Crystal structure of a calcium(II)–pyrroloquinoline quinone (PQQ) complex outside a protein environment

Pyrroloquinoline quinone (PQQ) is an important cofactor of calcium- and lanthanide-dependent alcohol dehydrogenases. The crystal structure of a Ca–PQQ complex (Ca3PQQ2·13H2O) is reported for the first time outside a protein environment.


Introduction
Pyrroloquinoline quinone (PQQ) is the redox cofactor of glucose dehydrogenase enzymes and alcohol dehydrogenases. In particular, the methanol dehydrogenase (MDH) enzymes, which catalyze the oxidation of methanol for the energy household of many methano-and methylotrophic microorganisms, have attracted attention recently. For proper functionality, a metal ion is needed, which acts as a Lewis acid and which is coordinated by PQQ and several amino acids in the enzymatic active site (Fig. 1). The Ca-dependent MDH, encoded by the mxaF gene, was first discovered by Anthony & Zatman (1964a,b).
After the structure of MDH was elucidated in 1978-79 (Duine et al., 1978;Westerling et al., 1979;Salisbury et al., 1979), the cofactor attracted much attention in the following years, with articles published concerning its total synthesis (Corey & Tramontano, 1981), redox chemistry (Eckert et al., 1982), metal coordination (Noar et al., 1985) and small-molecule interaction (van Koningsveld et al., 1985). Itoh and coworkers published several articles presenting the interaction of PQQ with Ca and other alkaline earth metals (Itoh et al., 1997(Itoh et al., , 1998, and the synthesis of model compounds, mimicking the active site of MDH (Itoh et al., 2000). Those publications contributed to a better understanding of the functionality and reactivity of PQQ. However, no crystal structures were presented in those studies, which would reveal in-depth structural information of PQQ-metal interactions. While no Ca-PQQ structure has been published to date, in addition, few other crystal structures exist for PQQ with other metals. Outside of the Ca-MDH network (Blake et al., 1994;Williams et al., 2005), several structures were published with sodium ISSN 2053-2296 (Ishida et al., 1989;Ikemoto et al., 2012;Ikemoto et al., 2017), with PQQ structural analogs and iron (Tommasi et al., 1995), with copper and terpyridine (terpy) as co-ligand (Nakamura et al., 1994), with copper and triphenylphosphine (Wanner et al., 1999), with ruthenium and terpy (Mitome et al., 2015), and with ruthenium, silver and terpy (Mitome et al., 2013). In 2014, Pol et al. reported a new kind of MDH, found in the extremophile Methylacidiphilum fumariolicum SolV (SolV), which is native to volcanic mudpots close to the Solfatara crater in Italy . This MDH turned out to be strictly dependent on lanthanides Lumpe et al., 2018;Bogart et al., 2015). While SolV was originally thought to be a biological curiosity, more and more organisms in all kinds of ecosystems were found to be lanthanide dependent in the following years, not restricted to such extreme environments like SolV (Keltjens et al., 2014;Ramachandran & Walsh, 2015;Taubert et al., 2015). This also pushed lanthanide bioinorganic chemistry as a new and emerging scientific field with several reviews published (Skovran & Martinez-Gomez, 2015;Cheisson & Schelter, 2019;Chistoserdova, 2019;Cotruvo, 2019;Daumann, 2019;Picone & Op den Camp, 2019;Semrau et al., 2018). Recently, also, the first crystal structure of a europium-PQQ complex outside the MDH network was published through a collaborative effort and was reported as an Eu 2 PQQ 2 structure (Lumpe et al., 2020) (Fig. 2). In light of those advances and the still scarce structural information available about PQQ-metal interactions, we present here the first crystal structure of a Ca-PQQ complex without the need of structural PQQ analogs or additional co-ligands. The molecular formula of the complex is Ca 3 PQQ 2 Á13H 2 O.

Crystal growth and analysis
Na 2 PQQÁH 2 O (32.8 mg, 0.08 mmol) was dissolved in H 2 O (12 ml). CaCl 2 Á2H 2 O (2.0 equiv., 23.6 mg, 0.16 mmol) was added as a solid. The metal addition led to precipitation of a pale-grey-brown solid, which was centrifuged, removed and analyzed as a 1:1 PQQ-Ca complex, as described in our previous article (Lumpe & Daumann, 2019). From the supernatant, consisting of a highly diluted aqueous mixture of Na 2 PQQ and CaCl 2 , small dark crystals, suitable for X-ray crystallography, grew over a period of several months. To obtain more crystalline material of better quality, a procedure from our recent publication (Lumpe et al., 2020) was implemented. Na 2 PQQÁH 2 O (24.2 mg, 61.8 mmol) was completely dissolved in H 2 O (4 ml) at 80 C in an ultrasonic bath. CaCl 2 Á2H 2 O (27.3 mg, 185.4 mmol, 3 equiv.) was dissolved in a small amount of water (0.2 ml) and was added to the Na 2 PQQ solution at 80 C, which caused precipitation of a grey-brown solid. The mixture was placed directly in a drying oven at 80 C, which was then switched off and the reaction mixture allowed to cool slowly. After 1 d, small dark crystals had grown between the bulk precipitate. The crystals grew in size over the next few days while consuming the surrounding bulk precipitate. Crystals suitable for X-ray diffraction analysis were then picked out of the reaction mixture. The crystal used for analysis was selected in paraffin oil to prevent dehydration and then placed and measured on a Mitegen Microloop. The crystals obtained from both methods showed the same structure depicted in Fig. 3 H 2.90, N 5.76; found: C 34.30, H 3.20, N 6.06. Crystals were picked out of the reaction mixture and then dried for 1 d on filter paper prior to elemental analysis.

Refinement
Crystal data, data collection and structure refinement details are summarized in Table 1. Four reflections have been omitted from the refinement. Three of them are hidden by the beam stop and show no intensity. A further omitted reflection of higher order (090) has a significantly higher F o 2 (63.72) compared to its F c 2 (1.09). This behaviour is observed quite often for reflections of higher order when multigraded X-ray mirrors are used as monochromators. All C-bound H atoms have been calculated in ideal geometry riding on their parent atoms, while the O-and N-bound H atoms were refined freely. Full details of the refinement strategy can be found in the embedded instruction file in the CIF.

Investigation of PQQ-Ca complexation
In our previous article, PQQ-metal complexes were reported with the trivalent lanthanides La 3+ , Eu 3+ and Lu 3+ , and with Ca 2+ (Lumpe & Daumann, 2019). Regardless of the excess of added metal salt, 1:1 complexes were identified by elemental analysis. While no further structural information could be provided in that study, we were recently able to verify the proposed stoichiometry by the crystal structure of an Eu-PQQ complex with the net formula Eu 2 PQQ 2 Á12H 2 O (Lumpe et al., 2020). The Eu 3+ ion is coordinated by PQQ in the same fashion as in MDH, with participation of N2, O4 and O5, in addition to the participation of O1 (Fig. 2). The latter residue is not utilized in the enzyme for metal coordination.
From a similar experimental approach using Ca 2+ instead of Eu 3+ , single crystals suitable for X-ray analysis were grown over a period of several days. Fig. 3 illustrates the composition of the asymmetric unit: the charges of two triply deprotonated PQQ units are balanced by three Ca 2+ ions supplemented by 13 water molecules. The structural motif depicted in Fig. 2 the formation of binuclear units by means of two PQQ ligands acting as linkers between the metal centres -is realized in Ca 3 PQQ 2 Á13H 2 O in a comparable fashion for two of the three Ca ions (Ca1 and Ca3). Ca1 is coordinated by PQQ in a similar fashion to Eu; however, the coordination sphere is completed by a carboxylate group of a nearby pyridine moiety of PQQ (instead of a carboxylate of a pyrrole ring). Ca3, on the other hand, uses the same pocket and residues as Eu; however, this interaction is assisted by a hydrogen bond of a Ca3-bound water molecule to the carboxylate group of the pyrrole ring ( Fig. 3b, green arrows). In the structure, these two types of alternating Ca1 and Ca3 units are connected via Ca1 into strands along [111]. The charge of the Ca 2 PQQ 2 unit is balanced by Ca2, which is coordinated solely by water molecules and carboxylate groups, however, never in the biologically relevant ONO pocket of PQQ. All N-H and O-H donor groups are involved in classical hydrogen bonds with either carboxylate groups, keto groups or water molecules, The structure of the active site from Ca-dependent MDH (PDB code 1w6s). Table 2 Hydrogen-bond geometry (Å , ). (19) 110 (2) Symmetry codes: (i) Àx þ 1; Ày þ 1; Àz þ 1; (ii) x À 1; y; z; (iii) Àx þ 1; Ày; Àz; (iv) x þ 1; y; z; (v) Àx þ 2; Ày; Àz; (vi) Àx þ 1; Ày; Àz þ 1; (vii) x; y À 1; z; (viii) x; y; z À 1; (ix) Àx þ 1; Ày þ 1; Àz; (x) Àx þ 2; Ày þ 1; Àz.

Figure 2
The crystal structure of the inversion-symmetric Eu 2 PQQ 2 complex. acting as acceptors establishing a three-dimensional network (see Table 2 for hydrogen-bond details).
Interestingly, while the elemental analysis of the initially precipitated (amorphous) solid showed a 1:1 Ca-PQQ stoichiometry (Lumpe & Daumann, 2019), the present structure from slowly crystallized material reveals a network of three different Ca 2+ ions and two differently-coordinated PQQ anionic ligands, resulting in a 3:2 stoichiometry. Also, in the Ca-PQQ structure, both PQQ molecules coordinate in the same fashion as in the MDH enzyme ( Fig. 1) (14) Symmetry codes: (i) Àx + 1, Ày + 1, Àz + 1; (ii) x À 1, y, z.  consisting of inversion-symmetric Ca 2 PQQ 2 2À pairs. Here, for clarity, all water molecules, except for that involved in intra-pair hydrogen bonds (green arrows), have been omitted. For symmetry codes, see Table 2.

Figure 4
Normalized IR absorption spectra of the Ca 3 PQQ 2 complex in black, the 1:1 Ca-PQQ precipitate in grey and the Eu 2 PQQ 2 complex in blue. Inset: closeup of the PQQ-related IR absorption peaks.
in the crystal structure, of which 11 directly coordinate to atoms Ca1-Ca3 and two water molecules (O28 and O29) have no direct coordination partners. Interestingly, elemental analysis of the dried crystalline material fits best to only 11 water molecules, most likely due to the disappearance of the two noncoordinating water molecules during the drying process. Ca1 and Ca2 show pentagonal-bipyramidal geometries, with coordination numbers (CNs) of 7 and Ca3 shows a distorted geometry with a CN of 8. All metal-to-ligand bond lengths and angles of Ca 3 PQQ 2 are given in Table 3, in addition to the values for Eu 2 PQQ 2 . The known PQQ-water adduct (diol in C5 position), which is formed to some extent in aqueous solution (Dekker et al., 1982), is not present in the complex, and this is in line with all known crystal structures of PQQ, to the best of our knowledge.
In the Eu 2 PQQ 2 complex, the Eu ions are coordinated in a similar fashion by PQQ. The bonds to Eu are up to 0.141 Å longer than to Ca1 and Ca3. The CN of Eu in the complex is 9, which corresponds to an ionic radius of 1.12 Å according to Shannon (1976), while the ionic radius of Ca is 1.06 Å for a CN of 7 and 1.12 Å for a CN of 8. Therefore, the larger bond lengths to Eu can hardly be explained by different ionic radii, which are overall similar, but by differences in the CNs and different participation in coordination of a second PQQ molecule.
The IR spectra of the precipitated Ca-PQQ amorphous solid, Eu 2 PQQ 2 and Ca 3 PQQ 2 crystals were recorded and compared (Fig. 4). The spectra can be roughly divided into two areas. While PQQ C O stretching vibrations of the carboxylate and quinone groups absorb in the range 1750-1600 cm À1 (Zhejiang Hisun Pharmaceutical Co. Ltd, 2020), the peaks with smaller wavenumbers are largely related to PQQ lattice vibrations. While the heights of the large absorption bands in the range 3600-2600 cm À1 are a direct result of the different amounts and coordination modes of cocrystallized water, the differences in the area 1750-1550 cm À1 further indicate the different coordination modes already depicted in the crystal structures.

Conclusion
We present here the first crystal structure of PQQ with the biologically relevant metal ion calcium. The complex consists of PQQ and the metal ion alone, unlike previously reported structures with other metal ions. Those complexes often needed additional co-ligands, which limited the use of the structures for comparison with the biologically active site. However, in particular, the use of methylated PQQMe 3 (with all three carboxyl groups esterified) prevented participation of (nonbiogenic) carboxyl groups in complexation. This is not the case in the presented structure, where calcium is coordinated by PQQ in the same pocket as in MDH, in addition to further carboxyl-group participation, spanning a three-dimensional coordination network. However, considering the few crystal structures of PQQ complexes reported over the years, we are confident that the presented structure will help to better explain the coordination behaviour of PQQ outside the MDH enzyme and help guide the design of mononuclear model complexes for these fascinating enzymes. program(s) used to solve structure: SHELXT2014 (Sheldrick, 2015a); program(s) used to refine structure: SHELXL2018 (Sheldrick, 2015b); molecular graphics: ORTEP-3 (Farrugia, 2012); software used to prepare material for publication: SHELXL2018 (Sheldrick, 2015b).

Special details
Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.