(II) complexes were recorded on Shimadzu LCMS-9050-QTOF mass spectrometer, while Fe(II) samples were recorded on Bruker Q-TOF compact spectrometer. Ligand concentration was 0.1 mM, M : L ratio was 1 : 1 and 1 : 2. All samples were prepared in LC-MS grade methanol : water mixture (50 : 50, w/w) and diluted with methanol before the injection. The spectra were recorded in the positive ion mode. For the Shimadzu LCMS-9050-QTOF, the parameters of the spectra acquisition were as follows: nebulizing gas: nitrogen, nebulizing gas flow: 3.0 L min⁻¹, drying gas flow: 10 L min⁻¹, heating gas flow: 10 L min⁻¹, interface temperature: 300 °C, desolvation line temperature: 400 °C, detector voltage: −2.02 kV, interface voltage: 4.0 kV, collision gas: argon, mobile phase: MeCN +0.1% HCOOH. Some of the parameters used on Bruker Q-TOF compact were: dry gas, nitrogen; T = 170 °C; capillary voltage: 4500 V; ion energy: 5 eV. The spectra were processed using LabSolutions software (Shimadzu, Kyoto, Japan), and Compass Data Analysis 4.0 software (Bruker Daltonics).

Potentiometric titrations

The potentiometric titrations were carried out on an automatic system consisting of Metrohm Titrando 905 titrator connected to Dosino 800 dosing system. InLab Semi-Micro (Mettler-Toledo) electrode was used to measure the potential during the experiments. The electrode was calibrated for hydrogen ion concentration by acid–base titration of 2 ml of 4 mM perchloric acid. Standardized Titripur® sodium hydroxide was used as a titrant for every titration. The stability constants of proton, Fe(II), Mn(II), and Zn(II) complexes with ligand L1, were calculated from the potentiometric data with SUPERQUAD³² and HYPERQUAD 2008³³ software. HYSS³⁴ software was utilized to prepare the speciation and competition plots, as well as to calculate the pM and K_d values. The samples contained ligand in 1 mM concentration, 0.1 M sodium perchlorate as an ionic strength, and 4 mM perchloric acid to adjust the pH to about 2.0. The Gran method was used to determine the exact ligand concentration. For the titrations of the metal complexes, M : L = 1 : 1.1. Standard deviations calculated by HYPERQUAD 2008 refer only to random errors. The hydrolysis constants for Fe(II), Mn(II), and Zn(II) were taken into account for the stability constants calculations (Table S1).

Nuclear magnetic resonance (NMR) spectroscopy

NMR experiments were performed using a Bruker Ascend™ 400 MHz spectrometer equipped with 5 mm automated tuning and a matching broadband probe (BBFO) with z-gradients. Samples utilized for NMR experiments ranged from 0.4 to 1.0 mM and were dissolved in a 90/10 (v/v) H₂O–D₂O solvent mixture. All NMR experiments were performed at 298 K in 5 mm NMR tubes. The 2D ¹H–¹³C heteronuclear correlation spectra (HSQC) were acquired using a phase-sensitive sequence employing Echo-Antiecho-TPPI gradient selection with a heteronuclear coupling constant J_XH = 145 Hz and shaped pulses for all 180° pulses on the f2 channel with decoupling during acquisition. Sensitivity improvement and gradients in back-INEPT were also employed. Relaxation delays of 2 s and 90° pulses of about 10 μs were applied for all experiments. Solvent suppression was achieved using excitation sculpting with gradients. The spin-lock mixing time of the ¹H–¹H TOCSY experiment was obtained with MLEV17. ¹H–¹H TOCSY experiments were performed using mixing times of 60 ms. ¹H–¹H ROESY spectra were acquired with spin-lock pulses duration in the range of 200–250 ms. The assignments of 1H and ¹³C were made by a combination of mono- and bi-dimensional and multinuclear NMR techniques: ¹H–¹H TOCSY, ¹H–¹³C HSQC, and ¹H–¹H ROESY, at different pH values. To avoid severe broadening of the signals, because of the paramagnetic character of Mn(II) and Fe(II), the NMR experiments were performed with the subsequent addition of a substoichiometric amount of metal ion to the ligand solution. All NMR data were processed using TopSpin (Bruker Instruments) software and analyzed using Sparky 3.11 and MestReNova 6.0.2 (Mestrelab Research S.L.) programs.

Circular dichroism (CD) spectroscopy

Jasco J-1500 CD spectrometer was used to record the CD spectra. Spectra were acquired in 180–250 nm range, with scanning speed 400 nm m⁻¹, optical path length: 0.01 cm. Seven accumulations of each spectra were acquired. Ligand concentration was 0.35 mM, M : L ratio was 1 : 1.1. Spectra Analysis (Jasco Inc.) software was utilized for data processing.

Electron paramagnetic resonance (EPR) spectroscopy

EPR was used to study the Mn(II) : L1 system. The EPR spectra at X-band frequency were acquired using Bruker ELEXSYS E500 CW-EPR spectrometer equipped with an NMR teslameter (ER 036TM) and a frequency counter (E 41 FC). Spectra were acquired with a microwave power of 10 mW, modulation amplitude of 1 mT, and modulation frequency of 100 kHz. The peptide concentration was 1 mM, and the metal : ligand molar ratio was 1 : 1.1. The spectra were acquired at room temperature. Data was processed with Doublet new (EPR OF S = 1/2) program by A. Ozarowski (National High Field Magnetic Laboratory, University of Florida, Gainesville, FL).

UV-Vis spectroscopy

Absorption spectra in 350–650 nm range were recorded using Jasco V-730 UV-Visible spectrophotometer. Scanning speed: 400 nm per minute, number of accumulations: 1, cuvette optical length: 0.1 cm. The colorimetric determination of Fe(II) concentration was based upon the formation of 1 : 3 M : L red Fe(II) complex with 1,10-phenanthroline (λ_max = 510 nm). Utilizing previously prepared calibration curve for 0.1–1.1 mM range, the concentration of the Fe(II) solution was determined based on the absorption at 510 nm. The excess of 1,10-phenanthroline (M : L = 1 : 5) was used to ensure the complete complexation of the metal ion.

Results and discussion

We have utilized several physicochemical methods to characterize the complexes formed between L1, Ac-VEHDPAHAET-CONH₂, and Fe(II), Mn(II), and Zn(II). Mass spectrometry provided information about the stoichiometry of the formed complexes. The potentiometric data were used to determine the stability constants and calculate the affinity of the ligands towards the studied metal ions. The NMR spectroscopy provided information about the mode of metal-binding at various pH values, CD spectra reflected the conformational changes of the systems, and EPR spectroscopy provided further confirmation of the formation of Mn(II) complexes.

L1 contains 5 amino acid residues able to deprotonate in the studied pH range (2–11), therefore, fully protonated form is [H₅L]²⁺. The positive charge is a result of the protonated imidazole rings in two His residues. This complex form dominates from the start of the titration at pH = 2.0, up to pH of about 4.10 (Fig. S1). The deprotonation of the Asp residue with the pK_a value of 3.45 leads to the formation of [H₄L]⁺ species. The subsequent deprotonation of the two Glu residues results in [H₃L] and [H₂L]⁻ formation in solution. The pK_a values determined for these deprotonations are 4.09 and 4.72. Two His residues are the last to deprotonate, with the pK_a values 6.40 and 7.10, leading to the formation of [HL]²⁻ species, and fully deprotonated [L]³⁻, which is the only ligand form present in solution above pH about 10.0. Determined pK_a values are consistent with the literature.³⁵ The speciation plot for L1 is shown in Fig. S1, while the determined stability constants are collected in Table 1.

Table 1 Stability constants (log β) and pK_a values of the ligand Ac-VEHDPAHAET-NH₂ ^a

Peptide	Species	log β^b	pKa ^c	Deprotonating residue
a T = 298 K, I = 0.1 M NaClO4, standard deviations on the last digit given in parentheses.b Overall stability constants (log β) expressed by the equation: β(HnL) = [HnL]/[L][H + ]^n.c Acid dissociation constants (pKa) expressed as: pKa = log β(HnL) − log β(Hn−1L).
L1	[H5L]^2+	25.76(2)	3.45	Asp
	[H4L]^+	22.31(2)	4.09	Glu
	[H3L]	18.22(2)	4.72	Glu
	[H2L]^−	13.50(1)	6.40	His
	[HL]^2−	7.10(2)	7.10	His

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Stoichiometry of the metal complexes

ESI-MS experiments provided insight into the stoichiometry of the complexes formed between L1 and Fe(II), Mn(II), and Zn(II). The mass spectra for all studied systems exhibit signals related to the complexes of 1 : 1 (M : L) stoichiometry only. The acquired mass spectra are shown in Fig. S2–S4. Comparison of the simulated and experimental isotopic distribution ensured a correct peak assignment. Table S2 contains the most abundant peaks related to the free ligand and metal complexes.

Iron complexes

Fe(II) complexes were studied in anaerobic conditions, to prevent the oxidation of the metal ion. The best complexation model, obtained by fitting the experimental data, contains six complex species in Fe(II) : L1 system (Table 2). NMR experiments were performed using a Fe(II) : L1 molar ratio of 0.33 : 1 at pH 4.5, 7.0, and 8.9, to probe the interaction between Fe(II) and the peptide under varying protonation states.

Table 2 Stability constants (log β) and pK_a values of the Fe(II) : L1 system^a

Peptide	Species	log β^b	pKa ^c	Deprotonating residue
a T = 298 K, I = 0.1 M NaClO4, standard deviations given in parentheses.b Overall stability constants (log β) expressed by the equation: β([FeHnL](^n+2)^+) = [[FeHnL]^(n+2)+]/[Fe(II)][[L]^n+][H^+]^n.c Acid dissociation constants (pKa) expressed as: pKa = log β([FeHnL]^(n+2)+) − log β([FeHn−1L]^(n+1)+)
L1	[FeH2L]^+	16.79(6)	5.59	His
	[FeHL]	11.20(7)	6.73	His
	[FeL]^−	4.47(6)	7.44	Namide or Owater
	[FeLH−1]^2−	−2.97(5)	—	2 × (Namide or Owater)
	[FeLH−3]^4−	−20.67(5)	10.04	Namide or Owater
	[FeLH−4]^5−	−30.71(6)	—

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The complexation starts above pH = 3.50, with [FeH₂L]⁺, which contains one Asp and two Glu residues in deprotonated form (Fig. 3). The deprotonation of the His residue leads to the formation of [FeHL] species. In the NMR spectra recorded at pH 4.5, only minor perturbations were observed, including very slight effects on the aromatic imidazole protons of the His residue and on Asp and Glu residues. These subtle changes are consistent with potentiometric data, which indicate that the predominant species in solution is [FeH₂L]⁺, accounting for less than 20% of the total Fe(II) at this pH. The limited spectral response therefore suggests weak or incomplete complex formation under acidic conditions, as the high fraction of free ligand (∼80%) is likely to mask small perturbations at the coordinating residues.

Fig. 3 Distribution diagrams of complexes formed in Fe(II) : L1 system. Species distribution calculated for NMR experimental conditions: [L]_tot = 1 mM; Fe(II) : L = 1 : 3.

Completely deprotonated species, [FeL]⁻, forms because of the second His residue deprotonation. The determined pK_a values for the imidazole nitrogen deprotonation are lower in Fe(II) complex species (5.59 and 6.73) compared to the free ligand (6.40 and 7.10), suggesting the involvement of His residues in iron coordination.

At pH 7.0, where multiple species coexist, with [FeL]⁻ being the dominant form, distinct and selective perturbations were detected across several residues (Fig. 4). In particular, significant changes were observed in the side-chain nuclei of both His3 and His7, specifically in Hα, Hβ, Hε1, and Hδ2, confirming direct Fe(II) coordination through the imidazole nitrogen atoms (Fig. 5 and Fig. S5).

Fig. 4 Structural representation of the Fe(II) : L1 complex at pH 7, showing nuclei experiencing significant paramagnetic effects upon Fe(II) binding. Severely broadened signals, mapped onto the structure, indicate residues in close proximity to the metal center and highlight those directly or indirectly involved in the coordination environment.

Fig. 5 Comparison of the selected aromatic region of the ¹H–¹H TOCSY spectra for the free peptide L1 (red) and the Fe(II) : L1 complex (black) at a 0.3 : 1 molar ratio, at pH 7. Signals exhibiting significant chemical shift perturbations upon Fe(II) coordination are highlighted in red.

Moreover, strong paramagnetic effects were evident for Asp4, with selective disappearance of the Hα, Hβ, and HN proton signals, suggesting involvement of its carboxylate side chain in metal binding. In addition, the γ protons signals of Glu2 and Glu9 also disappeared, indicating their probable participation in coordination through their carboxylic groups (Fig. S6). Additionally, signal loss for the Hβ–HN cross-peak of Ala8 was noted, likely due to its spatial proximity to the paramagnetic Fe(II) center. However, the presence of a still-visible Hα–HN cross-peak suggests that Ala8's backbone amide remains protonated and is not directly involved in coordination. Therefore, the coordination mode of Fe(II) in the completely deprotonated, [ML] form, is most likely {2 N_im, 3 COO⁻, H₂O}.

The last three species detected in the studied pH range, [FeLH₋₁]²⁻, [FeLH₋₃]⁴⁻, and [FeLH₋₄]⁵⁻, are most probably a consequence of the peptide backbone amide nitrogen or water molecule deprotonations. The possibility of Fe(II) coordination by amide groups has been documented in the literature for various ligands.^36–41 Such a possibility has been negated for low-spin Fe(II) complexes,⁴² however, in this work, L1 forms high-spin complexes with Fe(II). This is confirmed by the selective broadening and disappearance of the signals in NMR spectra, consistent with the paramagnetic character of the high-spin Fe(II).

Manganese complexes

The processing of the potentiometric data acquired for Mn(II) : L1 system resulted in a very similar model to that of Fe(II), lacking only the last complex form. The complex species exhibit the same protonation and similar values of the stability constants (Table 3 and Fig. 6).

Fig. 6 Distribution diagrams of complexes formed in Mn(II) : L1 system. Species distribution calculated for NMR experimental conditions: [L]_tot = 1 mM; Mn(II) : L = 1 : 50.

Table 3 Stability constants (log β) and pK_a values of the Mn(II) : L1 system^a

Peptide	Species	log β^b	pKa ^c	Deprotonating residue
a T = 298 K, I = 0.1 M NaClO4, standard deviations given in parentheses.b Overall stability constants (log β) expressed by the equation: β([MnHnL](^n+2)^+) = [[MnHnL]^(n+2)+]/[Mn(II)][[L]^n+][H^+]^n.c Acid dissociation constants (pKa) expressed as: pKa = log β([MnHnL]^(n+2)+) − log β([MnHn−1L]^(n+1)+)
L1	[MnH2L]^+	17.46(7)	6.32	His
	[MnHL]	11.14(9)	6.80	His
	[MnL]^−	4.34(6)	9.54	Owater
	[MnLH−1]^2−	−5.20(6)		2 × Owater
	[MnLH−3]^4−	−25.77(5)

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The first species forming in solution is [MnH₂L]⁺. NMR titration experiments were conducted by progressively adding sub-stoichiometric amounts of Mn(II) to the L1 peptide system at pH 4.5. Under these acidic conditions, only minor spectral perturbations were observed, consistent with potentiometric data indicating that the predominant species is [MnH₂L]⁺, which accounts for less than 20% of total Mn(II) in solution. This suggests limited interaction between Mn(II) and the peptide at low pH.

In contrast, at pH 7.0, where two species, [MnHL] and [MnL]⁻, coexist in equilibrium, significant and residue-specific perturbations were detected. The paramagnetic effects of Mn(II) were particularly pronounced for His3, His7, and Glu2, followed by Glu9 and Asp4, suggesting their involvement in Mn(II) coordination (Fig. 7, 8, and Fig. S8, S9).

Fig. 7 Comparison of the selected aromatic region of the ¹H–¹H TOCSY spectra for the free peptide L1 (red) and the Mn(II) : L1 complex (gold) at a 0.005 : 1 molar ratio, at pH 7. Signals exhibiting significant chemical shift perturbations upon Mn(II) coordination are highlighted in red.

Fig. 8 Comparison of the selected aromatic region of the ¹H–¹³C HSQC spectra for the free peptide L1 (red) and the Mn(II) : L1 complex (gold) at a 0.005 : 1 molar ratio, at pH 7. Signals exhibiting significant chemical shift perturbations upon Mn²⁺ coordination are highlighted in red.

These effects were further mapped in Fig. 9, where the ¹H and ¹³C nuclei exhibiting significant shifts are color-coded, highlighting the residues most affected by Mn(II) binding.

Fig. 9 Structural representation of the Mn(II) : L1 complex at pH 7, showing nuclei affected by significant paramagnetic broadening upon Mn(II) binding. The mapped nuclei reflect close spatial proximity to the paramagnetic center, highlighting key residues involved in, or adjacent to, the metal coordination environment.

The data indicate a coordination environment involving both His residues, both Glu residues, and Asp4, suggesting a multi-dentate binding mode stabilized by side-chain donors. The calculated pK_a values ascribed to His residues deprotonation (6.32 and 6.80) are slightly higher than in Fe(II) system, which might suggest weaker interactions between Mn(II) and the His residues.

A similar coordination pattern was observed at pH 9.0, where the dominant species is [MnL]⁻, with comparable residue-specific perturbations (Fig. S10 and S11). This means that Mn(II) coordination sphere in [ML] form is most likely the same as in the Fe(II) complex: {2 N_im, 3 COO⁻, H₂O}.

The deprotonation of water molecules leads to [MnLH₋₁]²⁻ and [MnLH₋₃]⁴⁻ formation. Similarly to Fe(II), [MnLH₋₂]³⁻ form could not be detected.

The EPR spectra recorded for Mn(II) : L1 at room temperature at various pH values exhibit a distinct six-lined pattern characteristic of Mn(II), for which both electron (S) and nuclear (I) spins are 5/2 (Fig. S12). The acquired spectra are very similar, regardless of the pH, characterized by a hyperfine coupling constant, A, of approximately 95 G, and a g-factor value of around 2.0. Such values are consistent with hexaaqua Mn(II).^43,44 Although EPR spectroscopy can be a quantitative method with the use of external EPR standard of known concentration,⁴⁵ the quantitative quality of EPR spectrometers is generally poor, because of the difficulty in maintaining identical resonance parameters for each measured sample.⁴⁶ The decrease in the intensity with rising pH is only slightly pronounced, suggesting a rather slow exchange of the water molecules around the metal ion and thus slow changes in the ligand field of the metal ion, reflected in no significant broadening of the signal.⁴⁷ This might suggest the presence of free Mn(II) ions throughout the entire studied pH range, consistent with the distribution diagram shown in Fig. 6.

Zinc complexes

Three Zn(II) complex forms could be detected by potentiometric titrations (Table 4 and Fig. 10). NMR analyses were conducted at pH 4.5, 7.0, and 9.1 using a 1 : 1 molar ratio of peptide to Zn(II).

Fig. 10 Distribution diagrams of complexes formed in Zn(II) : L1 system. Species distribution calculated for NMR experimental conditions: [L]_tot = 1 mM; Zn(II) : L = 1 : 1.

Table 4 Stability constants (log β) and pK_a values of the Zn(II) : L1 system^a

Peptide	Species	log β^b	Deprotonating residue
a T = 298 K, I = 0.1 M NaClO4, standard deviations are given in parentheses.b Overall stability constants (β) expressed by the equation: β(Zn(II)HnL) = [Zn(II)HnL]/([Zn(II)][L][H^+]^n).
L1	[ZnH2L]^+	17.26(7)	2 × His
	[ZnL]^−	5.21(4)	2 × Owater
	[ZnLH−2]^3−	−11.76(5)

---

The first species is [ZnH₂L]⁺, in which only the two His residues remain protonated. The stability constant for [ZnHL] could not be determined with a reliable standard deviation, most likely due to being a transient form, with scarce formation in solution. At pH 4.5, the NMR spectra of L1 in the presence of Zn(II) are essentially identical to those of the free ligand, showing no clear evidence of coordination. This observation is consistent with the distribution diagram, which indicates that more than 80% of Zn(II) remains unbound at this pH.

The deprotonation of two His residues leads to the formation of [ZnL]⁻ species. At pH 7.0, where the predominant species is [ZnL]⁻, strong and specific peptide–Zn(II) interactions were evident, as indicated by selective and significant chemical shift changes in residues containing potential donor atoms capable of coordinating metal ions (Fig. 11, Fig. 12, and Fig. S13, S14).

Fig. 11 Comparison of the aliphatic region of the ¹H–¹³C HSQC spectra for the free peptide L1 (red) and the Zn(II) : L1 complex (blue) at a 1 : 1 molar ratio, at pH 7. Signals exhibiting significant chemical shift changes upon Zn(II) coordination are highlighted in red arrows.

Fig. 12 Chemical shift perturbations in the Zn(II) : L1 complex at pH 7. Histograms displaying chemical shift changes (Δδ, ppm) for proton (a) and carbon (b) nuclei, respectively, upon Zn(II) coordination. (c) Structural representation of the Zn(II) : L1 complex with the most perturbed nuclei highlighted, ¹H in blue and ¹³C in red, corresponding to the largest Δδ values observed in panels (a) and (b).

Notably, Zn(II) coordination produced well-resolved chemical shift perturbations rather than signal broadening, indicating the formation of a defined and stable metal–ligand complex. This spectral behavior suggests a slow-to-intermediate exchange regime on the NMR timescale, characteristic of strong and specific interactions, rather than fast exchange processes typically associated with dynamic or weak binding events.⁴⁸ The most pronounced perturbations were observed for His3, His7, and Glu9, supporting their direct involvement in Zn(II) coordination. Additional but less significant shifts were observed for Asp4 and Glu2, which may be involved indirectly, possibly due to peptide conformational rearrangements upon metal binding. In particular, the spatial orientation of Asp4, positioned opposite to His3, may hinder direct coordination. Thus, Zn(II) coordination sphere in [ML] form could be either {2 N_im, COO⁻, H₂O} or {2 N_im, 2 COO⁻}, with at least one of Asp4 and Glu2 residues not participating in the metal-binding.

A similar coordination pattern was observed at pH 9.1 for the [ZnLH₋₂]³⁻ species, supported by comparable chemical shift variations in the HSQC and TOCSY spectra (Fig. S15 and S16). [ZnLH₋₂]³⁻ forms in solution as a consequence of the deprotonation of two water molecules. Above pH = 9, precipitation was observed, likely due to the hydrolysis of the metal ion, implying that the ligand does not bind the metal ion very tightly.

The secondary structure of the ligand and metal complexes

We have investigated the secondary structure exhibited by the free ligand L1 and its Fe(II), Mn(II), and Zn(II) complexes, by utilizing far-UV CD spectroscopy. For each system, we have recorded spectra in the 180–250 nm range at four pH values: 3.50, 5.50, 7.50, and 9.50. All spectra exhibit a negative band around 198 nm, which is characteristic of a random coil structure.⁴⁹ As the addition of the studied metal ions to the free ligand did not significantly alter the spectra, the presence of neither Fe(II), Mn(II), nor Zn(II) results in major conformational changes. Therefore, all studied systems exhibit mostly a random coil secondary structure. This is in partial agreement with the secondary structure displayed by the sequence of the model in the crystal of the YfeA protein (PDB: 6Q1C), in which site 2 is both in helical and in random coil conformation. The CD spectra are shown in Fig. S17.

Thermodynamic stability of the formed metal complexes

To compare the affinity of L1 towards Fe(II), Mn(II), and Zn(II), we have calculated the dissociation constant values (K_d), a factor frequently utilized to compare the ability of the biological ligands to bind the metal ions. Dissociation constant can be defined as: , where [M] refers to the concentration of the free metal ion, [L] refers to the concentration of all ligand species, and [ML] refers to the concentration of all metal–ligand complex species. In Table 5, we have collected the K_d values calculated for L1 and other reported in the literature model peptides containing His, Asp, and Glu residues, as well as transporting (FeoB, MntH, ZupT, YfeA, MtsA) and regulatory (Fur, MntR) proteins of the studied metal ions.

Table 5 Comparison of K_d [M] values for studied and biological ligands for Fe(II), Mn(II), and Zn(II)^a

Ligand	Fe(II)	Mn(II)	Zn(II)	Ref.
a Kd values calculated as: at pH = 7.0. [M] refers to the concentration of the free metal ion, [L] refers to the sum of the concentrations of all ligand species, and [ML] refers to the sum of the concentrations of all metal–ligand complex species.
L1 Y. pestis Site 2 YfeA	4.54 × 10^−5	1.22 × 10^−4	2.85 × 10^−5	This work
Ac-HDHDHDHDH-NH2	6.27 × 10^−6	8.41 × 10^−5	1.77 × 10^−7	30
Ac-HEHEHEHEH-NH2	1.88 × 10^−5	1.25 × 10^−4	1.49 × 10^−7	30
E. coli C-terminal FeoB	4.75 × 10^−7	7.02 × 10^−7	6.31 × 10^−8	28
E. coli Core CFeoB	1.88 × 10^−4	1.35 × 10^−4	1.07 × 10^−5	50
E. coli ExxE motif NFeoB	2.21 × 10^−3	1.31 × 10^−2	—	50
E. coli Fur	1.2 × 10^−6	2.4 × 10^−5	1.4 × 10^−10	51
S. pyogenes MtsA	4.3 × 10^−6	—	—	52
B. subtilis MntR	—	0.2 × 10^−6–2 × 10^−6	—	53
Y. pestis YfeA	—	1.78 × 10^−8	6.6 × 10^−9	21
T. pallidum TroA	—	7.1 × 10^−9	2.25 × 10^−8	54
D. radiodurans MntH	—	1.9 × 10^−4	—	55
Synechocystis ZnuA	—	—	7.3 × 10^−9	56

---

The calculated K_d values for L1 complexes fall within the range of model peptides containing similar binding residues, with a more significant difference observed for Zn(II) complexes, likely due to the presence of more histidine residues in the model peptides and preference of Zn(II) for poly-histidine coordination.^57–59 The affinity of L1, which contains two Glu and one Asp residues, towards Fe(II) and Mn(II) ions is comparable to that of the model peptide Ac-HEHEHEHEH-NH₂ and is lower than peptide containing Asp residues. It is in agreement with the statement that the type of acid present in the sequence is of great importance to the stability of the complex, and Mn(II) and Fe(II) ions prefer Asp to Glu.³⁰ In comparison with other Fe(II) and Mn(II) transporters, the affinity of L1 towards the studied metal ions falls within the lower range, suggesting rather labile metal binding. The affinity of the peptidic model of YfeA site 2 towards Mn(II) and Zn(II) determined in this work is significantly lower than reported before for the full-length protein.²¹ However, this data most likely refers to the main metal-binding site, not site 2, as only a 1 : 1 (M : L) stoichiometry of binding was found in the ITC experiments. Described in the later work through crystallographic experiments, site 2, due to being surface-located and utilizing water molecules to complete the coordination sphere, is believed to bind the metal ions in a much more labile fashion.^22,23 This is consistent with relatively low affinities determined by us for the site 2 YfeA peptidic model. It must be noted that the peptide system cannot fully reflect the complicated web of interactions present in the full-length protein; thus, the affinity of site 2 in native YfeA could differ, albeit most likely not dramatically, from the values presented in this work. Since site 2 is surface-oriented, it does not exhibit as many interactions with the rest of the protein as the deep-buried main metal-binding site. The affinity of L1 towards Zn(II) being higher than that of Fe(II) and Mn(II) might explain why it was determined to be the preferred metal ion for site 2 in crystallographic experiments. In light of YfeA not being able to transport Zn(II), most likely the protein employs mechanisms such as conformation changes to ensure the binding of the proper metal ion.⁶⁰ However, that does not need to be the case, as Zn(II) has been shown to interfere with YfeA-mediated Fe(II) transport;²¹ therefore, strategies utilized by the protein might not be sufficient to prevent zinc binding. Furthermore, site 2 may not be directly involved in metal transfer from YfeA to the transmembrane components of the YfeABCD system, but rather plays a role in potential protein–protein interactions.²³ In that case, binding the proper metal ion substrate might not be as crucial as in the main metal-binding site.

Conclusions

We have characterized the metal-binding properties of the peptidic model of site 2 of Y. pestis YfeA. All of the studied metal ions are bound by the two histidine residues, with varying involvement of aspartic and glutamic acid residues. The affinity of the site 2 model towards Fe(II), Mn(II), and Zn(II) is relatively low, suggesting rather labile binding, consistent with the site being surface-oriented. While site 2 has been shown to be involved in protein–protein interactions, the possibility of the transfer of the metal ion between site 2 and the main metal-binding site of YfeA has not been clarified yet. Thanks to the labile binding at site 2, this region of the protein could act as the initial metal-binding site in YfeA, with the subsequent metal ion transfer to the main metal-binding site. However, with a distance of over 26 Å between the sites, metal translocation could be challenging.²³ The peptidic model of YfeA site 2 exhibited only a slightly higher affinity for Zn(II) over Fe(II). Interestingly, no crystal structure of YfeA with Fe(II)-bound site 2 has been published, suggesting that the differences in conditions between peptide solution studies and full-length protein crystallization studies prevent the iron binding. This could be due to the differences in the conformation of the peptidic model of site 2 versus the conformation of the site in the native protein. Nevertheless, our findings enhance the understanding of the metal-binding properties of site 2 and are a valuable contribution to the still underexplored topic of Fe(II) coordination chemistry.

Conflicts of interest

The authors declare no conflicts of interest related to this study.

Data availability

The authors state that the data used to prepare the manuscript will be stored in RODBUK Cracow Open Research Data Repository of University of Wroclaw.

All supporting data are provided in the Supplementary Information, including hydrolysis constants, ESI-MS data, and NMR and CD spectra of the metal–peptide systems across different conditions. Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d6dt00422a.

Acknowledgements

We acknowledge the Polish National Science Centre (NCN, 2022/47/I/ST4/02354) for financial support. M. O. was supported by the Polish National Science Centre (UMO-2021/43/D/ST4/01231). The results were obtained within the frame of COST Action CA18202, NECTAR – Network for Equilibria and Chemical Thermodynamics Advanced Research, supported by COST (European Cooperation in Science and Technology).

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