Direct Detection and Characterization of Bioinorganic Peroxo Moieties in a Vanadium Complex by ¹⁷O Solid-State NMR and Density Functional Theory

Abstract

Electronic and structural properties of short-lived metal-peroxido complexes, which are key intermediates in many enzymatic reactions, are not fully understood. While detected in various enzymes, their catalytic properties remain elusive because of their transient nature, making them difficult to study spectroscopically. We integrated ¹⁷O solid-state NMR and density functional theory (DFT) to directly detect and characterize the peroxido ligand in a bioinorganic V(V) complex mimicking intermediates non-heme vanadium haloperoxidases. ¹⁷O chemical shift and quadrupolar tensors, measured by solid-state NMR spectroscopy, probe the electronic structure of the peroxido ligand and its interaction with the metal. DFT analysis reveals the unusually large chemical shift anisotropy arising from the metal orbitals contributing towards the magnetic shielding of the ligand. The results illustrate the power of an integrated approach for studies of oxygen centers in enzyme reaction intermediates.

1. Introduction

Reactions catalyzed by metalloenzymes often consist of several metal-based, short-lived catalytic intermediates that allow the enzymes to efficiently perform energetically unfavorable chemical reactions. Elucidating the electronic, chemical and structural properties of these reaction intermediates is key to understanding biological catalytic processes. During enzymatic catalysis, a reaction is initiated by the binding of the substrate and/or cofactor molecules to the active site of the protein. For example, dioxygen is a cofactor for oxygenase enzymes while hydrogen peroxide cofactor acts as an oxidant in several peroxidases. The O-O bond of the cofactor is activated when the substrate is present in the active site of the enzyme. For such enzymes, this bond activation can take place via the formation of transient peroxo or hydroperoxo intermediates [1]. Being short-lived, these intermediates are difficult to detect and, consequently, crystal structures of peroxo species have been characterized only for very few enzymes, such as superoxo and alkyl peroxo intermediates in iron dependent homoprotochatechuate dioxygenase and peroxo intermediate in Rieske cis-diol dioxygenase [2, 3], as well as a peroxo intermediate of vanadium-dependent chloroperoxidase (VCPO)[4] (Figure 1). Thus, although speculated to be present in many enzymatic reactions, the electronic and structural properties of peroxo intermediates largely remain elusive.

Figure 1.

Crystal structures of (a) NH₄[VO(¹⁷O₂)(H₂O)(C₅H₃N(COO)₂] [5], (b) peroxo intermediate of vanadium chloroperoxidase (pdb code: 1IDU) [4] and, (c) the superoxo intermediate of homoprotocatechuate dioxygenase (pdb code: 2IGA) [3].

Electronic properties of enzymatic oxygen centers have been most commonly probed indirectly by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies [6–13]. However, direct detection of ¹⁷O centers in catalytic reaction intermediates is rare. Although NMR is a viable tool for investigating oxygen centers, ¹⁷O detected NMR measurements suffer from low sensitivity. This is because the only NMR active isotope of oxygen, ¹⁷O, has a mere 0.037% natural abundance. Furthermore, this isotope has a low gyromagnetic ratio (¹⁷O Larmor frequency is 67.78 MHz at 11.7 T), necessitating ¹⁷O enrichment in most samples, for efficient detection. Finally, owing to its large quadrupole moment (¹⁷O is a spin-5/2 nucleus, Q = −2.558 × 10⁻³⁰ m²), ¹⁷O NMR spectra are often moderately broadened. The broadening arises from the second-order quadrupolar interaction, which dominates ¹⁷O NMR spectra, and which cannot be completely removed by magic angle spinning (MAS). Despite these challenges, ¹⁷O solid-state NMR (SSNMR) spectra have been reported in several small organic and biological molecules,[14–18] in oxygen containing transition metal coordination complexes [19–22], as well as in protein-ligand complexes, such as hemoglobin- and myoglobin-¹⁷O₂ states [23], [C¹⁷O]myoglobin [24], egg-white avidin-[¹⁷O₂]biotin and ovo-transferrin-Al^III-[¹⁷O₄]oxalate [25]. In several reports, it has been demonstrated that data acquisition at high magnetic fields alleviates the low-sensitivity and resolution loss from anisotropic nuclear interactions in ¹⁷O spectra, since the magnitude of second order quadrupolar interaction diminishes with increasing applied magnetic field [18].

In this report, we present ¹⁷O SSNMR investigation into a V⁵⁺-peroxido complex mimicking the peroxo intermediates found in mononuclear non-heme enzymes, vanadium-dependent haloperoxidases (VHPO) [26]. Similar intermediates, if trapped in enzymatic reactions, are often present at low concentrations and are thus challenging to detect spectroscopically. Therefore, biomimetic complexes that are typically chemically more stable compared to their natural counterparts are often suitable substitutes for characterization of related chemical species found in enzymatic cycles.

2. Materials and Methods

2.1. Preparation of HS003.

Ammonium oxoperoxo(pyridine-2,6-dicarboxylato) vanadate(V) hydrate, NH₄[VO(¹⁷O₂)(H₂O)(C₅H₃N(COO)₂)]. xH₂O (x ≈1.3) or HS003 with ¹⁷O labeled peroxo ligand was prepared as described previously [5, 26]. The ¹⁷O isotope enrichment of the peroxido ligand was 45%.

2.2. NMR Spectroscopy.

¹⁷O detected static and MAS NMR spectra were acquired on a Bruker 19.96 T AVIII spectrometer outfitted with a 1.6 mm HXYD probe manufactured by PhoenixNMR. The spectral width was set to 2 MHz and 90°pulse (4.4 μs) for solids obtained from ¹⁷O resonance of H₂O was used for static experiment. Static QCPMG[27] spectrum was acquired with the spikelet frequency set to 3.7 kHz. The MAS spectra of HS003 were collected using the Hahn echo[28] experiment (90°-τ−180°) with delay τ set to 68 and 198 μs for 40 and 20 kHz spinning speeds, respectively. A pulse of 1.47 μs corresponding to liquid 30° flip angle at radio frequency (rf) field of 57 kHz was used. The ¹⁷O shifts were referenced using to ¹⁷O resonance of H₂O as a secondary reference (0 ppm). The spectra were processed in TopSpin using exponential apodization of 1000 Hz.

2.3. Numerical Simulations.

Least-square fitting of NMR spectra was performed using the SOLA routine in TopSpin software by Bruker BioSpin. The Haeberlen−Mehring−Spiess convention was used for chemical shift parameter notation: the three principal components of the chemical shift tensor, δ_xx, δ_yy, and δ_zz, and the isotropic component δ_iso, are defined according to ∣δ_yy − δ_iso∣ ≤ ∣δ_xx − δ_iso∣ ≤ ∣δ_zz − δ_iso∣, and δ_iso = (δ_xx + δ_yy + δ_zz)/3; the reduced anisotropy, δ_σ = δ_zz − δ_iso; and the asymmetry parameter η_σ = (δ_yy − δ_xx)/(δ_zz − δ_iso). The quadrupolar tensor is defined by the EFG tensor parameters as C_Q = eQV_ZZ/h and η_Q = (V_xx − V_YY)/V_ZZ, where ∣V_ZZ∣ ≥ ∣V_YY∣ ≥ ∣V_XX∣ are the three components of the quadrupolar coupling tensor, e is the electronic charge, Q is the nuclear quadrupole moment, and h is Planck’s constant.

2.4. DFT Calculations.

Two crystal structures of HS003 have been reported in the literature which include crystallographic water molecule (CCDC number: 1236862) or a NH₄⁺ counterions (CCDC number: 233791). DFT calculations were performed using the Gaussian09 software package [29] using the crystal structure of HS003 with and without crystallographic water molecules and NH₄⁺ counterion [5]. NMR parameter calculation was performed by the GIAO method using B3LYP hybrid functional and 6–311G(d,p) and 6–311++G(d,p) basis sets. For comparison of DFT computed principal components of chemical shielding with experimental measurements, the following standard notation was used: σ₃₃ ≥ σ₂₂ ≥ σ₁₁. The reference shielding value to convert DFT computed shielding to chemical shift was chosen such that a trend line correlating the calculated shielding tensor components and the corresponding experimental chemical shift values has zero intercept. Kong et al. [17] have used the same methodology to obtain σ_ref of 290.0 ppm for ¹⁷O chemical shift calculations of platinum-carboxylate complexes, which is similar to σ_ref = 294.34 ppm used in the present work.

3. Results and Discussion

3.1. ¹⁷O Solid-State NMR Spectra and NMR Parameters of NH₄[VO(¹⁷O₂)(H₂O)(C₅H₃N(COO)₂]

For the V(V) peroxido complex, NH₄[VO(¹⁷O₂)(H₂O)(C₅H₃N(COO)₂] (herein referred to as HS003), studied in this report, we have detected a single powder pattern in ¹⁷O NMR spectrum which is indicative of a single ¹⁷O site. This indicates that both oxygen centers of the peroxido ligand are identical, as they are in the X-ray crystal structure. Density functional theory (DFT) calculations were conducted to gain insights into the contributions of the chemical shift tensor and the effect of metal-ligand bonding on NMR parameters. .

Figure 2a shows static ¹⁷O NMR spectra of HS003 acquired at 19.96 T using quadrupolar Carr-Purcell Meiboom-Gill (QCPMG) [27] pulse sequence (see Materials and Methods for experimental details). However, the singularities in the static spectrum are not defined well enough to uniquely determine the chemical shift anisotropy (CSA) and quadrupolar interactions of the peroxido ligand.

Figure 2.

¹⁷O NMR spectra of HS003 at 19.96 T. (a) QCPMG experiment,¹⁷O MAS spectra (colored lines) together with the best-fit simulated traces (gray). (b) Hahn echo experiment at 20 kHz MAS and, (c) Hahn echo experiment at 40 kHz MAS. The simulations of the experimental spectra were performed using the best-fit parameters as given in Table 1. The inset shows the structure of HS003 with the isotopically ¹⁷O labeled oxygen atoms.

The 20 and 40 kHz MAS spectra of HS003 acquired using the Hahn-echo experiment are presented in Figure 2b and c, respectively. Simultaneous least-squares fitting of the static and MAS spectra allowed for the extraction of unique parameters describing the chemical shift tensor, (δ_σ, η_σ, following the Haeberlen-Mehring-Spiess convention[30]) and quadrupolar tensor (C_Q, η_Q); these are summarized in Table 1. These simulations, performed considering a single ¹⁷O site, are in good agreement with the experimental spectra suggesting that the two isotopically labeled oxygen atoms of the peroxido ligand have similar magnetic environments, as anticipated from the X-ray studies.[5] The simulations yield δ_iso = 620 ppm, δ_σ = 440 ppm, η_σ = 0.15, C_Q = 15.5 MHz and η_Q = 0.55, α = 14, β = 10 and γ = 46, where α, β, γ represent the Euler angles denoting the relative orientation between the chemical shift and quadrupolar tensors. ¹⁷O isotropic solution shifts of ca. 600 ppm for the peroxido ligand in vanadium monoperoxido complexes and δ_iso = 593 ppm for peroxide bound to vanadium bromoperoxidase have been reported [31]. Therefore, the isotropic chemical shift for HS003 observed herein is consistent with the values reported previously for vanadium peroxido complexes of similar structure.

Table 1.

Experimental and DFT computed NMR parameters of the [¹⁷O]peroxido ligand in HS003

	δ11	δ22	δ33	δσ	ησ	CQ	ηQ
Exp	1040	435	385	440	0.15	15.5[b]	0.55
DFT	1288[a]	633[a]	395[a]	516	0.46	−16.1	0.8

^[a]Determined from DFT chemical shielding values and a reference shielding. The intercept of the correlation plot between experimental shift and calculated shielding was chosen as the reference value.

^[b]The sign of C_Q cannot be determined experimentally and the reported value reflects experimentally measured ∣C_Q∣

3.2. Density Functional Theory Calculations of ¹⁷O NMR Parameters

¹⁷O NMR parameters are sensitive probes of the local chemical environment and bonding. For example, ionic interactions of oxygen centers with metal ions have been shown to affect ¹⁷O NMR parameters in biological systems [32, 33]; and large negative ¹⁷O isotropic shifts have been reported for transition metal bound oxygen-containing ligands [34–37]. ¹⁷O chemical shifts in crystalline amino acids have also been demonstrated to correlate linearly with bond distance and angles [38] and variable-temperature measurements have been used to examine the dynamics and measure ¹H-¹⁷O couplings allowing for the measurements of O-H bond distances in barium chlorate monohydrate [39]. DFT calculations on bioinorganic complexes can help to correlate the experimental NMR parameters with local coordination environment and geometry of the observed nuclei [40–43]. ¹⁷O NMR spectra of oxygen atoms directly bonded to paramagnetic metal ions have been reported by Kong et al. [22] This work is particularly relevant in the context of enzymatic peroxo intermediates which typically have the peroxido moiety is ligated to a paramagnetic metal cofactor. DFT calculations qualitatively predicted the paramagnetic chemical shift tensors, demonstrating the feasibility of ¹⁷O NMR studies and quantum chemical calculations for investigation of metalloprotein active sites. Recently, Kong et al. have observed large differences in ¹⁷O isotropic chemical shifts between metal and non-metal bound oxygen atoms for carboxylate platinum complexes [17]. On the basis of the analysis of the experimental and DFT-calculated chemical shift tensors, the authors attribute this result to the interaction of the lone electron pair of oxygen with Pt(II) orbitals. Large variation in chemical shift tensors between metal and non-metal bound oxygen atoms suggest that similar to platinum carboxylate complexes, vanadium d-orbitals in HS003 may also contribute to the magnetic shielding of the peroxido ligand. To gain more insight into these contributions, DFT calculations were performed on the crystal structure of HS003 using gauge including atomic orbitals (GIAO). The magnitude of computed C_Q was larger than the experimental measurement, consistent previous reports that have demonstrated that electric field gradient tensors are systematically overestimated by DFT calculations [44]. The calculated NMR shift parameters (Table 1) agree reasonably well with the experimental measurements. These calculations allowed for the determination of the relative orientation of the chemical shift and the quadrupolar tensors. Figure 3 shows the orientation of the principal components of the chemical shielding (green) and quadrupolar tensors (blue) relative to the molecular frame. Interestingly, the largest chemical shift tensor component (δ₃₃^DFT = 1288 ppm) points towards the V-O bond and the smallest component (δ₁₁^DFT = 395 ppm) makes an angle of 7.7° with the O-O bond. The smallest component of the quadrupolar tensor points towards the V-O bond, while the largest component is along the O-O bond and makes an angle of 5.4° with the O-O bond. The orientation of the largest component of the quadrupolar tensor along the O-O bonds indicates a large gradient in the charge distribution along the O-O bond of the peroxido ligand.

Figure 3.

Left: Relative orientation of the quadrupolar and the chemical shift tensors in the molecular frame of HS003. Right: Contour plots of selected occupied (left) and unoccupied (right) molecular orbitals contributing to the chemical shift tensor of the peroxido ligand in HS003 from DFT calculations. The dashed lines indicate symmetry allowed transformations of the angular momentum that can introduce non-zero paramagnetic shielding. The red/green lobes indicate regions of positive/negative parity.

Based on Ramsey’s formalism [45], nuclear magnetic shielding can be expressed as a combination of the contributions from the core (diamagnetic term, σ_d) and delocalized (paramagnetic term, σ_p) electrons. σ_d, is isotropic and originates from the ground state wavefunctions of a given nucleus. The paramagnetic term, on the other hand, gives rise to the anisotropic components of shielding and originates from the interaction between the ground and excited state wavefunctions. Previous reports have demonstrated that the paramagnetic shielding of carbon- and nitrogen- bound ¹⁷O nuclei originates from oxygen lone pair electrons that occupy high-energy orbitals [46–49]. However, the orientation of the largest component of chemical shift tensor along the V-O bond in HS003 indicates the participation of vanadium orbitals in the paramagnetic shielding of the peroxido oxygens. This observation suggests that the chemical shift tensor and its orientation can provide information regarding the nature of the metal-peroxido bond. To further investigate this, molecular orbitals predicted by GIAO-DFT calculations and their contributions to the chemical shielding were considered.

Figure 3 shows the four highest-energy occupied orbitals and representative four lowest-energy unoccupied orbitals associated with the peroxido ligand. The occupied MO^Occ-2 (MO # 70 from DFT calculations) can be described as an admixture of O p_x (or p_y) and V d_xy (or d_x2-y2) orbitals. Although d-orbitals of V⁵⁺ in HS003 are formally empty, partial vanadium-character of this doubly occupied orbital suggests delocalization of electron density onto empty metal d-orbitals. According to Ramsey’s formalism, paramagnetic shielding from such an orbital arises due to symmetry-allowed angular momentum transformations from this ground state to excited electronic states, the magnitude of which is inversely dependent on the energy gap between the ground state and the excited electronic state under consideration. Jameson et al. have described a general formalism for calculations of paramagnetic terms of the magnetic shielding for p- and d-orbitals [50]. According to their explicit expressions, angular momentum transformations of MO^Occ-2 to MO^Unocc-3, 6 and 7 are symmetry allowed, and the interaction of MO^Occ-2 with these orbitals will introduce non-zero contributions to paramagnetic shielding. MO^Unocc-3 (MO # 74 from DFT calculation) is an admixture of ¹⁷O p_z and ⁵¹V d_yz (or d_xz) orbitals while MO^Unocc-6 (MO # 77 from DFT calculation) and 7 (MO # 78 from DFT calculation) are combinations of ¹⁷O p_x (or p_y) and ⁵¹V d_xy (or d_x2-y2) orbitals. Interaction of MO^Occ-2 with MO^Unocc-6 and 7 will contribute to shielding along all principal axes directions, but its interaction with MO^Unocc-3 will only contribute to shielding along the axes oriented towards the V-O bond. Furthermore, MO^Unocc-3, being the lowest energy excited state, will introduce larger contribution to σ_p compared to MO^Unocc-6 and 7 because the magnitude of σ_p is inversely proportional to the energy gap between the ground and the excited electronic orbitals. Therefore, interaction of p-orbitals of the peroxido ligand with the vanadium d-orbitals determines the magnitude and orientation of the paramagnetic component of chemical shielding.

¹⁷O isotropic chemical shift of ca. 200 ppm have been reported for free H₂O₂ in solution [31] while the isotropic shifts for metal-bound vanadium peroxide complexes are ca. 600 ppm. This significant difference in the chemical shifts originates due to the contribution from the metal d-orbitals to the paramagnetic chemical shift as described above. Owing to the lack of such contributions in free H₂O₂, if similar measurements were to be performed on enzymatic samples, such species, which may be present in excess, can easily be distinguished from metal-bound peroxido ligands.

Further evidence of the contribution of vanadium orbitals towards magnetic shielding comes from the natural bond orbital (NBO) and natural chemical shift (NCS) analysis using DFT calculations. The largest diamagnetic and paramagnetic contributions to all three principal axes, along with the corresponding natural localized molecular orbital (NLMO), are reported in Table 2. Oxygen core s-electrons are responsible for the isotropic diamagnetic term and vanadium orbitals contribute to the paramagnetic term along all three principal axes. It is noteworthy that small admixtures of vanadium d-orbitals to oxygen p-orbitals introduce large paramagnetic shielding. σ₁₁ and σ₃₃ originate due to the polarization of the lone pair of electrons on the peroxido ligand by delocalized electronic spin on vanadium d-orbitals, while V-O bonding orbitals contribute to σ₂₂.

Table 2.

The diamagnetic and paramagnetic terms of magnetic shielding along principal axes and the contributing atomic orbital based on NCS analysis from DFT calculations.

σp (ppm)	σd (ppm)	NLMO
σ11
0	276	17O s core e−
−1065	46	94.3% 17O p, 4% 51V d
σ 22
0	276	17O s core e−
−574	43	70.3% 17O p, 28.3% 51V d
σ 33
0	276	17O s core e−
−446	42	94.3% 17O p, 4% 51V d

Moderate resolution X-ray crystal structures, particularly for large biological molecules, can have uncertainties in the position of hydrogen atoms. To examine the effect of this uncertainty on computed NMR parameters, DFT calculations were performed using both the crystal structures of HS003 and the structures generated by optimizing hydrogen atom positions while restricting the motions of heavy atoms. Geometry optimization of proton positions did not introduce significant differences in the computed CSA values (Table 3) indicating that for this complex, the position of protons does not affect the calculated NMR parameters. X-ray structures of HS003 have been reported in presence NH₄⁺ ions (CCDC number: 233791) and with crystallograhic water molecules (CCDC number: 1236862). Such crystallographic molecules, when in close contact with the ligand, may also contribute to the observed chemical shift anisotropy. To examine the effect of the presence of such crystallographic molecules, GIAO-DFT calculations were performed on crystal structures with and without NH₄⁺ counter-ion and crystallographic water molecule (see Supporting Information Figure S1 for structures). The results of these calculations are summarized in Table 3. Although, the inclusion of water molecule did not significantly affect the calculated CSA values, consideration of NH₄⁺ counter-ion in the calculations introduced ca. 22% error when compared to experimental measurements.

Table 3.

GIAO-DFT computed ¹⁷O NMR chemical shift anisotropy for structures of HS003 with and without counter-ion and hydrogen atom geometry optimization.

Basis Set	X-ray Structure (σσ[a], ησ)			Proton Optimization (σσ[a], ησ)
	No ion[b]	with NH4+[c]	with H2O[b]	No ion[b]	with NH4+[c]	with H2O[c]
6-311g(d,p)	516, 0.45	580, 0.45	513, 0.46	517, 0.45	587, 0.45	517, 0.46
6-311++g(d,p)	518, 0.51	554, 0.49	488, 0.52	495, 0.51	560, 0.49	492, 0.52

^[a]in ppm.

^[b]CCDC number: 1236862

^[c]CCDC number: 233791

4. Conclusions

To summarize, in this work we have investigated the electronic properties of the peroxido ligand in a V⁵⁺-peroxido complex HS003, using ¹⁷O solid-state NMR spectroscopy. High-resolution, high-field ¹⁷O NMR spectra acquired for this complex, which mimics the short-lived peroxo intermediate detected in the catalytic cycles of non-heme enzymes, allowed for the determination of anisotropic NMR parameters. DFT calculations reproduced the experimental NMR parameters and provided insights into the origin of the large magnitude of the chemical shift tensor. These quantum mechanical calculations reveal that the magnitude of the largest component of the chemical shift tensor, which is oriented along the V-O bond, can be attributed to the contributions from vanadium d-orbitals. These results indicate that the chemical shift tensor is a sensitive reporter of the nature of the metal-peroxido bond in catalytic intermediates. Enzymatic peroxo intermediates, detected in several Fe and Mn enzymes are paramagnetic. Kong et al. have recently demonstrated that high-resolution ¹⁷O spectra can be obtained from oxygen atoms coordinated to paramagnetic metal centers [22]. These findings, combined with the present work, which illustrates the strength of the integrated ¹⁷O SSNMR and DFT approach, suggest that investigations of enzymatic peroxido complexes and their biomimetic counterparts can provide invaluable information regarding the nature of the metal-peroxido bond in peroxo intermediates.

Highlights.

• ¹⁷O NMR spectroscopy of biologically relevant vanadium peroxo complex is presented

• Density functional theory calculations demonstrate that the ¹⁷O NMR parameters are sensitive to the interaction of the peroxo moiety with the metal d-orbitals