Studies of Redox Cofactor Pyrroloquinoline Quinone and Its Interaction with Lanthanides(III) and Calcium(II)

Article abstract of DOI:10.1021/acs.inorgchem.9b00568

Recently it was discovered that lanthanides are biologically relevant and found at the centers of many bacterial proteins. Poorly understood, however, is the evolutionary advantage that certain lanthanides might have over calcium at the center of methanol dehydrogenase enzymes bearing redox cofactor PQQ. Here, we present a straightforward method to obtaining clean PQQ from vitamin capsules. Furthermore, we provide full NMR, IR, and UV-vis spectroscopic characterizations of PQQ. We conducted NMR experiments with the stepwise addition of diamagnetic and paramagnetic lanthanides to evaluate the binding to PQQ in solution. This study provides a deeper understanding of PQQ chemistry and its interaction with lanthanides.

Full text of DOI:10.1021/acs.inorgchem.9b00568

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Studies of Redox Cofactor Pyrroloquinoline Quinone and Its
Interaction with Lanthanides(III) and Calcium(II)
Henning Lumpe and Lena J. Daumann*
̈
̈
Department of Chemistry, Ludwig-Maximilians-Universitat Munchen, Butenandtstr. 5-13, 81377 Munich, Germany
S
* Supporting Information
ABSTRACT: Recently it was discovered that lanthanides are
biologically relevant and found at the centers of many bacterial
proteins. Poorly understood, however, is the evolutionary
advantage that certain lanthanides might have over calcium at
the center of methanol dehydrogenase enzymes bearing redox
cofactor PQQ. Here, we present a straightforward method to
obtaining clean PQQ from vitamin capsules. Furthermore, we
provide full NMR, IR, and UV−vis spectroscopic character-
izations of PQQ. We conducted NMR experiments with the
stepwise addition of diamagnetic and paramagnetic lantha-
nides to evaluate the binding to PQQ in solution. This study
provides a deeper understanding of PQQ chemistry and its
interaction with lanthanides.
INTRODUCTION
■
Oxidoreductases are a group of enzymes which transfer redox
equivalents from or to substrates.1 While nicotinamide or flavin
are commonly found cofactors in such enzymes,2 in 1964
Anthony and Zatman discovered an enzyme with an
alternative, noncovalently bound redox cofactor,3 which was
later determined to be pyrroloquinoline quinone (PQQ,
methoxatin). PQQ-containing enzymes form the oxidoreduc-
tase subgroup of quinoproteins and are an essential part of the
energy metabolism of many organisms.2
Figure 1. Active site visualization of Ln-MDH from strain SolV,
including cofactor PQQ, central metal Ln, and surrounding amino
acid residues.4
Redox cofactor PQQ is activated by a Lewis acid.5 For more
than 30 years, it was thought that calcium was the only metal
ion that had this function in nature. However, many methanol-
oxidizing bacteria can in fact produce two types of MDH: the
traditional one that has been studied intensively for more than
50 years uses calcium, and a second one, which has only
recently been discovered, uses lanthanides instead of calcium.
Lanthanides have long been thought to have no biological
relevance but are now firmly established as the natural metal
ion cofactors in MDH enzymes that are encoded by the xoxF
gene.4,6 Phylogenetic analysis shows that this lanthanide-
dependent class of MDH is, in fact, more widespread in nature
and might be evolutionarily older than its calcium counterpart
and perhaps the major pathway for methanol oxidation in
methylotrophic bacteria growing on methane or methanol.7
The first structure of a Ln-MDH was obtained from the XoxF-
MDH isolated from strain Methylacidiphilum fumariolicum
SolV and was reported by Pol et al.4 The active site (Figure 1)
contains a nine-coordinate lanthanide ion, surrounded by
cofactor PQQ, three carboxyl groups from Asp299, Asp301 (a
residue that is lacking in Ca-MDH), and Glu172 as well as an
amide from Asn256. This MDH X-ray structure was refined
with Ce3+(PDB 4MAE). Structures of a Eu-MDH (PDB
6FKW) from the same organism and a La-MDH (PDB
6DAM) from Methylomicrobium buryatense 5GB1C are now
also available, showing similar active site arrangements.8 In Ca-
MDH, PQQ is coordinated in the same way by the Lewis acid
(Site 1, Figure 2).9 Many bacteria possess genes for both MDH
enzymes encoded by mxaF (Ca-MDH) and xoxF (Ln-MDH)
and can switch between them depending on Ln availability;
this has been termed the “lanthanide switch”.10 It has been
shown that Methylorubrum extorquens AM1 preferentially
expresses Ln-MDH even with only nanomolar concentrations
of lanthanides and a 20 μM concentration of Ca in the
cultivation medium.11 The trivalent lanthanides are better
Lewis acids than calcium, and it has been suggested that they
pose an advantage in the redox cycling of PQQ.12
Furthermore, it has been shown that not all lanthanides
Received: February 26, 2019
© XXXX American Chemical Society
A
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correlation (HMQC) and heteronuclear multiple bond connectivity
(HMBC) experiments were used to assign each resonance in the
spectra. Experiments were performed by default with 16 scans for 1H
and 4000 scans for 13C experiments and were increased to up to 64
1
scans for H and 10 000 scans for 13C for correct assignments and
side-product analysis. Solid state NMR was performed at room
temperature with a Bruker Avance III (500 MHz, 11.74 T) with
1
magic-angle spinning (MAS) in 4 mm rotors. H with single pulse
excitation, 13C with cross-polarization over 1H. Scan delays (d1): 13C,
16 s; H, 128 s. Chemical shifts are reported in units of ppm relative
to TMS, indirectly measured with 0.1% TMS in CDCl3. All NMR
spectra are included in the Supporting Information.
Figure 2. PQQ numbering scheme according to Unkefer et al.14 and
1
possible binding sites in solution, adapted from Nakamura et al.15
Stepwise Addition of Lanthanide(III) and Calcium(II) Salts
to PQQ. Metal-induced NMR shifts were used to examine the
position of coordinating metals to PQQ in solution. PQQ shows good
solubility in polar solvents such as H2O (pH >7), MeOH, DMSO,
and DMF but not in MeCN. While metal addition to aqueous
solutions of PQQ leads to the precipitation of a 1:1 complex,
solutions in methanol give a hemiketal adduct as the predominant
species, which complicates analysis. In DMSO, significant solvent
complexation can also occur and is comparatively stronger than in
other solvents ([Ln(DMSO)8]3+. For a summary, see Cotton and
Harrowfield.28 Therefore, DMF-d7 was the solvent of choice for
coordination experiments. Unless otherwise noted, the NMR tube
contained PQQ (9 mg, 27 μmol), which was dissolved by sonication
at 50 °C for 5 min. LiClO4 (16 mg, 0.15 mmol) for controlled ionic
strength and Ln/Ca salts were always added as solids and were
dissolved by sonication at 50 °C for 5 min. In the case of very small
amounts of metal (0.01 equiv), a stock solution was prepared in
DMF-d7 (0.01 mg/μL), which was used for metal addition (0.01
equiv equals ∼10 μL of stock solution (Table S1)). 1H and 13C NMR
spectra were recorded within 1 h after the metal addition.
Experiments with larger amounts of PQQ (50 mg, 151 μmol) in
DMF-d7 (0.6 mL) were performed in the same way, but higher
temperatures (65 °C) were necessary to ensure complete dissolution.
All NMR experiment conditions can be found in Table S1, and shifts
can be found in Tables S2−S4. FT-infrared spectroscopy (IR) was
carried out with a Jasco FT/IR-460 Plus with an ATR diamond plate.
IR data can be found in Table S5 and Figures S3 and S4. UV−vis
spectroscopy of the different PQQ derivatives was conducted with an
Agilent 8453 diode array spectrophotometer in a 1-cm-path-length
quartz Suprasil cuvette. The following stock solutions were prepared:
PQQ in DMF or DMSO (4 mM). A blank was recorded with 990 μL
of solvent (DMF/DMSO/H2O), followed by the addition of 10 μL of
the respective PQQ stock solution (4 mM) to yield a final
concentration of 4 μM. For measurements in H2O, the PQQ stock
solution in DMF was used, leading to a total DMF concentration of
1% in the cuvette. Spectra were recorded instantly after mixing. UV−
vis spectra can be found in Figure S5. UV−vis spectroscopy of PQQ
(1) in DMF or water with lanthanide chloride and calcium chloride
was performed on an Epoch 2 plate reader from Biotek using a 96-
well quartz microplate from Hellma. In DMF, measurements included
190 μL of a PQQ stock solution in DMF (42.1 μM) and 10 μL of
DMF or 10 μL of LnCl3·nH2O (Ln = La−Lu, n = 7 for La−Ce and n
= 6 for Pr−Lu, except for Sm and Dy, where n = 0) or CaCl2·2H2O in
DMF (0.8 mM) to yield a final concentration of 40 μM for both PQQ
and the metal salts (1:1) (Figure 6). In a subsequent experiment, 2 μL
of DMF and 2 μL of LnCl3·nH2O (Ln = La−Lu, except Pm) or
CaCl2·2H2O in DMF (40 mM) were added to the respective well,
resulting in a final PQQ concentration of 39.6 μM and a PQQ to
metal ratio of 1:11. A blank was recorded in pure DMF (200 μL), and
the resulting absorbance was subtracted from the spectra. Additional
UV−vis spectra can be found in Figures S10 and S11. Measurements
in water included 180 μL of a PQQ stock solution in H2O (278 μM),
20 μL of H2O or 18 μL of H2O, and 2 μL of LnCl3·nH2O (Ln = La−
Lu, n = 7 for La−Ce and n = 6 for Pr−Lu, except Sm and Dy, where n
= 0) or CaCl2·2H2O in H2O (25 mM) to yield a final concentration
of 250 μM for both PQQ and metal salts (1:1) (Figure S7). A solvent
blank was recorded and subtracted from the respective spectrum.
Job’s plot measurements included PQQ as well as LuCl3 (anhydrous)
promote methanol oxidation in Ln-MDH equally.4,8a,10d,11b,13
For the native metalloenzyme isolated from SolV, it has been
shown that the enzyme functions more efficiently with early
lanthanides (La−Nd).13a Bacteria also prefer early lanthanides
for growth and show improved uptake of these elements.4 13c
,
The lanthanide switch, the advantage over calcium, and why
some Ln are better than others are currently not yet fully
understood.
Cofactor PQQ was first isolated by Anthony and Zatman16
and was later determined to be a quinone by Duine et al.17
Salisbury et al.18 reported the first crystal structure of the
acetone adduct and proposed the structure of PQQ, shown in
Figure 2. The unusual orthoquinone structure of PQQ,
detected in the EPR measurements of Duine et al., led them
to propose the name pyrrolo-quinoline quinone for this new
prosthetic group.19 In successive years, PQQ was extensively
studied regarding total synthesis,20 adduct formation with
nucleophiles,21 redox behavior,22 and metal ion interaction
(Ca2+)23 with several reviews published.2,24 PQQ has also been
proposed to be a vitamin for humans; however, these claims
remain doubtful.25 Previously, it was reported that PQQ
without the surrounding enzyme pocket is capable of the
coordination of metal ions at three positions (Figure 2). This is
supported by several crystal structure determinations (site 126
(Cu), site 227 (Ru), and site 315 (Cu)). For Ca2+, no crystal
structure exists with PQQ by itself, but studies of
trimethylester derivative PQQTME 2 in MeCN suggest a
coordination mode similar to that of MDH.23 In light of the
discovery of lanthanide-dependent metalloenzymes, we have
investigated the metal ion coordination behavior of PQQ in
solution by NMR and UV−vis spectroscopy using lanthanides
and calcium. Our studies suggest that in solution the
coordination of lanthanides occurs in the same position as in
MDH (site 1) and that even if PQQ acts as a tridentate ligand
(C5O, N6, C7−CO2H) the Ln-PQQ complexes undergo fast
exchange in solution. In this study, we have investigated
diamagnetic trivalent lanthanides La and Lu as well as
calcium(II) and compared them with paramagnetic lantha-
nides Ce, Pr, Sm, Eu, Tb, Er, and Tm.
EXPERIMENTAL SECTION
■
1
Nuclear Magnetic Resonance (NMR). H NMR and 13C NMR
spectra were recorded, unless otherwise stated, at room temperature
or 0 °C with Jeol ECP 270 (400 MHz), Jeol ECX 400 (400 MHz),
and Bruker Avance III (400 MHz) spectrometers operating at 400
MHz for proton nuclei and 100 MHz for carbon nuclei. Low-
temperature measurements (−50 °C) were performed on a Varian
NMR System (400 MHz). 1H chemical shifts are reported in units of
ppm relative to DMSO-d6 (δH = 2.50) and DMF-d7 (δH = 8.03). 13
chemical shifts are given in units of ppm relative to DMSO-d6 (δH
C
=
central line of septet: δC = 39.52) and DMF-d7 (central line of triplet:
δC = 163.15). The software used for data processing was MestReNova
version 11.0. Two-dimensional heteronuclear multiple quantum
B
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stock solutions in DMF (200 μM, Figure S9) or in water (250 μM,
Figure S8) and were conducted using a plate reader (Epoch 2). For
the PQQ stock solution in water, a concentrated DMF solution was
prepared first (25 mM), which was diluted with water to the final 250
μM concentration (including 130 mM DMF). The LuCl3 stock
solution in water was treated with the same amount of DMF (130
mM). Both water- and DMF-based measurements included x μL of
PQQ + y μL of LuCl3 (x = 200, 190, 180, ...., 0) (y = 200 − x), a total
of 20 different ratios. For data processing, the corrected extinction of
PQQ was calculated at 343 nm (data analysis in water) or 435 nm (in
DMF) and plotted against the mole fraction of added lutetium(III).
The stepwise addition (0.2 equiv steps) of metal salts to a fixed
concentration of dissolved PQQ included stock solutions of PQQ
(333.33 μM) in water and a 5 mM stock solutions of LaCl3·7H2O,
LuCl3·6H2O, or CaCl2·2H2O in water. Each well contained 200 μL
total volume, including 150 μL of PQQ + x μL of metal salt + y μL of
water (x = 0, 2, 4, ..., 50) (y = 50 − x), resulting in 250 μM PQQ with
increasing amounts of metal salts (0.2 equiv per 2 μL added) (Figures
5 and S6).
Elemental Microanalyses (EA). (C, H, N) were analyzed with a
vario EL element analyzer. Inductively coupled plasma optical
emission spectroscopy (ICP-OES) was conducted with a Varian-
Vista instrument with autosampler and was used for determining
lanthanide and calcium contents in precipitates. Samples were
digested in hot nitric acid and then diluted with Millipore water to
a final HNO3 concentration of 3%. The following wavelengths (nm)
were used for metal content determination: La (333.749, 408.671),
Eu (381.967, 397.197), Lu (291.139), and Ca (393.366, 396.847).
The values shown are averaged. DFT calculations were performed
with Gaussian 09, revision D.01.29 All calculations were performed
using the B3LYP functional,30 and the 6-31g(d) basis set was used.
Solvent effects were modeled using the conductor-like polarizable
continuum model (CPCM) with the default setting for the universal
force field (UFF).31 Magnetic properties (NMR shifts) were
calculated by the gauge-independent atomic orbital (GIAO)
method.32 Calculated structures and shifts can be found in Table
S6. Thermogravimetric analysis (TGA) was performed on a TGA
4000 system from PerkinElmer using Al2O3 crucibles, and the plots
can be found in Figure S13. ESI mass spectra of PQQ were recorded
with a Thermo Finnigan LTQ FT ultra Fourier transform ion
cyclotron resonance mass spectrometer with acetonitrile/water as the
carrier solvent.
DMSO-d6): δ/ppm = 8.61 (s, 1H, 8), 7.08 (d, J = 1.4 Hz, 1H, 3). IR
(neat): ṽ
/cm−1 = 3303, 2360, 2171, 1668, 1603, 1541, 1499, 1339,
1230, 905, 712. EA: Found C 35.72; H 3.39; N 5.93. Calcd for
C14H4N2Na2O8.5.5H2O (PQQ disodium salt) (373.98): C 35.53; H
3.19; N 5.92. The isolated PQQNa2 was converted to the fully
protonated form according to a literature procedure.33 In brief, crude
PQQNa2 (1.12 g, 2.99 mmol) was dissolved in water (500 mL) and
heated to 70 °C. Concentrated HCl (2.3 mL) was added, and the
mixture was stirred at 70 °C for 24 h. The resulting precipitate was
filtered, washed with 2 M HCl, and dried in vacuo. The product (1.02
g, 3.09 mmol, 91%) was isolated as a bright red solid. Total yield over
two purification steps: 85%. IR (neat): ṽ
/cm−1 = 1742 (m), 1699
(m), 1640 (s) (CO); 1506 (m) (CN); 1191 (s), 1263 (s), 1315
(s) (CC); 865 (m) (C−N). EA: Found: C, 48.04; H, 2.50; N, 7.93.
Calcd for [C14H6N2O8· 1.15 H2O]: C, 47.92; H, 2.38; N, 7.98. HRMS
(ESI− , H2O/MeCN): m/z calcd for [C14H5N2O8]−: 329.0051;
found: 329.0051.
PQQ-Metal Complex Precipitation from Water. PQQNa2 (30
mg, 0.08 mmol as received from Doctor’s Best PQQ capsules) was
dissolved in H2O (12 mL). LaCl3·7H2O (A: 0.5 equiv, 14.9 mg, 0.04
mmol/B: 1.0 equiv, 29.8 mg, 0.08 mmol/C: 2.0 equiv, 59.5 mg, 0.16
mmol) was added as a solid, resulting in immediate turbidity and a
color change from dark red to light brown. The suspension was
centrifuged (5 min at 4500 rpm in a Heraeus Megafuge 8R benchtop
centrifuge with a swinging bucket), and the colorless supernatant was
removed. To wash the resulting pellet, water was added (12 mL), and
the suspension was first vortex mixed and then centrifuged (same
configuration), followed by the removal of the supernatant. This
washing step was repeated two more times. The pellet was then
transferred to a Schlenk flask and dried overnight under high vacuum
to afford A: 26 mg, B: 23 mg, and C: 43 mg of a brown powder. EA
(CHNLa): (A) Found C, 30.42; H, 2.44; N, 5.17; La, 25.94. (B)
Found: C, 31.21; H, 2.34; N, 5.22; La 22.97. (C) Found: C, 30.14; H,
2.50; N, 5.19; La 25.18. Calcd for PQQLa·5H2O [C14H13LaN2O13]:
C, 30.23; H, 2.36; N, 5.04; La, 24.98. The experiments were repeated
with CaCl2·2H2O (0.5, 1.0, 2.0, and 3.0 equiv), EuCl3·6H2O (1.0 and
3.0 equiv), and LuCl3·6H2O (1.0 and 3.0 equiv), each indicating a
PQQ·M·5H2O complex as well. (IR data are available in the
Supporting Information, Figure S5.) We have attempted to obtain
mass spectra of the M-PQQ complexes; however, we mostly observed
PQQ (1) and the geminal diol (3, Figure S1).
Materials. Metal salts were purchased from abcr Germany (LnCl3·
nH2O-Ln, 99.9% = Nd, Sm, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu;
Ln(NO3)3·nH2O-Ln, 99.99% = La, Lu), Sigma-Aldrich (LnCl3·nH2O-
Ln, 99.99% = La, Ce, Pr, Eu), Alfa Aesar (LiClO4, 99%; Ca(NO3)3·
4H2O, 99.9995%), and VWR (CaCl2·2H2O, 99%). The water of
crystallization of the lanthanides (nH2O) was analyzed by elemental
microanalysis prior to experiments and is given in the respective
experimental descriptions. Deuterated solvents were purchased from
Sigma-Aldrich (DMF-d7, 99.5%) and EurIsotop (DMSO-d6, 99.8%).
PQQ was either used from a commercial source (Fluorochem Ltd.
Hadfield, 97% purity. (EA) Found: C, 47.81; H, 3.02; N, 7.98. Calcd
for [C14H6N2O8·1.4H2O]: C, 47.31; H, 2.5; N, 7.88) or conveniently
(and cheaply) extracted from Doctor’s Best Science-Based Nutrition
PQQ capsules, as described below, and transferred from its disodium
salt to the fully protonated form, which gave PQQ in high purity.
Milli-Q-grade water (pH 5.5), received from a Millipore Synergy UV
system from Merck (Darmstadt, Germany), was used for all
experiments.
Isolation and Purification of PQQ. PQQ was extracted from
Doctor’s Best Science-Based Nutrition BioPQQ capsules containing
PQQ (as PQQ disodium salt), cellulose, and modified cellulose. The
capsules (60 × 20 mg of PQQ = 1.20 g, 3.21 mmol) were emptied,
and the powder was suspended in water (250 mL). While the PQQ
sodium salt was soluble in water, the cellulose which remained as a
solid was filtered off and washed several times with water until the
filtrate was colorless. The water was subsequently removed from the
filtrate under reduced pressure to give the disodium salt of PQQ as a
brown powder (1.12 g, 2.99 mmol, 93%). 1H NMR (400 MHz,
RESULTS AND DISCUSSION
■
Study of PQQ Metal Ion Interactions in Water. After
precipitation of PQQ the water equivalent present in the solid
could not be removed by drying at 100 °C or under high
vacuum. To exclude the possibility that the solid could be the
water adduct of PQQ (3, a geminal diol can result from the
hydration of the quinone 1 at the C5 position, Figures 2 and 3)
solid state NMR was performed, which indicated pure PQQ
(1) and can be found in the Supporting Information (Figure
S2A). IR spectra of disodium salt and purified fully protonated
PQQ can be found in the Supporting Information (Figure S3,
Table S5). The solubility of PQQ in water is limited to its
(partially) deprotonated form (pKa = 0.30 (N6), 1.60 (C7-
CO2H), 2.20 (C9-CO2H), 3.30 (C2-CO2H), 10.30 (N1)).34 In
addition, PQQ will form the geminal diol (3) in a molar ratio
of 2(1):1(3) in water, as observed with NMR by Duine et al.35
In DMF, PQQ will form 3 to some extent when traces of water
are present or introduced by the addition of metal salts
containing waters of crystallization (Figure S1A). On the basis
of DFT calculations (Table S6), we propose that this addition
is taking place at the C5 carbon. The formation of a diol is
evidenced by a new resonance at 91.7 ppm stemming from a
diol moiety in position 5. The formation of 3 is also supported
by ESI mass spectrometry in a H2O/MeCN mixture where
both PQQ (1, m/z = 329.0051, [C14H5N2O8]−) and PQQ-
C
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Figure 3. Structures of the different PQQ species and PQQ adducts
referred to in this study.
Figure 4. Overlayed IR spectra of PQQ (red) and the PQQ-La
complex (green) between 1800 and 1400 cm−1.
H2O (3, m/z = 347.0157 [C14H7N2O9]−) are observed (Figure
S1B). Previously, Zheng and Bruice also showed with
calculations that (methanol) adducts at C5 of PQQ are
energetically favored.36 When trivalent lanthanide ions or
calcium(II) were added to an aqueous, concentrated solution
of PQQNa2 (pH >7), a 1:1 PQQ−metal complex precipitated.
Elemental microanalysis of the solids revealed similar
stoichiometries, regardless of the amount of added metal salt.
Analysis suggested the formation of a PQQ·M·5H2O complex
(M = Ca2+, La3+, Eu3+, Lu3+), although the exact coordination
mode in the solid state could not be ascertained. TGA analysis
of the solids (PQQ + Eu or La) did not give a clear indication
of whether the five water molecules were bound tightly to the
metal ion in the complex or were present as cocrystallized
water molecules or whether one of them was covalently bound
to PQQ to form 3. TGA analysis showed a continuous weight
loss to 84% on heating to 100 °C, which fits with the
elimination of the five water molecules (Figure S13). The
compound then gradually lost three CO2 molecules until it was
heated to 500 °C (Figure S13). Numerous attempts to
recrystallize the solid or obtain single crystals suitable for X-ray
crystallography were unsuccessful. IR spectra of solid PQQ
showed seven bands between 1743 and 1583 cm−1 which can
be attributed to the CO stretching vibrations of the carboxyl
and quinone groups (Figure 4).37
and 1521 cm−1(Figure 4). In the IR spectra of Fe2+ and Fe3+
complexes with structural PQQ analogues, the bands
associated with the coordinated carboxyl groups (C7) are
much more strongly shifted, or disappear completely, in
comparison to noncoordinating carboxyl or ester groups
(C9).39 Hence, a clear statement about the coordination
mode of lanthanides solely by IR data remains difficult, but the
data indicate a participation of all three carboxyl groups in a
three-dimensional coordination network in the solid state with
different coordination modes.
An analysis of the PQQ-lanthanide complexes in water was
difficult because the precipitation of Ln-PQQ complexes
limited the investigations to UV−vis spectroscopy at low
concentrations. Furthermore, as mentioned above, PQQ forms
at least two species (1 and 3) in aqueous solution. Upon metal
ion addition, there are then at least four different species
present: 1, 3, and M-1, and M-3. The UV−vis spectra of PQQ
with the stepwise addition of lanthanum(III) or lutetium(III)
in unbuffered water is shown in Figure 5. Both lanthanides
induce a decrease in the 330 and 478 nm transitions of PQQ
(mixture of 1 and 3) and give rise to an additional absorption
feature at 375 (La) or 380 (Lu) nm, respectively. With the
same amount of calcium(II), the changes are less pronounced
and the new feature is not as red-shifted and appears at 357
nm. The absence of clear isosbestic points and overlapping
transitions and the presence of more than two species
complicates the analysis of the stoichiometry in solution
using Job’s method (Figure S8).40
Study of PQQ Metal Ion Interactions in Nonaqueous
Solvents. The coordination chemistry of PQQ was previously
examined with several transition metals as well as sodium ions
in both the solid state and in solution. Depending on the metal
and the coligands, all three possible binding sites of PQQ can
be occupied: With structural PQQ analogues (benzoquino-
lines) and Fe2+,39 or with C9-decarboxy PQQ and Cd2+,41 or
with Cu2+,41 coordination was observed in site 1 (Figure 1).
With Cu2+, PQQ and 2,2′-bipyridine (bipy) or terpyridine
(terpy) coordination also took place in site 1,42 as well as with
Cu2+, PQQ and terpy,15 and with Cu+, PQQTME and PPh3 as
coligands also in site 1.26 With Cu+, PQQ and terpy,
A complex of PQQ with Ti4+ in binding site 1, previously
described by Dimitrijevic et al,.38 led to a splitting of the
quinone signal into two features (1643 to 1653 and 1636
cm−1) and an intensity reduction of one of the carbonyl
stretches (1744 cm−1) due to coordination to C5O and C7−
CO2H while the other carbonyl signals remained unaffected. It
is important to note that the affected carboxyl group was
denoted by Dimitrijevic et al.38 to be an ester, stemming from
an impurity in their PQQ sample. However, their PQQ data
match that obtained in the present work (from biological
synthesis and fermentation, a highly purified sample), making
the previous ester assignment doubtful and the feature at 1744
cm−1 more likely to be a carboxylic acid stretch. In our study,
the coordination with lanthanum(III) led to a strong shift of all
seven carboxyl and quinone absorption bands and the
appearance of a broad, poorly resolved feature between 1734
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coordination took place in sites 1 and 3,43 and with Ru2+, PQQ
and bipy in site 2.44 With Na+, coordination was observed in all
three sites.45
Itoh et al. described a Ca2+ coordination with trimethyl ester
PQQTME (2) in MeCN solution, where shifts of the proton
and carbon NMR resonances indicated coordination in site
1.23,46 However, not all PQQ signals were mentioned, and no
indication was given of the PQQ/metal ratio. An ESI mass
spectrometry, UV−vis spectroscopy, and in silico study of the
interaction of the uranyl ion and Ca2+ with PQQ suggested
binding in site 1.47 In 2018, Schelter described a La3+
coordination in site 1 with an MDH active site model ligand
containing a structural PQQ analogue (benzoquinoline
quinone).48 Here, we examine the interaction of PQQ (1)
with lanthanides for the first time directly, without the help of
structural analogues or methylester species PQQTME (2). As
mentioned above, water proved to be problematic for metal
coordination experiments as a result of the precipitation of a
poorly soluble complex at higher concentrations, limiting the
analysis in water to UV−vis spectroscopy. PQQ shows good
solubility in MeOH, DMF, and DMSO and for the PQQNa2
salt also in H2O (UV−vis spectra are included in the SI, Figure
S5 and Table S7). Because PQQ is known to form hemiketal
adducts in methanol (5, 6),35a and water adducts (to yield 3),
additional coordination experiments were conducted in DMF.
UV−vis experiments with a stepwise addition of lanthanides
and calcium ions to PQQ are shown in Figures 6 and S9−S11.
Figure 6. UV−vis spectra of PQQ in DMF (40 μM) with 1 equiv of
LnCl3·nH2O (Ln = La, Gd, Lu) or CaCl2·2H2O, measured directly
after metal addition. For spectra of the complete Ln series, see Figure
S10.
The UV−vis spectrum of PQQ (1) in DMF is only slightly
influenced by Ca2+, leading to a decrease in the intraligand
absorption bands at 334 and 447 nm. Throughout the
lanthanide series, the absorption intensity is steadily reduced
upon addition of 1 equiv of Ln (La3+ to Gd3+), accompanied by
a small but steady shift toward longer wavelengths. The effects
of metal ions on PQQ in DMF are much weaker than in water.
However, coordination to Ln in DMF clearly influences the
electronic structure of PQQ. From Tb3+ onward, the red shift
is more pronounced, with the maximum absorbance now being
at 340 nm. However, the intensity change of the absorption
(upon Tb3+ to Lu3+ addition) is less drastic as with the earlier
Figure 5. UV−vis spectra of PQQ in H2O (250 μM) with increasing
equivalents of LnCl3·nH2O (Ln = La, Lu) or CaCl2·2H2O, directly
measured after metal addition. For spectra of the complete Ln series
and data showing the addition of up to five metal equivalents, see
Figures S6 and S7.
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lanthanide-series ions (Figure S10). We can only speculate as
to why such a clear break between early and late lanthanides
appears, but a decrease in the coordination number, caused by
the lanthanide contraction, is common for lanthanide
complexes and could also be of relevance here.49 Kaim et al.
had previously observed a shift from 364 and 434 to 378 and
488 nm upon binding of Cu+ to the trimethylester derivative
(2) in CH2Cl2.26 Our experiment in DMF shows that the
interaction of 1 with Ln follows a pattern that can be attributed
to the different properties of the lanthanides (decrease in ionic
radii and increase in Lewis acidity) caused by the lanthanide
contraction. Spectra were recorded again after 15 min and with
a total PQQ/metal ratio of 1:11 (Figures S10 and S11),
leading to a further increase in the observed effects: red shift
and absorption intensity change. The addition of water (10 μL,
5 vol %) to the DMF solution decreases the intraligand
absorption bands even further but causes only a slight red shift
(Figure S10 and S11). Because of the up to 7 equiv of water in
the lanthanide salts, not only metal coordination but also
water-adduct formation has to be taken into account. This is
also further elaborated on in the NMR section below. Because
lutetium generated the strongest shift in the UV−vis spectrum
in DMF, we attempted to analyze the binding of this
lanthanide using the method of continuous variation (Job’s
plot)50 in the absence (to avoid the formation of 3) and
presence of traces of water.40 Because the most pronounced
change in the absorption spectrum of PQQ, without other
overlapping transitions, was in the region of 435 nm, we used
this wavelength for our analysis. As mentioned above, PQQ
can add water, and thus we have to assume the presence of
multiple PQQ species before even adding metal ions. We thus
collected the data in dry DMF and with anhydrous LuCl3. The
data collected directly after mixing PQQ with Lu3+ suggest a
PQQ/Lu 1:1 complex (mole fraction ∼0.5, Figure S9).
Surprisingly, the absorbance changed slightly over time (within
15 min), shifting the maximum of Job’s plot to 0.6, which
could indicate a different stoichiometry and/or the presence of
a dynamic process/formation of another species. We want to
emphasize here that the complex nature of multiple species of
PQQ in solution clearly hampers a straightforward analysis of
stoichiometry in solution and that PQQ does add traces of
water at the C5 position, leading to the formation of a water
adduct (3). When using LuCl3·6H2O instead of anhydrous
lutetium salt, the shift over time was stronger, clearly indicating
an effect of water on PQQ. Furthermore, the curvature was
stronger in DMF, indicating a low binding affinity of PQQ
(Figure S9). We have also explored the possibility of learning
about the coordination of lanthanides using the hypersensitive
transitions that some Ln exhibit.51 As can be seen in Figure
S12, the hypersensitive transitions of the Nd3+ ion are visible
before and after PQQ addition. The transition at 578 nm gains
some intensity; however, within this experimental setup, no
meaningful insight into the nature of the coordination sphere
of Nd was obtained. To investigate the site of coordination in
solution further, we conducted NMR experiments on 1 with
the stepwise addition of different lanthanides. Full NMR data
of 1 in DMSO are included in the Supporting Information
(Table S4); however, this solvent is known to be more strongly
coordinating (to lanthanides)28 and 1 is less soluble in DMSO,
hence DMF was used to investigate the interaction with metal
ions. Initial investigations included Ca2+ and diamagnetic Ln
salts La3+ and Lu3+, with chlorides and nitrates as counterions
with increasing amounts of added metal salts from 1 to 10
equiv and with and without controlled ionic strength (LiClO4).
For all three metals and regardless of the counterion employed,
1
the resulting shift for both H and 13C experiment was very
small, with the exception of C9 (Figure 2, para to Npyr). In
addition, new resonances appeared in both 1H and 13C
experiments, especially with increasing amounts of added
metals. Because of the presence of up to seven waters of
crystallization per added lanthanide equivalent, the formation
of a water adduct is plausible. In fact, the addition of water
itself (20 equiv, 9.8 μL) to a solution of 1, CaCl2(10 equiv),
and LiClO4 in DMF further increased the intensity of the new
signal sets, confirming the presence of the water adduct (3).
Even a sample of purified 1 showed minute traces of additional
signals due to trace amounts of residual water. A recrystallized
pure sample of 1, which contained 2 equiv of DMF crystal
solvent but no traces of water, did not show such additional
1
signals in the H NMR recorded in dry DMF (Figure S14).
This demonstrates that PQQ readily adds water in nonaqueous
solvents even when only minute traces are present, confirming
our conclusions from UV−vis spectroscopy. On the basis of
DFT NMR-shift calculations, this addition is proposed to take
place at the C5 carbon (Table S6). In contrast to the
experiments with PQQTME (2) described by Itoh,23 where
one coordination site is blocked (site 3, see Figure 2), a clear
conclusion solely based on resonance shifts as to where metal
coordination takes place with the diamagnetic metal ions and
PQQ (1) proved to be challenging. Interestingly, besides the
discussed water resonances, no new resonances which could be
attributed to complex formation appeared. Thus, temperature-
dependent NMR experiments with La(NO3)3 (0.5 equiv) were
conducted at r.t., 0 °C, and −50 °C (the lowest accessible
temperature within our experimental setup), but regardless of
the temperature, only one signal set was visible for PQQ,
suggesting a fast exchange in solution even at low temper-
atures. However, all resonances were further shifted depending
on the temperature (1H and 13C: C9′, C7′, C2′, and C5a
downfield, with all other signals upfield).
Experiments with lanthanides and 2,6-dipicolinic acid (dpa)
reported by Piguet and co-workers showed a similar
phenomenon:52 excess ligand, added to an already-formed
[Lu(dpa)3] complex in D2O, gave distinct NMR signals for
free and bound ligands, which merged to one signal set at
higher temperatures. Hence, −50 °C is likely not low enough
to affect a signal separation in our experiments. While no
strong metal-induced shifts could help with the assignment of
the coordination position, we recognized a broadening of some
resonances with increasing amounts of lanthanum and lutetium
but not with calcium. Especially with lutetium, the addition of
1 equiv caused resonances C7′, C7, and C9 to almost disappear
in the noise, and exponential line broadening (6.5 Hz) had to
be used to make them visible (Figure 7). To further study the
coordination mode of PQQ to biologically relevant metals in
solution, paramagnetic lanthanides were used.53 Shifts of the
light paramagnetic Ln-chlorides (Ce, Pr, Sm, Eu) for 1H
experiments with 0.5 equiv of metal salt were expectedly
stronger than the ones from diamagnetic Ln (0.04−0.19 (H1),
0.05−0.62 (H8), 0.01−0.11 (H3)), and all signals became
broadened, especially the H8 signal (Eu > Pr > Ce > Sm,
Tables S1−S3). With increasing amounts of metal salt (0.5−
3.0 equiv), all signals were further shifted and broadened (8 >
1 > 3). Shifts of 13C experiments were in the same range as
those from diamagnetic Ln; however, resonances that
decreased in intensity or completely disappeared due to strong
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Article
resonances of the water adduct (3), no additional resonances
were observed. However, taking together the observation that
certain 13C resonances were unaffected (C1a, C3a, and C3)
while others (especially C5, C7′, and C7) decreased in intensity
or completely disappeared because of broadening, our
experiments support the binding of lanthanides to PQQ in
solution in the biologically relevant coordination pocket (site
1, Figure 2), as was also proposed for the uranyl ion by Peyton
and co-workers.47
CONCLUSIONS
■
Although PQQ has been known and studied for over 50 years,
many analytical details of species have been only sparingly
reported and often relied on the use of the trimethyl ester. For
the first time, we report full NMR, IR, and UV−vis
characterization of PQQ and its water adduct. In addition,
the interaction of PQQ in solution with biologically relevant
metal ions (lanthanides and calcium) has been investigated,
and these studies suggest that the coordination of lanthanides
in nonaqueous solvents takes place in the biologically relevant
pocket (site 1).4,9 We further show that even if lanthanides
have similar chemical properties, the subtle differences in ionic
radii across the series impact the electronic structure as
evidenced in the UV−vis spectrum of PQQ. These results will
aid the development of PQQ-based model systems and further
our understanding of lanthanide-dependent enzymes.
Figure 7. Stacked 13C spectra of 1 in DMF-d7 (27.2 μmol) with 1.0
equiv of LaCl3·7H2O or LuCl3·6H2O showing the broadening of some
resonances after the addition of these diamagnetic metal ions.
broadening (especially C5, C7′, and C7) were induced by lower
metal ion concentrations. With 0.5 equiv, certain resonances
were already undetectable (Figure 8). With Pr and Eu, more
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.9b00568.
NMR data for PQQ (ZIP)
NMR data for diamagnetic lanthanides (ZIP)
NMR data for paramagnetic lanthanides (ZIP)
Tables of NMR conditions and observed resonances, IR
spectra and data, additional UV−vis spectra and data of
PQQ in different solvents and with lanthanides,
calculated 13C NMR shifts of water adduct, mass
spectrum of PQQ, TGA plots, and additional exper-
imental procedures (PDF)
Figure 8. Stacked 13C spectra of 1 in DMF-d7 (27.2 μmol) with 0.5
equiv of PrCl3·6H2O, showing the disappearance of some resonances
after metal addition.
resonances decreased in intensity or disappeared than with Ce
and Sm (Table S3), probably stemming from differences in the
electronic structure and the magnetic susceptibility tensor of
the lanthanides.53a,c,54 The resonances, which disappeared,
were partially different (Table S3) and were affected by the
amount of added metal.
The only resonances which remained unaffected in these
experiments were C1a, C3a, and C3. With the heavier,
paramagnetic Ln-chlorides of Tb, Er, and Tm, 0.01 equiv of
metal salt led to the above-mentioned changes. Because of the
commonly known pseudocontact shift of paramagnetic
compounds,53c,55 the spectral width of 13C experiments was
increased to −300 to 600 ppm.52 However, no additional
resonances were detected in this range. It is still possible that,
because of the paramagnetically induced broadening, the
resonances were indeed shifted to low field or high field but
were too broad to be observed. Experiments were also repeated
with larger amounts of PQQ (50 mg, 143.2 μmol) with 1 equiv
of CeCl3 or PrCl3, but besides the now clearly visible
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: lena.daumann@lmu.de.
ORCID
Lena J. Daumann: 0000-0003-2197-136X
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
H.L. and L.J.D. gratefully acknowledge a Sachbeihilfe from the
Deutsche Forschungsgemeinschaft (DFG) (project no.
392552271) and support from the Center for Integrated
Protein Science Munich (CIPSM), SFB 749, and the LMU.
̈
We thank Dr. Jorg Stierstorfer for assistance with the TGA
measurements.
G
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(12) Bogart, J. A.; Lewis, A. J.; Schelter, E. J. DFT Study of the
Active Site of the XoxF-Type Natural, Cerium-Dependent Methanol
Dehydrogenase Enzyme. Chem. - Eur. J. 2015, 21 (4), 1743−1748.
(13) (a) Lumpe, H.; Pol, A.; Op den Camp, H. J. M.; Daumann, L. J.
Impact of the lanthanide contraction on the activity of a lanthanide-
dependent methanol dehydrogenase - a kinetic and DFT study.
Dalton Trans. 2018, 47 (31), 10463−10472. (b) Wehrmann, M.;
Billard, P.; Martin-Meriadec, A.; Zegeye, A.; Klebensberger, J.,
Functional Role of Lanthanides in Enzymatic Activity and Transcrip-
tional Regulation of Pyrroloquinoline Quinone-Dependent Alcohol
Dehydrogenases in Pseudomonas putida KT2440. mBio 2017, 8 (3),
DOI: 10.1128/mBio.00570-17. (c) Shiller, A. M.; Chan, E. W.; Joung,
D. J.; Redmond, M. C.; Kessler, J. D. Light rare earth element
depletion during Deepwater Horizon blowout methanotrophy. Sci.
Rep. 2017, 7, 10389.
(14) Houck, D. R.; Hanners, J. L.; Unkefer, C. J.; van Kleef, M. A.
G.; Duine, J. A. PQQ: Biosynthetic studies in Methylobacterium AM1
andHyphomicrobium X using specific 13C labeling and NMR. Antonie
van Leeuwenhoek 1989, 56 (1), 93−101.
(15) Nakamura, N.; Kohzuma, T.; Kuma, H.; Suzuki, S. Synthetic
and Structural Studies on Copper(II) Complexes Containing
Coenzyme PQQ and Terpyridine. Inorg. Chem. 1994, 33 (8),
1594−1599.
(16) Anthony, C.; Zatman, L. J. The microbial oxidation of
methanol: The prosthetic group of the alcohol dehydrogenase of
Pseudomonas sp. M27: a new oxidoreductase prosthetic group.
Biochem. J. 1967, 104 (3), 960−969.
(17) Westerling, J.; Frank, J.; Duine, J. A. The prosthetic group of
methanol dehydrogenase from hyphomicrobium X: Electron spin
resonance evidence for a quinone structure. Biochem. Biophys. Res.
Commun. 1979, 87 (3), 719−724.
(18) Salisbury, S. A.; Forrest, H. S.; Cruse, W. B. T.; Kennard, O. A
novel coenzyme from bacterial primary alcohol dehydrogenases.
Nature 1979, 280, 843.
REFERENCES
■
(1) Nomenclature Committee of the International Union of
Biochemistry and Molecular Biology (NC-IUBMB). http://www.
sbcs.qmul.ac.uk/iubmb/enzyme/EC1/ (visited Oct 16, 2018).
(2) Duine, J. A.; Frank, J.; Jongejan, J. A. Enzymology of
Quinoproteins. Advances in Enzymology and Related Areas of Molecular
Biology; John Wiley & Sons, Inc.: New York, 2006; pp 169−212.
(3) (a) Anthony, C.; Zatman, L. J. The Microbial Oxidation of
Methanol. Biochem. J. 1964, 92 (3), 609−614. (b) Anthony, C.;
Zatman, L. J. The Microbial Oxidation of Methanol - 2. The
methanol-oxidizing enzyme of Pseudomonas sp. M27. Biochem. J.
1964, 92 (3), 614−621.
(4) Pol, A.; Barends, T. R. M.; Dietl, A.; Khadem, A. F.; Eygensteyn,
J.; Jetten, M. S. M.; Op den Camp, H. J. M. Rare earth metals are
essential for methanotrophic life in volcanic mudpots. Environ.
Microbiol. 2014, 16 (1), 255−264.
(5) Anthony, C. Quinoprotein-catalysed reactions. Biochem. J. 1996,
320 (3), 697−711.
(6) Martinez-Gomez, N. C.; Vu, H. N.; Skovran, E. Lanthanide
Chemistry: From Coordination in Chemical Complexes Shaping Our
Technology to Coordination in Enzymes Shaping Bacterial
Metabolism. Inorg. Chem. 2016, 55 (20), 10083−10089.
(7) Semrau, J. D.; Di Spirito, A. A.; Gu, W.; Yoon, S., Metals and
Methanotrophy. Appl. Environ. Microbiol. 2018, 84 (6),
DOI: 10.1128/AEM.02289-17.
(8) (a) Jahn, B.; Pol, A.; Lumpe, H.; Barends, T.; Dietl, A.;
Hogendoorn, C.; Op den Camp, H.; Daumann, L. Similar but not the
same: First Kinetic and Structural Analyses of a Methanol
Dehydrogenase Containing a Europium Ion in the Active Site.
ChemBioChem 2018, 19, 1147. (b) Good, N. M.; Vu, H. N.; Suriano,
C. J.; Subuyuj, G. A.; Skovran, E.; Martinez-Gomez, N. C.
Pyrroloquinoline Quinone Ethanol Dehydrogenase in Methylobacte-
rium extorquens AM1 Extends Lanthanide-Dependent Metabolism to
Multicarbon Substrates. J. Bacteriol. 2016, 198 (22), 3109−3118.
(c) Keltjens, J. T.; Pol, A.; Reimann, J.; Op den Camp, H. J. M. PQQ-
dependent methanol dehydrogenases: rare-earth elements make a
difference. Appl. Microbiol. Biotechnol. 2014, 98 (14), 6163−6183.
(9) (a) Blake, C. C. F.; Ghosh, M.; Harlos, K.; Avezoux, A.;
Anthony, C. The active site of methanol dehydrogenase contains a
disulphide bridge between adjacent cysteine residues. Nat. Struct. Mol.
Biol. 1994, 1 (2), 102−105. (b) Williams, P. A.; Coates, L.;
Mohammed, F.; Gill, R.; Erskine, P. T.; Coker, A.; Wood, S. P.;
Anthony, C.; Cooper, J. B. The atomic resolution structure of
methanol dehydrogenase from Methylobacterium extorquens. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 2005, 61 (1), 75−79.
(10) (a) Chu, F.; Beck, D. A. C.; Lidstrom, M. E. MxaY regulates the
lanthanide-mediated methanol dehydrogenase switch in Methylomi-
crobium buryatense. PeerJ 2016, 4, No. e2435. (b) Chu, F.; Lidstrom,
M. E. XoxF Acts as the Predominant Methanol Dehydrogenase in the
Type I Methanotroph Methylomicrobium buryatense. J. Bacteriol. 2016,
198 (8), 1317−1325. (c) Good, N. M.; Walser, O. N.; Moore, R. S.;
Suriano, C.; Huff, A. F.; Martinez-Gomez, N. C. Investigation of
lanthanide-dependent methylotrophy uncovers complementary roles
for alcohol dehydrogenase enzymes. Bio Rxiv 2018, 329011.
(d) Masuda, S.; Suzuki, Y.; Fujitani, Y.; Mitsui, R.; Nakagawa, T.;
Shintani, M.; Tani, A. Lanthanide-Dependent Regulation of Methylo-
trophy in Methylobacterium aquaticum Strain 22A. MSphere 2018, 3
(1), No. e00462-17. (e) Zheng, Y.; Huang, J.; Zhao, F.;
Chistoserdova, L. Physiological Effect of XoxG(4) on Lanthanide-
Dependent Methanotrophy. mBio 2018, 9 (2), No. e02430-17.
(11) (a) Martinez-Gomez, N. C.; Skovran, E. In Methane
Biocatalysis: Paving the Way to Sustainability. Kalyuzhnaya, M. G.,
Xing, X.-H., Eds.; Springer International Publishing: Basel, Switzer-
land, 2018. (b) Vu, H. N.; Subuyuj, G. A.; Vijayakumar, S.; Good, N.
M.; Martinez-Gomez, N. C.; Skovran, E. Lanthanide-Dependent
Regulation of Methanol Oxidation Systems in Methylobacterium
extorquens AM1 and Their Contribution to Methanol Growth. J.
Bacteriol. 2016, 198 (8), 1250−1259.
(19) Duine, J. A.; Frank, J.; Verwiel, P. E. J. Structure and Activity of
the Prosthetic Group of Methanol Dehydrogenase. Eur. J. Biochem.
1980, 108 (1), 187−192.
(20) (a) Corey, E. J.; Tramontano, A. Total synthesis of the
quinonoid alcohol dehydrogenase coenzyme (1) of methylotrophic
bacteria. J. Am. Chem. Soc. 1981, 103 (18), 5599−5600. (b) Jongejan,
J. A.; Bezemer, R. P.; Duine, J. A. Synthesis of 13C- and 2H-labelled
PQQ. Tetrahedron Lett. 1988, 29 (30), 3709−3712. (c) Glinkerman,
C. M.; Boger, D. L. Catalysis of Heterocyclic Azadiene Cycloaddition
Reactions by Solvent Hydrogen Bonding: Concise Total Synthesis of
Methoxatin. J. Am. Chem. Soc. 2016, 138 (38), 12408−12413.
(21) Itoh, S.; Ogino, M.; Fukui, Y.; Murao, H.; Komatsu, M.;
Ohshiro, Y.; Inoue, T.; Kai, Y.; Kasai, N. C-4 and C-5 adducts of
cofactor PQQ (pyrroloquinolinequinone). Model studies directed
toward the action of quinoprotein methanol dehydrogenase. J. Am.
Chem. Soc. 1993, 115 (22), 9960−9967.
(22) Itoh, S.; Kawakami, H.; Fukuzumi, S. Electrochemical Behavior
and Characterization of Semiquinone Radical Anion Species of
Coenzyme PQQ in Aprotic Organic Media. J. Am. Chem. Soc. 1998,
120 (29), 7271−7277.
(23) Itoh, S.; Kawakami, H.; Fukuzumi, S. Model Studies on
Calcium-Containing Quinoprotein Alcohol Dehydrogenases. Cata-
lytic Role of Ca2+ for the Oxidation of Alcohols by Coenzyme PQQ
(4,5-Dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic
Acid). Biochemistry 1998, 37 (18), 6562−6571.
(24) (a) Jongejan, A.; Machado, S. S.; Jongejan, J. A. The
enantioselectivity of quinohaemoprotein alcohol dehydrogenases:
mechanistic and structural aspects. J. Mol. Catal. B: Enzym. 2000, 8
(1), 121−163. (b) Duine, J. A. The PQQ story. J. Biosci. Bioeng. 1999,
88 (3), 231−236.
(25) (a) Akagawa, M.; Nakano, M.; Ikemoto, K. Recent progress in
studies on the health benefits of pyrroloquinoline quinone. Biosci.,
Biotechnol., Biochem. 2016, 80 (1), 13−22. (b) Felton, L. M.;
Anthony, C. Role of PQQ as a mammalian enzyme cofactor? Nature
2005, 433, E10.
H
DOI: 10.1021/acs.inorgchem.9b00568
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(26) Wanner, M.; Sixt, T.; Klinkhammer, K.-W.; Kaim, W. First
Experimental Structure of a 1:1 Metal Complex with a PQQ Cofactor
Derivative outside Dehydrogenase Enzymes. Inorg. Chem. 1999, 38
(11), 2753−2755.
(27) Mitome, H.; Ishizuka, T.; Shiota, Y.; Yoshizawa, K.; Kojima, T.
Heteronuclear RuIIAgI Complexes Having a Pyrroloquinolinequi-
none Derivative as a Bridging Ligand. Inorg. Chem. 2013, 52 (5),
2274−2276.
(28) Cotton, S. A.; Harrowfield, J. M. Lanthanides: Solvation. In
Encyclopedia of Inorganic and Bioinorganic Chemistry; John Wiley &
Sons, Ltd.: 2011.
Kirchhoff, J. R.; Hudson, R. A. High-Performance Liquid Chromato-
graphic Separation and pH-Dependent Electrochemical Properties of
Pyrroloquinoline Quinone and Three Closely Related Isomeric
Analogues. Biochem. Biophys. Res. Commun. 1995, 212 (1), 41−47.
(35) (a) Duine, J. A.; Frank, J.; Verwiel, P. E. J. Characterization of
the Second Prosthetic Group in Methanol Dehydrogenase from
Hyphomicrobium X. Eur. J. Biochem. 1981, 118 (2), 395−399.
(b) Dekker, R. H.; Duine, J. A.; Frank, J.; Verwiel, P. E. J.;
Westerling, J. Covalent Addition of H2O, Enzyme Substrates and
Activators to Pyrrolo-quinoline Quinone, the Coenzyme of
Quinoproteins. Eur. J. Biochem. 1982, 125 (1), 69−73.
(29) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson,
G. A.; Nakatsuji, H.; X. Li, M. C.; Marenich, A.; Bloino, J.; Janesko, B.
G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; Ortiz, J. V.;
Izmaylov, A. F.; Sonnenberg, J. L.; Williams-Young, D.; Ding, F.;
Lipparini, F.; Egidi, F.; Goings, J.; Peng, B.; Petrone, A.; Henderson,
T.; Ranasinghe, D.; Zakrzewski, V. G.; Gao, J.; Rega, N.; Zheng, G.;
Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.;
Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.;
Throssell, K.; Montgomery, J. A.; Jr, J. E. P.; Ogliaro, F.; Bearpark, M.;
Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Keith, T.;
Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J.
C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Millam, J. M.; Klene, M.;
Adamo, C.; Cammi, R.; Ochterski, J. W.; Martin, R. L.; Morokuma,
K.; Farkas, O.; Foresman, J. B.; Fox, D. J. Gaussian 09, Revision A.02;
Gaussian, Inc.: Wallingford, CT, 2016.
(30) (a) Becke, A. D. Density-functional thermochemistry. III. The
role of exact exchange. J. Chem. Phys. 1993, 98 (7), 5648−5652.
(b) Lee, C.; Yang, W.; Parr, R. G. Development of the Colle-Salvetti
correlation-energy formula into a functional of the electron density.
Phys. Rev. B: Condens. Matter Mater. Phys. 1988, 37 (2), 785−789.
(c) Petersson, G. A.; Al-Laham, M. A. A complete basis set model
chemistry. II. Open-shell systems and the total energies of the first-
row atoms. J. Chem. Phys. 1991, 94 (9), 6081−6090. (d) Petersson, G.
A.; Tensfeldt, T. G.; Montgomery, J. A. A complete basis set model
chemistry. III. The complete basis set-quadratic configuration
interaction family of methods. J. Chem. Phys. 1991, 94 (9), 6091−
6101. (e) Stephens, P. J.; Devlin, F. J.; Chabalowski, C. F.; Frisch, M.
J. Ab Initio Calculation of Vibrational Absorption and Circular
Dichroism Spectra Using Density Functional Force Fields. J. Phys.
Chem. 1994, 98 (45), 11623−11627. (f) Vosko, S. H.; Wilk, L.;
Nusair, M. Accurate spin-dependent electron liquid correlation
energies for local spin density calculations: a critical analysis. Can. J.
Phys. 1980, 58 (8), 1200−1211.
(31) (a) Barone, V.; Cossi, M. Quantum Calculation of Molecular
Energies and Energy Gradients in Solution by a Conductor Solvent
Model. J. Phys. Chem. A 1998, 102 (11), 1995−2001. (b) Cossi, M.;
Rega, N.; Scalmani, G.; Barone, V. Energies, structures, and electronic
properties of molecules in solution with the C-PCM solvation model.
J. Comput. Chem. 2003, 24 (6), 669−681.
(32) (a) Cheeseman, J. R.; Trucks, G. W.; Keith, T. A.; Frisch, M. J.
A comparison of models for calculating nuclear magnetic resonance
shielding tensors. J. Chem. Phys. 1996, 104 (14), 5497−5509.
(b) Wolinski, K.; Hinton, J. F.; Pulay, P. Efficient implementation
of the gauge-independent atomic orbital method for NMR chemical
shift calculations. J. Am. Chem. Soc. 1990, 112 (23), 8251−8260.
(33) Ikemoto, K.; Mori, S.; Mukai, K. Synthesis and crystal structure
of pyrroloquinoline quinol (PQQH2) and pyrroloquinoline quinone
(PQQ). Acta Crystallogr., Sect. B: Struct. Sci., Cryst. Eng. Mater. 2017,
73 (3), 489−497.
(36) Zheng, Y.-J.; Bruice, T. C. Conformation of coenzyme
pyrroloquinoline quinone and role of Ca(2+) in the catalytic
mechanism of quinoprotein methanol dehydrogenase. Proc. Natl.
Acad. Sci. U. S. A. 1997, 94 (22), 11881−11886.
(37) (a) GRAS Exemption Claim for Pyrroloquinoline Quinone
(PQQ) Disodium Salt Zhejiang Hisun Pharmaceutical Co. Ltd.:
h t t p s : / / w w w . f d a . g o v / d o w n l o a d s / F o o d /
IngredientsPackagingLabeling/GRAS/NoticeInventory/ucm508141.
pdf (visited Jan 11th 2019), 2016. (b) Nakamoto, K. Infrared and
Raman Spectra of Inorganic and Coordination Compounds, Part B:
Applications in Coordination, Organometallic, and Bioinorganic Chem-
istry, 6th ed.; John Wiley & Sons, Inc.: 2009; Chapter 1.
(38) Dimitrijevic, N. M.; Poluektov, O. G.; Saponjic, Z. V.; Rajh, T.
Complex and Charge Transfer between TiO2 and Pyrroloquinoline
Quinone. J. Phys. Chem. B 2006, 110 (50), 25392−25398.
(39) Tommasi, L.; Shechter-Barloy, L.; Varech, D.; Battioni, J. P.;
Donnadieu, B.; Verelst, M.; Bousseksou, A.; Mansuy, D.; Tuchagues,
J. P. Synthesis of Pyrroloquinolinequinone Analogs. Molecular
Structure and Moessbauer and Magnetic Properties of Their Iron
Complexes. Inorg. Chem. 1995, 34 (6), 1514−1523.
(40) Renny, J. S.; Tomasevich, L. L.; Tallmadge, E. H.; Collum, D.
B. Method of continuous variations: applications of job plots to the
study of molecular associations in organometallic chemistry. Angew.
Chem., Int. Ed. 2013, 52 (46), 11998−12013.
(41) Noar, J. B.; Rodriguez, E. J.; Bruice, T. C. Synthesis of 9-
decarboxymethoxatin. Metal complexation of methoxatin as a possible
requirement for its biological activity. J. Am. Chem. Soc. 1985, 107
(24), 7198−7199.
(42) Suzuki, S.; Sakurai, T.; Itoh, S.; Ohshiro, Y. Preparation and
characterization of ternary copper(II) complexes containing coen-
zyme PQQ and bipyridine or terpyridine. Inorg. Chem. 1988, 27 (4),
591−592.
(43) Nakamura, N.; Kohzuma, T.; Kuma, H.; Suzuki, S. Synthetic
and structural studies on copper (II) complexes containing coenzyme
PQQ and terpyridine. Inorg. Chem. 1994, 33, 1594.
(44) Schwederski, B.; Kasack, V.; Kaim, W.; Roth, E.; Jordanov, J.
Ambidentes Verhalten des “neuen Vitamins” Methoxatin (Cofaktor
̈
PQQ) gegenuber Metallen: Koordinative Stabilisierung der Pyrrolid-
und der Semichinon-Form. Angew. Chem. 1990, 102 (1), 74−76.
(45) Ishida, T.; Doi, M.; Tomita, K.; Hayashi, H.; Inoue, M.;
Urakami, T. Molecular and crystal structure of PQQ (methoxatin), a
novel coenzyme of quinoproteins: extensive stacking character and
metal ion interaction. J. Am. Chem. Soc. 1989, 111 (17), 6822−6828.
(46) Itoh, S.; Kawakami, H.; Fukuzumi, S. Modeling of the
Chemistry of Quinoprotein Methanol Dehydrogenase. Oxidation of
Methanol by Calcium Complex of Coenzyme PQQ via Addition-
Elimination Mechanism. J. Am. Chem. Soc. 1997, 119 (2), 439−440.
(47) VanEngelen, M. R.; Szilagyi, R. K.; Gerlach, R.; Lee, B. D.;
Apel, W. A.; Peyton, B. M. Uranium exerts acute toxicity by binding
to pyrroloquinoline quinone cofactor. Environ. Sci. Technol. 2011, 45
(3), 937−942.
(48) McSkimming, A.; Cheisson, T.; Carroll, P. J.; Schelter, E. J.
Functional Synthetic Model for the Lanthanide-Dependent Quinoid
Alcohol Dehydrogenase Active Site. J. Am. Chem. Soc. 2018, 140 (4),
1223−1226.
(49) Cotton, S. A.; Harrowfield, J. M. Lanthanides: Coordination
Chemistry. Encyclopedia of Inorganic and Bioinorganic Chemistry; John
Wiley & Sons, Ltd: 2011.
(34) (a) Kano, K.; Mori, K.; Uno, B.; Kubota, T.; Ikeda, T.; Senda,
M. Voltammetric determination of acid dissociation constants of
pyrroloquinoline quinone and its reduced form under acidic
conditions. Bioelectrochem. Bioenerg. 1990, 24 (2), 193−201.
(b) Kano, K.; Mori, K.; Uno, B.; Kubota, T.; Ikeda, T.; Senda, M.
Voltammetric and spectroscopic studies of pyrroloquinoline quinone
coenzyme under neutral and basic conditions. Bioelectrochem. Bioenerg.
1990, 23 (3), 227−238. (c) Zhang, Z. P.; Tillekeratne, L. M. V.;
I
DOI: 10.1021/acs.inorgchem.9b00568
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(50) Job, P. Formation and Stability of Inorganic Complexes in
Solution. Annali di Chimica Applicata 1928, 9, 113−203.
(51) (a) Henrie, D. E.; Fellows, R. L.; Choppin, G. R.
Hypersensitivity in the electronic transitions of lanthanide and
actinide complexes. Coord. Chem. Rev. 1976, 18 (2), 199−224.
(b) Karraker, D. G. Hypersensitive transitions of six-, seven-, and
eight-coordinate neodymium, holmium, and erbium chelates. Inorg.
Chem. 1967, 6 (10), 1863−1868.
(52) Ouali, N.; Bocquet, B.; Rigault, S.; Morgantini, P.-Y.; Weber, J.;
Piguet, C. Analysis of Paramagnetic NMR Spectra of Triple-Helical
Lanthanide Complexes with 2,6-Dipicolinic Acid Revisited: A New
Assignment of Structural Changes and Crystal-Field Effects 25 Years
Later. Inorg. Chem. 2002, 41 (6), 1436−1445.
(53) (a) Vonci, M.; Mason, K.; Suturina, E. A.; Frawley, A. T.;
Worswick, S. G.; Kuprov, I.; Parker, D.; McInnes, E. J. L.; Chilton, N.
F. Rationalization of Anomalous Pseudocontact Shifts and Their
Solvent Dependence in a Series of C3-Symmetric Lanthanide
Complexes. J. Am. Chem. Soc. 2017, 139 (40), 14166−14172.
(b) Poh, A. W. J.; Aguilar, J. A.; Kenwright, A. M.; Mason, K.; Parker,
D. Aggregation of Rare Earth Coordination Complexes in Solution
Studied by Paramagnetic and DOSY NMR. Chem. - Eur. J. 2018, 24
(60), 16170−16175. (c) Suturina, E. A.; Mason, K.; Geraldes, C. F. G.
C.; Kuprov, I.; Parker, D. Beyond Bleaney’s Theory: Experimental and
Theoretical Analysis of Periodic Trends in Lanthanide-Induced
Chemical Shift. Angew. Chem., Int. Ed. 2017, 56 (40), 12215−12218.
(54) Blackburn, O. A.; Edkins, R. M.; Faulkner, S.; Kenwright, A. M.;
Parker, D.; Rogers, N. J.; Shuvaev, S. Electromagnetic susceptibility
anisotropy and its importance for paramagnetic NMR and optical
spectroscopy in lanthanide coordination chemistry. Dalton Trans.
2016, 45 (16), 6782−6800.
(55) (a) McConnell, H. M.; Robertson, R. E. Isotropic Nuclear
Resonance Shifts. J. Chem. Phys. 1958, 29 (6), 1361−1365.
(b) Bleaney, B. Nuclear magnetic resonance shifts in solution due
to lanthanide ions. J. Magn. Reson. 1972, 8 (1), 91−100.
J
DOI: 10.1021/acs.inorgchem.9b00568
Inorg. Chem. XXXX, XXX, XXX−XXX