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Stepwise hydration of [CH3COOMg]+ studied
by cold ion trap infrared spectroscopy: insights
into interactions in the magnesium channel
selection filters†
Hikaru Takayanagi,‡ab Jean-Xavier Bardaud, §‡c Keisuke Hirata,adValérie Brenner, ¶c Eric Gloaguen, §*c Shun-ichi Ishiuchi *ad andMasaaki Fujii *abe
The magnesium channel controls Mg2+ concentration in the cell and plays an indispensable role inbiological functions. The crystal structure of the Magnesium Transport E channel suggested that Mg2+
hydrated by 6 water molecules is transported through a selection filter consisting of COO groups on two
Asp residues. This Mg2+ motion implies successive pairing with OOC-R and dissociation mediated by watermolecules. For another divalent ion, however, it is known that RCOO   Ca2+ cannot be separated evenwith 12 water molecules. From this discrepancy, we probe the structure of Mg2+(CH3COO )(H2O)4–17 clusters by measuring the infrared spectra and monitoring the vibrational frequencies of COO with the help ofquantum chemistry calculations. The hydration by (H2O)6 is not enough to induce ion separation, andReceived 3rd March 2023,
Accepted 16th August 2023

partially-separated or separated pairs are formed from 10 water molecules at least. These results suggest

DOI: 10.1039/d3cp00992k

that the ion separation between Mg2+ and carboxylate ions in the selection-filter of the MgtE channel not
only results from water molecules in their first hydration shell, but also from additional factors including

rsc.li/pccp

water molecules and protein groups in the second solvation shell of Mg2+.

1. Introduction
Ion channels are membrane proteins that selectively permeate
specific ions and play an important role in biologicalsystems.1–4 The mechanism of ion selectivity depends on thetype of ion channel, but the simplest understanding is acompetition between the ion affinity of the channel and theaLaboratory for Chemistry and Life Science, Institute of Innovative Research,
Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama,226-8503, Japan. E-mail: ishiuchi.s.aa@m.titech.ac.jp, mfujii@res.titech.ac.jpbSchool of Life Science and Technology, Tokyo Institute of Technology,
4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa, 226-8503, Japanc
LIDYL, CEA, CNRS, Université Paris Saclay, CEA Saclay, Bât 522,
Gif-sur-Yvette 91191, France. E-mail: eric.gloaguen@cnrs.frdDepartment of Chemistry, School of Science, Tokyo Institute of Technology,
2-12-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japane
IRFI/IPWR, Institute of Innovative Research, Tokyo Institute of Technology,
4259 Nagatsuta-cho, Midori-ku, Yokohama, 226-8503, Japan† Electronic supplementary information (ESI) available: Experimental Methods;
Theoretical methods; Structures. See DOI: https://doi.org/10.1039/d3cp00992k‡ Contributed equally.
§ Present address: Université Paris-Saclay, CNRS, ISMO, 91400 Orsay, France.
¶ Present address: Direction de la recherche fondamentale, Commissariat àl’énergie atomique et aux énergies alternatives, CEA Saclay – 91191 Gif-surYvette, Bât 530 – p 319B, France.

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solvation of the ion. For example, in the case of potassium, theselective filter motif at the channel opening has several carbonyl groups, which are responsible for ion selection.1,2 This ion
selection is achieved because the interaction energy of the
carbonyl group with the potassium ion is slightly more favorable than the hydration energy of the potassium ion, whereas
the sodium ion cannot enter the ion channel because its
hydration energy is more favorable. Further detailed understanding at the molecular level has been gained by using X-ray
diffraction,1,2 2D infrared spectroscopy,5 molecular dynamics
simulations,6–8 and bottom-up approaches in which the interactions between parts of the selection filter and metal ions arestudied by cold ion trap laser spectroscopy.9–12
The magnesium channel is one of the ion channels that
controls Mg2+ concentration in the cell.13–17 The magnesium
ion is the most popular divalent ion in biological cells and isessential for life because of its role in biological functions, such
as RNA and protein formation, catalysis by enzymes and soon.18 Recently, the crystal structure of Magnesium Transport E(MgtE), a type of Mg2+ channel, has been revealed by Nureki’s
group19–21 raising questions about Mg2+ transport mechanismand selectivity still under investigation.22 According to thisreport, the selection filter responsible for selectivity consistsof two COO groups on two Asp residues. This anionic moiety

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Fig. 1 (a) Mg2+(H2O)6 transportation through the –COO selection filterand (b) full contact ion pair formation with 6 H2O. The former corresponds
to separated ion pair of R–COO Mg2+ by hydration (Solvent separated Ion
Pair in solution chemistry).

does not bind directly to Mg2+, but binds to the water moleculeclad Mg2+ in an octahedral arrangement, thus exerting ion
selectivity. In the crystal structure analysis, 13 water molecules
are found in the channel, but only 6 in the vicinity of Mg2+,
which is interpreted as the transportation of Mg2+ hydrated by
6 water molecules, Mg2+(H2O)6, i.e. corresponding to a complete
first hydration shell for this ion. The transportation of Mg2+
hydrated by its first solvation shell seems to be preferred over
the transportation of the isolated divalent ion (see Fig. 1a).21
Indeed, the hydration shell appears necessary to avoid the strong
interactions that can occur between the divalent ion and thechannel,10 and contributes to smooth transport.
Here, we would like to raise a new viewpoint on Mg2+
transportation in MgtE. With a small number of water molecules, Mg2+ and R–COO can bind to form a full contact ion
pair because of their strong electrostatic interaction (seeFig. 1b), but such a contact has not been observed in the crystal
structure.21 Instead, the transportation of Mg2+(H2O)6 throughthe –COO selection filter involves two separated ion pairs
between Mg2+ and two opposite carboxylate groups where ions
are separated by hydration (see Fig. 1a). One may think that thesecond –COO may help to keep such a centred position of
Mg2+, however the presence of a two charge system neverguarantees such an equilibrium at the centre of the two charges
like Fig. 1a. In general, such a two-charge centre system may
also lead to a double minimum potential, which is the reason
why e.g. a lithium cation prefers to bind to one of the oxygen of
the carboxylate group instead of making two equal interactions
with the oxygen atoms (bidentate binding) in crystals23 as wellas in solution.24 Such a double minimum potential would put
the Mg2+ cation in one side, like in Fig. 1b.
Such solvent induced ion pair separation has been extensively studied by gas-phase laser spectroscopy and ab initio
calculations on solvated clusters, such as Li+I and Cs+I ,25
Mg2+HO ,26 Na+AcO ,27 Na+Cl ,28,29 Mg2+RCOO ,30 Ca2+
RCOO ,30–32 Ba2+AcO 32 and LiCl2 .33 These studies show that
5 or 6 solvent molecules are barely enough to separate pairs
made of monovalent ions. For divalent ions, however, noseparation has been observed for the cluster size investigated.
Recent results on RCOO   Ca2+ ion pairs31 demonstrated thateven 12 water molecules are not able to separate these ions.
Although the chemical properties of Ca2+ and Mg2+ are not thesame,34 both are divalent ions capable of binding strongly to
RCOO . Then, it is legitimate to wonder whether six watermolecules are intrinsically enough to separate –COO and Mg2+

23924 | Phys. Chem. Chem. Phys., 2023, 25, 23923–23928

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or not. If not, additional factors must help to keep these ionsseparated in the channel as observed by XRD.21
In order to solve the discrepancy between Mg2+(H2O)6 transport in MgtE and the accumulated knowledge of ion pair
separation by solvent molecules, we have applied cold ion trap
spectroscopy35–39 to hydrated Mg2+(CH3COO ) clusters, which
serve as minimum model systems of the selection filter in
MgtE. Infrared spectra can sensitively probe the coordinationand H-bonds in hydrated Mg2+(CH3COO ) clusters. With thehelp of quantum chemistry calculations, we can discuss
whether a solvent-separated ion pair state which corresponds
to the Mg2+(H2O)6 transport, is formed from Mg2+(CH3COO )by hydration of six water molecules or not.

2. Methods
The experimental procedure is described in a previous article
except for an additional quadruple ion mass spectrometer(Q-MS) before the first ion trap, which generate hydrated
clusters (Fig. S1, ESI†). [CH3COOMg]+ is introduced into
vacuum from electrospray (ESI) source (2.5  10 4 M inmethanol solution). The ions are collected by ion funnel andintroduced into the first Q-MS. The mass-selected ions aretrapped by the first clustering ion trap40,41 at 130 K, in whichwater vapor and He buﬀer gas are filled. The hydrated ions,(CH3COOMg)+(H2O)n are further mass-selected by the second
Q-MS, and then are introduced into the cryogenic quadruple
ion trap (QIT) at 4 K. The QIT was cooled to 4 K by a closed cycle
He refrigerator. A buﬀer gas (He/H2 (20%)) was injected into the
QIT via a pulsed nozzle, and H2 were able to attach the hydrated
complex ions. Then, a tunable IR laser (OPO/OPA: LaserVision)
was introduced into the ion trap and scanned over the range of
CO stretching vibrational range (1300 to 1800 cm 1). When thefrequency of the IR laser is resonant to vibrational levels of
H2-tagged clusters, H2 is dissociated. Then, the infrared photodissociation (IRPD) spectra, which correspond to the IR absorption spectra, can be measured by monitoring the H2-lossed ion
intensity as a function of the IR laser wavenumber (H2-tagging
method). Here, the fragment ions were measured by time-offlight mass spectrometer (TOF-MS), and ion signal is digitized
and integrated in PC via a digital oscilloscope. Following theinvestigation of [CH3COOMg]+ (H2O)n (n = 1–5) by DePalmaet al.,30 we measured the infrared spectra of [CH3COOMg]+(H2O)n from n = 4 to 17, and discussed the change of ion pairsolvation state by stepwise hydration with the help of theoretical calculations (see ESI†).
We performed two steps of theoretical calculations. The first
calculations are simpler one to estimate the vibrational signatures that indicate the solvation condition of Mg2+ from
CH3COO , such as full-contact ion pair state (bidentate ionpair), dissociated state (solvent-shared ion pair) and semidissociated state (monodentate ion pair) structures. For thispurpose, IR spectra of (CH3COOMg)+(H2O)6 are calculated bydensity functional theory (DFT) calculations at CAM-B3LYP-D3/
6-311+G(d,p)42–45 level by using Gaussian 16. Optimized

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structures of three different bonding states are reported for(CH3COOMg)+(H2O)6 by the DFT calculations without dispersion correction and the theoretical spectra are not shown.46 We
build the three initial structures according to the previous
work, and the geometries are optimized by the dispersion
corrected DFT calculations. The calculations give several conformations for a single ionic condition (such as the dissociatedstate). The conformational difference is relatively the minor
part in relation to the ionic condition, so that we obtained thetheoretical IR spectra for three typical ionic conditions. The
obtained harmonic vibrational frequencies were scaled by
0.985. The performance and limitation of this well-known
approach used to compare experimental and theoretical frequencies can be found e.g. in Section 5 of ref. 47 and ref.
therein.
The RI-B97-D3/dhf-TZVPP42,48,49 level of calculation (TURBOMOLE package50) was used for quantitative comparison withexperimental results obtained on (CH3COOMg)+(H2O)n clusters for n = 10 and 14. The potential energy surface of thesesystems being too complex for an exhaustive structural exploration, only a sample of selected structures was considered forgeometry optimization (see ESI† for detail). These initial structures were either built from educated guesses, or randomly
chosen among previously optimized structures of analogue[CH3COONa](H2O)n (n 4 14) clusters24,51 by replacing Na+ by
Mg2+ and removing water molecules in excess. These initialstructures were optimized by the DFT calculations and vibrational frequencies were calculated at the same level. Optimized
structures were sorted according to the type of ion pair formed,
i.e. either bidentate or monodentate contact ion pairs, or
solvent-shared ion pairs, thus defining three families of structures for each cluster size. Their Gibbs energy are reported on
Fig. S2 (ESI†) and frequencies and intensities are reported on
Tables S1–S6 (ESI†). Then, an averaged spectrum for eachfamily and each size was obtained by averaging energyweighted frequencies and intensities of each individual structures according to a procedure detailed in Section S4 (ESI†).
Finally, an arbitrary spectral width of 6 cm 1 was chosen inorder to model each vibrational transition by a Gaussianfunction, and present their sums as full theoretical spectra
for comparison with the experimental ones.

3. Results and discussion
Prior to spectroscopic measurements, DFT calculations (CAMB3LYP-D3/6-311+G(d,p)) were performed on [CH3COOMg]+
(H2O)6 to estimate the vibrational signatures of each type ofion pair (see Section S3 in ESI†). We optimized three typicalstructures of [CH3COOMg]+(H2O)6 in which COO– binds to
Mg2+ either as a full-contact ion pair state (bidentate), a
partially-separated state (monodentate), or a separated state(no coordination). The obtained theoretical spectra and structures are shown in Fig. 2. In the full-contact ion pair state, fromthe low wavenumber side, each band is assigned to dCH, vcoo,svcoo
and dHOH. However, in separated and partial-dissociatedasThis journal is © the Owner Societies 2023

Paper

Fig. 2 Calculated stick spectra of [CH3COOMg]+(H2O)6 for full-contact,
partially-separated and separated ion pairs with the IRPD spectrum of[CH3COOMg]+(H2O)6.

states, vcoo
and vcoo
are no more observed and replacedsaswith two decoupled vco modes, one of which being observed
at 1430–1450 cm 1 instead of B1510 cm 1 for vcoo
in the fullascontact ion pair. Thus, a transition at 1510 cm 1 is a signature
specific to bidentate ion pairs. The intensities of the vco modes
at 1430–1450 cm 1 are enhanced significantly in the separated
state and mildly in the partially-separated, and can thus be
used to monitor the formation of these states. Similarly, theenhancement of vco near 1550 cm 1 can be an indicatorof the separated state, although it may overlap with vcoo
inas
the case of a mixture of different types of structures. Fromthese expectations, we measured the infrared spectra of[CH3COOMg]+(H2O)n.
Fig. 3 shows the infrared spectra of [CH3COOMg]+(H2O)n(n = 4–17). The spectra of n = 4 and 5 reproduce those of aprevious report,30 and the spectral changes from n = 4 to n = 9
are relatively minor. These spectra can be assigned to the fullcontact ion pair state according to our calculations. They areindeed consistent with the corresponding theoretical spectrum
for n = 6 in Fig. 2, where the observed vibrational bands are
assigned to dCH, vcoo
, vcooand dHOH as indicated in the figure.
s
asAmong the minor changes in this series, the general blue shiftof vcooresults from the motion away from the cation in fullascontact, induced by hydration by an increasing number ofwater molecules as already observed in the case of Ca2+.31,32
The blue shift of dHOH between n = 6 and n = 9 suggests theformation of the second hydrated shell from the analogy to theinfrared spectra of [CD3CD2COOCa]+(H2O)n (n r 12),31 inwhich the ion pair COO   Ca2+ keeps the full contact structure. From these infrared spectra, we conclude that Mg2+ is infull contact with COO in [CH3COOMg]+(H2O)6, and thus thatthe solvent-separated ion pair (see Fig. 1a), which is involved in
the transportation of Mg2+(H2O)6 in MgtE in solution, is
intrinsically not stable enough to be formed with only six, orPhys. Chem. Chem. Phys., 2023, 25, 23923–23928 | 23925

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Fig. 3 Infrared(n = 4–17).

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photodissociation

spectraof[CH3COOMg]+(H2O)neven nine water molecules. In contrast, the XRD study21 clearlyshows the presence of the Mg2+(H2O)6 of octahedral hydration
structure separated from COO . Then, the formation of theseparated Mg2+(H2O)6 must be attributed to additional factors,
such as the presence of the second COO group and/or thesecond hydration shell. As the XRD study reported the presenceof the 13 water molecules in the channel, the effect of thesecond hydration shell should be considered. Indeed, for Mg2+
in water, a priori 6 water molecules form the first shell and thenext 12 molecules the second one.21 According to the Nureki’s
crystal structure, the second shell in the Mg2+ channel is filledby 7 water molecules and 4 oxygen atoms in the peptide
framework, instead of 12 water molecules.
The eﬀect of higher hydration can be explored by the
infrared spectra in Fig. 3. The spectrum changes significantly
when the 10th water molecule is added to the cluster (see Fig. 3
for the change from n = 9 to 10). In the dCH range at around1430 cm 1, the intensity of one band (green) is significantly
enhanced. In contrast, vcoo
becomes weaker. Such a clears
spectral change reveals a notable modification of the hydration
condition of the (Mg2+  COO ) ion pair. This spectral feature
keeps going up to n = 13, before another clear spectral changetakes place at n = 14 where vcoo
is no more observed, leavings
onlyvco-like bands. If we take the simple analogy of the calculated structures for n = 6, the disappearance of vcoo
from n = 13sto n = 14 strongly suggests the complete transition from fullcontact ion pairs to partially-separated or separated ones.
Similarly, the spectral change from n = 9 to n = 10 suggeststhat [CH3COOMg]+(H2O)10–13 clusters are a mixture of fullcontact ion pairs with partially-separated or separated ones.
These interpretations are further examined by more sophisticated simulations for two critical cluster sizes n = 10 and 14.
By investigating a set of structures representative of the diversity encountered in these clusters, the simulated spectra enable
us to study the influence of the partially-filled second hydration

23926 | Phys. Chem. Chem. Phys., 2023, 25, 23923–23928

Fig. 4 RI-B97-D3/dhf-TZVPP mode-dependent scaled harmonic vibrational spectra compared to IRPD spectra for n = 10 (a) and n = 14 (b)shell. These energy-weighted averaged spectra are presented inFig. 4 (see ESI† for details). It should be noted that the labels of
the vibrational bands in Fig. 2 and 3 are slightly diﬀerent from
Fig. 4 because the description of the vibrational motions interms of internal coordinates changes with the size of theclusters. The former figures present calculated systems with
only 6 water molecules, while the latter show an average ofvarious conformations for n = 10 and 14 clusters. The correspondence between the assignments in Fig. 2 and 4 are
explained in ESI.†First, according to the theoretical spectra for n = 10 (Fig. 4a),the whole experimental spectrum is consistent with the coexistence of only two types of structures: (i) a full contact ion pairwhose vibrational pattern can be continuously tracked fromn = 4 (Fig. 3), the poor agreement near 1500 cm 1 only resulting
from the insuﬃcient description of the vcoo
and dCH3s30
coupling by harmonic frequency calculations. (ii) a partiallyseparated ion pair, which is assigned to the new spectralfeatures popping up at n = 10, e.g. the blue-most componentof the vcoo
band (blue band) and the main red-most featureas(green band) in the vcoo
spectral region. In addition, bothsthese ion pairs are isoenergetic (ESI†), supporting their simultaneous observation at this cluster size. In turn, separated ion
pairs present the worst vcoo
frequency and energy agreementa(ESI†). For n = 14, the specific signature of the full contact ion
pair disappears (red band in Fig. 3 and 4), in agreement withThis journal is © the Owner Societies 2023

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the significant increase in their relative energy compared ton = 10 (ESI†). In parallel, the triplet on the green band is most
likely explained by the coexistence of several structures. According to calculated vcoo
bands (Fig. 4b), partially separated ionaspairs best agree with the experiment, suggesting that the IR
spectrum results mainly from a mixture of several partiallyseparated ion pairs. Alternatively, the appearance of separated
ion pairs should be accompanied by a new red-shiftedvcoo
band, which is not observed. By comparing n = 10 andasn = 14 spectra, however, a modest red-shift of vcoo
(B3 cm 1)
asis observed and could be interpreted as the manifestation of thepresence of separated ion pairs as minor species.

4. Conclusions

Paper
transportation of Mg2+ in MgtE, where separated ion pairs arefound in XRD of the crystal, may require hydration by at least
14 water molecules. In other sense, Mg2+ would be transported
without help of the circulstance generated by the whole protein
if Mg2+ is hydrated enough. Interestingly, the XRD analysis21 of
the channel shows 13 water molecules, which echo our conclusion. Overall, these results highlight the strength of ourbottom-up approach using solvated ion pair clusters. They
reveal the importance of various contributions of the molecularenvironment in Mg2+ transportation, which have not been
discussed yet. The transportation of other divalent ions suchas Ca2+ should also be studied with a similar approach in the
future.

Author contributions

2+

Mg (H2O)6 transportation through RCOO ion selection filtersin MgtE corresponds to the separation of (RCOO )2Mg2+ ion
pairs by 6 water molecules if we focus on the local structure.
From accumulated knowledge on ion pair solvation, we pointed
out that contact ion pairs in the channel could compete withseparated ion pairs. This hydration-induced separation of
RCOO   Mg2+ and its dependence on the number of watermolecules has been explored by cold ion trap IR laser spectroscopy in which the separation of the ion pair can be detected by
the shift of IR frequencies for specific sizes of [CH3COOMg]+(H2O)n(n = 4–17) clusters. With the help of quantum chemistrycalculations, the shifts of the vibrational bands in the 1300–
1800 cm 1 range were interpreted along this series. The natureof the ion pair, i.e. full contact, partially-separated and separated, is monitored by following the vibrational frequencies
and intensities of the CO2 stretch marker bands. No clear
change of these vibrational bands has been observed
from n = 4 to 9, which demonstrates that the hydration by
six water molecules cannot disturb the strong interaction
RCOO   Mg2+. Consequently, additional factors other thanthese six water molecules have to be invoked to promote thetransportation of Mg2+. Note that the presence of another
RCOO does not explain the central transportation path
between the pair of RCOO automatically because two charge
center produces the double minimum potential in which Mg2+
ion have to be located at one of RCOO , not at the center. The
results suggest that the transportation of hexa-hydrated Mg2+
have to be rationalized not only by the interaction to the pair of
RCOO but also by the additional factors, such as the circulstance generated by other part of the channel and/or higher
hydration. Considering additional water molecules in the second solvation shell of Mg2+, the change of the marker bands
revealed significant structural changes upon hydration fromn = 9 to n = 10, and n = 13 to n = 14. From the more sophisticatedquantum chemistry calculations, the former change in thevibrational spectrum is rationalized by the formation ofpartially-separated ion pairs, while the latter is consistent withthe disappearance of the full contact ion pair and the dominant
formation of several partially-separated ion pairs together
with minor separated ion pairs. It may suggest that theThis journal is © the Owner Societies 2023

E. G., S. I. and M. F. designed the project. H. T., K. H. and S. I.
conducted experiment with simple calculations, whereas
J. X. B., V. B. and E. G. performed sophisticated level calculations. The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.

Conflicts of interest
There are no conflicts to declare.

Acknowledgements
The authors are grateful to Dr Michel Mons for his interest in
this study and his advice. This study has been made possible
thanks to the following grants: KAKENHI (JP20H00372,
JP20K20446, JP21H04674, JP21K14585) and the Core-to-coreprogram (JPJSCCA20210004) of JSPS, World Research HubInitiatives in Tokyo Institute of Technology, the Cooperative
Research Program of the ‘‘Network Joint Research Center for
Materials and Devices’’ from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and the
RIKEN Pioneering Project, ‘‘Fundamental Principles Underlying the Hierarchy of Matter: A Comprehensive Experimental
Study’’; ANR grants IONPAIRS ANR-16-CE29-0017 and VAPOBIO
ANR-20-CE29-0016. The computations were in part performedat the Research Center for Computational Science, Okazaki,
Japan (21-IMS-C109).

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