and High-pH Mo(V) Species in Respiratory ... - ACS Publications

Article pubs.acs.org/IC

Elucidating the Structures of the Low- and High-pH Mo(V) Species in Respiratory Nitrate Reductase: A Combined EPR, 14,15N HYSCORE, and DFT Study Julia Rendon,† Frédéric Biaso,† Pierre Ceccaldi,†,‡,§ René Toci,‡ Farida Seduk,‡ Axel Magalon,‡ Bruno Guigliarelli,† and Stéphane Grimaldi*,† †

Aix Marseille Univ, CNRS, BIP, Marseille, France Aix Marseille Univ, CNRS, LCB, Marseille, France

‡

S Supporting Information *

ABSTRACT: Respiratory nitrate reductases (Nars), members of the prokaryotic Mo/W-bis Pyranopterin Guanosine dinucleotide (Mo/W-bisPGD) enzyme superfamily, are key players in nitrate respiration, a major bioenergetic pathway widely used by microorganisms to cope with the absence of dioxygen. The two-electron reduction of nitrate to nitrite takes place at their active site, where the molybdenum ion cycles between Mo(VI) and Mo(IV) states via a Mo(V) intermediate. The active site shows two distinct pH-dependent Mo(V) electron paramagnetic resonance (EPR) signals whose structure and catalytic relevance have long been debated. In this study, we use EPR and HYSCORE techniques to probe their nuclear environment in Escherichia coli Nar (EcNar). By using samples prepared at different pH and through different enrichment strategies in 98Mo and 15N nuclei, we demonstrate that each of the two Mo(V) species is coupled to a single nitrogen nucleus with similar quadrupole characteristics. Structure-based density functional theory calculations allow us to propose a molecular model of the low-pH Mo(V) species consistent with EPR spectroscopic data. Our results show that the metal ion is coordinated by a monodentate aspartate ligand and permit the assignment of the coupled nitrogen nuclei to the Nδ of Asn52, a residue located ∼3.9 Å to the Mo atom in the crystal structures. This is confirmed by measurements on selectively 15N-Asn labeled EcNar. Further, we propose a Mo-O(H)···HN structure to account for the transfer of spin density onto the interacting nitrogen nucleus deduced from HYSCORE analysis. This work provides a foundation for monitoring the structure of the molybdenum active site in the presence of various substrates or inhibitors in Nars and other molybdenum enzymes.

■

INTRODUCTION

Mo atom, is characterized by a remarkable structural and functional diversity.2,3 The respiratory nitrate reductase Nar is widely distributed in prokaryotes, allowing anaerobic respiration using nitrate as terminal electron acceptor. This membrane-bound heterotrimeric complex (NarGHI) couples the oxidation of membrane quinols at a periplasmically oriented Q site to the cytoplasmic two-electron reduction of nitrate.4,5 During turnover, NarGHI translocates protons across the membrane, contributing to the transmembrane proton gradient that drives ATP synthesis. The NarG catalytic subunit has the Mo-bisPGD cofactor and a [4Fe4S] cluster (FS0). NarH harbors four [Fe-S] clusters with the distal cluster being a [3Fe-4S] type (FS4). Finally, the cytoplasmically exposed NarGH subunits are connected to the membrane-integral NarI, which has two b-type hemes. The metal cofactors form a chain of electron transfer relays from the quinol oxidation site in NarI to the molybdenum atom, which

Nitrate reductases are mononuclear molybdenum enzymes responsible for the initial reductive steps in the global biogeochemical nitrogen cycle, according to the reaction NO3− + 2e− + 2H+ = NO2− + H 2O

(E°′7 = + 420 mV) (1)

Beside eukaryotic nitrate reductases primarily involved in nitrogen assimilation, prokaryotic nitrate reductases serve several purposes including respiration, dissimilation, or assimilation with distinct subtypes (Nar, Nap, Nas).1 The activity of the latter relies on the presence of a single molybdenum atom at their active site coordinated by four sulfur atoms originating from two pyranopterins substituted by a guanine dinucleotide (PGD), forming the Mo-bisPGD cofactor. Prokaryotic nitrate reductases therefore belong to the Mo/W-bisPGD enzyme superfamily (also commonly referred to as dimethylsulfoxide reductase family), which, in spite of housing a similar molybdenum cofactor (Moco) with a limited range of structure in the first coordination sphere of the © XXXX American Chemical Society

Received: December 22, 2016

A

DOI: 10.1021/acs.inorgchem.6b03129 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

aiso(17O) = 0.24 mT, that is, 6.6 MHz).7 The lpH-type signal exists as a series of complexes with different anion ligands (Cl−, F−, NO3−, NO2−, ClO3−, H2PO4−, HCO2−) that have specific and slightly different EPR parameters with aiso(1H) ranging between 28 and 36 MHz.14 However, the variations of the gvalues between these species remain correlated to the lpH and hpH signals, suggesting that the first coordination sphere of the Mo(V) ion is unchanged in these different species. Further, we can conclude that these anions do not directly bind to the metal ion.2,15 The catalytic relevance of the hpH and lpH Mo(V) species is still to be resolved.16 Initial studies suggested a simple acid− base conversion between the two species.9 By comparing the pKa of interconversion between high- and low-pH species (pKa ≈ 8.3) with enzyme activity profile, it was first proposed that only the lpH species is catalytically active.9 However, this pKa value was determined in the presence of chloride, and it was subsequently shown that anion binding modifies the hpH/lpH transition.7 Later, a pH dependence study of the redox potentials of the Mo(V) species performed in absence of contaminating anions has suggested that both could be involved in the catalytic cycle.17 Moreover, this study showed that substitution of the NarG FS0 cluster ligand His50 by Cys also affects the pKa of the transition, while the structural organization of the Moco and the FS0 centers is not modified,18 indicating that this pKa value is likely dependent on the H-bond network around the Moco. This idea has recently been corroborated by new experiments, using a 98Moenrichment strategy of EcNarGH, demonstrating that these forms are not related by a simple acid−base equilibrium.16 To date, the respective detailed structures of the spectroscopically detected lpH and hpH Mo(V) species remain to be elucidated as well as their relationship with the structural data obtained by X-ray crystallography. Indeed, resolving the structure of the Moco site in its various redox states is clearly needed to achieve a better understanding of Nar functioning. Amino acids beyond the first coordination sphere of the molybdenum and/or pyranopterins are expected to play an important role for tuning enzyme reactivity.19−23 Detailed structural information about the catalytic site can be obtained from detection and analysis of the hyperfine interactions (hfi) of the unpaired electron located on the Mo(V) with surrounding paramagnetic nuclei. In particular, high-resolution EPR spectroscopic techniques such as ESEEM and ENDOR spectroscopies have been used to provide detailed information on weak hfi of nuclei from substrates, products, inhibitors, and/ or amino acids that may be relevant for catalysis (for reviews, see refs 15 and 24). These techniques have been combined with various isotope enrichment strategies in low-abundancy isotopes such as 2H, 13C, 17O, 33S, 35,37Cl, 77Se, and 199,201Hg and, in some cases, with density functional theory (DFT) calculations, to extend the range of accessible nuclei (i.e., mainly the highly abundant 1H, 14N, and 31P isotopes). This experimental approach provided a detailed characterization of the structure of various Mo(V) forms found in mononuclear molybdenum enzymes, mostly from the sulfite oxidase or from the xanthine oxidase families (for recent reviews, see refs 1, 15, and 24). In contrast, few studies have focused on Mo-bisPGD enzymes using similar strategies. In this paper, we report on the development of a similar approach to identify the structure of the lpH and hpH Mo(V) species in Nar by using high-resolution EPR. In particular, we used HYSCORE spectroscopy to characterize the Mo(V)

cycles between the Mo(VI), Mo(V), and Mo(IV) oxidation states during the catalytic cycle.4 The crystal structure of the EcNarG subunit has been solved for both the entire NarGHI complex5 or the soluble NarGH form.6 While both crystal structures reveal bis-dithiolene coordination to the Mo atom, they also show two different conformations of the Asp222 ligand: the NarGHI structure depicts bidentate coordination through the δ1 and δ2 O atoms of the Asp222 carboxylate side chain, whereas the same residue in the NarGH structure is monodentate, the Mo first coordination sphere being there completed by an inorganic oxygen atom (Figure 1). Moreover, while Asp222 is hydrogen-

Figure 1. Mo active site structures determined by X-ray crystallography in (A) NarGHI, pdb code: 1Q165 and (B) NarGH, pdb code: 1R27.6 Dash lines show the H-bond between His546 and Asp222 side chains discussed in the text.

bound to the Nε of the conserved His546 residue in the NarGH crystal structure,6 this H-bond is absent in the bidentate NarGHI structure.5 In addition, one PGD cofactor is bicyclic with an open pyran ring in the NarGH structure,6 whereas the two PGD cofactors are in the usual tricyclic pyranopterin state in that of NarGHI.5 Altogether, these results are difficult to reconcile with those obtained from extended Xray absorption fine structure (EXAFS) studies indicating a mono-oxo Mo(VI) site and call for additional experiments to solve this apparent discrepancy.7,8 However, these structural variations suggest that the flexibility of the aspartate coordination and the pyran ring opening and closing could play a role in catalysis, especially for proton transfer.6 Such questions can be related to the various Mo(V) species detected in Nars by electron paramagnetic resonance (EPR) spectroscopy. Indeed, all bacterial Nars investigated in detail thus far by EPR spectroscopy show the coexistence of two characteristic pH-dependent Mo(V) signals associated to functional enzymes,9 namely, a “low pH” (lpH) signal (g1,2,3 ≈ 2.001, 1.986, 1.964) and a “high pH” (hpH) signal (g1,2,3 ≈ 1.987, 1.981, 1.962).10−12 The lpH Mo(V) signal shows a resolved anisotropic hyperfine splitting due to a single exchangeable proton, most likely from a Mo-OH hydroxo moiety,13,14 with hyperfine coupling constants A1,2,3,iso(1H) = (1.13, 0.85, 0.90, 0.96) mT [i.e., A1,2,3,iso(1H) = (31.6, 23.6, 24.7, 26.6) MHz] in EcNar, whereas no splitting is resolved on the hpH Mo(V) signal. In addition, the presence of at least one exchangeable proton in the active site of the hpH form of NarGHI having a near-isotropic coupling to a proton with aiso(1H) = 0.34 mT (i.e., 9.4 MHz) was estimated by simulating the observed decrease of the EPR linewidths of the Mo(V) hpH EPR signal (assuming a single coupled proton) that occurs upon solvent deuteration.14 A slight broadening of the line shape of the hpH Mo(V) signal (not visible on the lpH form) was also observed with nitrate reductase exchanged into 17Oenriched water, corresponding to a small hyperfine coupling B


Article

Inorganic Chemistry

added to enable the redox equilibration. Oxidation was performed by injecting small volumes of a solution of 10 mM K3Fe(CN)6. Samples were anaerobically frozen in liquid nitrogen after equilibration. EPR Measurements. All EPR spectra were measured at 50 K to avoid the contribution from the faster-relaxing FeS clusters present in the enzyme.30 X-band continuous wave (cw) EPR experiments were performed on a Bruker EleXsys E500 spectrometer equipped with an ER4102ST standard rectangular Bruker EPR cavity fitted to an Oxford Instruments helium flow cryostat. Two-dimensional (2D) HYSCORE experiments were performed on the same samples using a Bruker EleXsys E580 spectrometer equipped with an ER4118X-MD5 dielectric resonator and an Oxford Instruments CF 935 cryostat. This four-pulse experiment (π/2-τ-π/2-t1-π-t2-π/2-τ-echo) was employed with appropriate phase-cycling schemes to eliminate unwanted features from the experimental electron spin-echo envelopes. The intensity of the echo after the fourth pulse was measured with varied t2 and t1 and constant τ. The length of a π/2 pulse was 12 ns, and the length of a π pulse 22 ns. HYSCORE data were collected in the form of 2D time-domain patterns containing 256 × 256 points with steps of 16 ns. Spectra were recorded at the magnetic field value corresponding to the maximum intensity of the Mo(V) signal measured in a two-pulse field sweep electron spin-echo sequence (π/2-τ-π-τ-echo). Typical accumulation times for HYSCORE spectra are between 6 and 19 h depending on the Mo(V) concentration and on the desired signal-to-noise ratio. HYSCORE spectra were processed using Bruker’s Xepr software. Relaxation decays were subtracted (fitting by polynomial functions) followed by zero-filling and tapering with a Hamming window, before 2D Fourier transformation, which finally gives the spectrum in frequency domain. Processed data were then imported into Matlab (The MathWorks Inc., Natick, MA) for plotting. HYSCORE spectra are shown in absolute value mode and are presented as contour plots together with the skyline projection on the two frequency axes. Spin quantitation experiments indicated that the Mo(V) signals in the samples typically account for 0.4 ± 0.1 spin/molecule, consistent with previously published values.9,16 Nuclear Quadrupole and Hyperfine Parameters. Mo(V) ion has a 4d1 electron configuration that confers it an electron spin S = 1/2. While EPR signatures of Mo(V) ions in molybdoenzymes exhibit a relatively small g-tensor anisotropy, they are generally wellresolved, because of the sharpness of the corresponding EPR lines. Their typical linewidths of ∼0.2 mT are mainly due to inhomogeneous broadening from unresolved hyperfine interactions, preventing the detection of weak magnetic interactions of the unpaired electron spin S with nearby nuclei having nonzero nuclear spin I. In principle, this information can be recovered using high-resolution EPR methods such as HYSCORE spectroscopy. In this work, we target the I = 1 14N nuclei of the protein environment of the lpH and hpH Mo(V) species and determine the nuclear quadrupole interaction (nqi) parameters (κ, η) from analysis of 14N HYSCORE spectra. Their definition is recalled in the Supporting Information. They are valuable in reporting on the symmetry of the electronic environment at the nucleus and on the chemical group housing the nitrogen. They can therefore be used for the identification and characterization of the interacting nitrogen atom, as exemplified by our previous studies on the nitrogen-containing Hbond donor to the semiquinone intermediates stabilized in the NarGHI quinol oxidation site.31,32 In the present work, the knowledge of the 14N quadrupolar parameters inferred from spectral analysis is used to distinguish between nitrogen nuclei from pyranopterins or from the protein environment and makes it possible to assign the detected nucleus to a particular amino acid residue. Hyperfine interaction (hfi) parameters aiso and T describe the isotropic component (Fermi contact term) and the anisotropic one (dipolar term) in the approximation of axially symmetric hyperfine interaction. Whereas aiso is related to the electron spin density at the interacting nucleus, T is very sensitive to the geometry of the interaction, as detailed in the Supporting Information. Spectral Simulations. Numerical simulations of EPR and HYSCORE spectra were performed using the EasySpin package

nitrogen environment. Notably, only two studies reported on the detection of 14N interactions in xanthine oxidase25 and dimethyl sulfoxide (DMSO) reductase,26 which were however not analyzed in detail, and none were substantiated by 15N enrichment. Therefore, the potential role of the Mo(V) nitrogen environment in the catalytic mechanism largely remains to be addressed experimentally. In this work, we used 14,15N HYSCORE spectroscopy in combination with simple 98Mo or double 98Mo/15N isotope enrichment of purified EcNarGH prepared at both acidic and basic pH to detect weakly coupled 14,15N interactions to the hpH and lpH Mo(V) species. Combined with quantitative spectral simulation, this approach allows us to specifically characterize the nitrogen environment of the two pH-dependent species. Using DFT calculations based on available structural and EPR spectroscopic data, we propose a structural model for the lpH Mo(V) species involving coordination of the metal by the monodentate ligand Asp222 and a hydroxo group. The latter is found to make a hydrogen bond with the terminal nitrogen atom from a nearby asparagine residue (Asn52), directly identified as the source of the nitrogen HYSCORE signals detected for both hpH and lpH Mo(V) species using 98Moenriched/selectively 15N-Asn-labeled Nar. This work establish for the first time in Nar enzymes a clear relationship between the spectroscopically detected Mo species and the structures deduced from X-ray crystallographic studies. Our results provide a foundation for monitoring the structure of the molybdenum active site in the presence of various substrates or inhibitors and in variants of Nar or other molybdenum enzymes.

■

MATERIALS AND METHODS

Preparation of 98 Mo-Enriched and 15 N/ 98 Mo Doubly Enriched NarGH. Escherichia coli cells from the nitrate reductasedeficient JCB4023 strain transformed with plasmid pNarGHHis6J were grown under aerobic conditions in M9 minimal medium supplemented with Mohr salt (10 μM), Na98MoO4 (5 μM, isotope purity ∼98% Oak Ridge National Laboratory, US), and with NH4Cl (18.7 mM). Alternatively, 15NH4Cl at the same concentration was used for the production of uniformly 15N/98Mo-enriched enzymes. To allow selective labeling of the NarGH complex with 15N-labeled asparagine, the JCB4023 strain was rendered auxotrophic for this amino acid through the deletion of both the asnA and asnB genes. The asnA gene was inactivated in the JCB4023 strain using P1 transduction from the JW3722 strain of the Keio collection.27 The kanamycin-resistance cassette was further eliminated from the JCB4023asnA::Kan transductant strain with the use of the pCP20 plasmid encoding the FLP recombinase.28 Using the same procedure, the JCB4023asnA strain was used subsequently for inactivation of the asnB gene using the JW0660 strain. Growth was performed as described above provided that asparagine was supplied at a final concentration of 100 mg/L. Uniformly 15N-labeled asparagine (isotopic purity 98%) was purchased from Eurisotop (Saint-Aubin, France). NarGH was purified in one step by affinity chromatography, as described earlier.29 Buffers used were CAPS (pH 10), Tricine (pH 8.5), or MES (pH 6.0 or 6.2), at 50 mM each. All solutions contained ethylenediaminetetraacetic acid (EDTA; 1 mM). As-prepared samples were used for experiments performed at pH 10 and 8.5, while samples prepared in MES buffer were redox poised at +400 mV (98Moenriched NarGH) or +370 mV (15N/98Mo-doubly enriched NarGH) to increase the contribution of the lpH species relative to that of the hpH form.17 For this purpose, a redox titration experiment was performed in an anaerobic cell flushed with argon. A cocktail of mediators (10 μM each of 2,5-dimethyl-p-benzoquinone, 2,6dichlorophenol indophenol, 2,3,4,5-tetramethyl-p-phenylenediamine, 1,4-benzoquinone, and N,N′-dimethyl-p-phenylenediamine) was C


Inorganic Chemistry

■

(release 5.0.12) that works under Matlab (The MathWorks, Inc., US).33,34 EPR spectra were simulated by adding the contribution of two independent Mo(V) species. Anisotropic spectral broadening was taken into account through unresolved hyperfine couplings (HStrain function) with full width at half-maximum (fwhm, in MHz) of Gaussian lines along the three principal axes adjusted to (23 ± 2, 16 ± 2, 23 ± 2) and (18 ± 2, 16 ± 2, 22 ± 4) for the lpH and hpH Mo(V) species, respectively. These values were used for both 98Mo-enriched or 15N/98Mo-doubly enriched NarGH samples. Simulations of the lpH Mo(V) species were performed by considering an additional proton hyperfine coupling tensor with principal axes assumed to be collinear to those of the Mo(V) g-tensor.14,16 For the simulation of HYSCORE spectra, the limited excitation bandwidth (approximated by 1/tp) of the microwave pulses (with length tp) was explicitly taken into account. When appropriate, Euler angles (α, β, γ), which describe the orientation of the Ã or P̃ tensors in the molecular frame,33 analogous to the g̃ frame, were used. Typical errors for the determination of the proportions of the two Mo(V) forms using numerical simulations are ±5% and ±15% for cw EPR and HYSCORE spectra, respectively. Theoretical Calculations. Mo(V) principal g-values, 1H and 14,15 N hfi and nqi tensors were calculated using the ORCA quantum chemistry package (ver. 3.0.1) at the DFT level of theory.35 Geometry optimizations were performed in vacuo using the B3LYP hybrid functional (the Becke’s three parameters hybrid exchange functional with 20% of Hartree−Fock admixture and the Lee−Yang−Parr nonlocal correlation functional) and the zero-order regular approximation Hamiltonian (ZORA) for relativistic components.36 The ZORA-recontracted versions of the def2-SVP basis sets were used for all elements with addition of effective core potentials for molybdenum.37 The resolution of identity38 with the appropriate auxiliary basis sets39 was used to accelerate the calculations. Starting coordinates for the different structural models describing the molybdenum active site were based on the X-ray crystal structure of Ec NarGH (PDB 1R27).6 Two kinds of models for the molybdenum active site were used: (i) in a first step, small models containing ∼60 atoms were used. These include the metal ion inserted into a modified cofactor in which the two pyranopterins are truncated at the pyrazine ring, and the Asp222 ligand also truncated at the αC. While simplified, these models have the advantage to distinguish unique structural possibilities from one another by evaluating the overall fit of their calculated magnetic resonance parameters to each other and to the experimental results, within reasonable computational time. These parameters are indeed particularly sensitive to the nature and the geometry of the atoms and molecules present within the first coordination sphere of Mo(V). Similar truncated molecular models have been very helpful for interpreting Mo(V) EPR spectroscopic data obtained on complex enzymes in term of structural models, as exemplified in previous studies on sulfite oxidase,40−42 mitochondrial amidoxime reducing component,43 dimethyl sulfoxide reductase,44 and periplasmic nitrate reductase;45 (ii) larger models containing ∼100 atoms were used in a second step, which include the entire pyranopterins (without the guanosine diphosphate moiety) and the nearest residues around the metal, that is, Asp222, Asn52, and the amide bond between Val578 and Gly579. The following geometric constraints were applied during optimization phase to prevent unrealistic conformations: (i) the SSSS dihedral angle, where the first two sulfur atoms belong to the proximal (P) pterin, whereas the two later belong to the distal (Q) one,46 was kept fixed during the geometry optimization for all models; (ii) the molybdenum and nitrogen atoms of Asn52 and Gly579 were kept frozen at the crystallographic positions in larger models. The g-, hfi, and nqi tensors were calculated by running single-point calculations on optimized structures, using the all-electron def2-TZVPP basis set in its scalar relativistic recontraction. For larger models, single-point calculations were performed employing the conductor-like screening model (COSMO)47 with a solvent dielectric constant ε = 4.0.

Article

RESULTS AND DISCUSSION EPR Analysis of Mo(V) in 98Mo-Enriched and 98Mo/15N Doubly Enriched NarGH. The cw EPR spectra of 98Moenriched NarGH or 15N/98Mo-doubly enriched NarGH prepared at pH ≈ 6 (hereafter referred to as lpH-14N and lpH-15N, respectively) are presented in Figure 2A,B,

Figure 2. Experimental (continuous lines) and simulated (red dotted lines) MoV EPR spectra in 98Mo-enriched (A, C) and 98Mo/15N doubly enriched NarGH (B, D). Samples were prepared at pH 6.0 (A), 6.2 (B), 8.5 (C), and 10 (D). Spectral simulations were obtained by summing the individual contribution of the lpH and hpH MoV species in the following proportions: (A) 92% lpH and 8% hpH MoV, (B) 86% lpH and 14% hpH MoV, (C) 27% lpH and 73% hpH MoV, (D) 16% lpH and 84% hpH MoV. Measurement conditions: temperature, 50 K; microwave power, 0.25 mW (A, D), 4 mW (B, C); modulation amplitude, 0.4 mT (A−C), 0.2 mT (D); modulation frequency, 100 kHz; microwave frequency, 9.481 47 GHz (A), 9.481 18 GHz (B), 9.481 04 GHz (C), 9.477 10 GHz (D); number of scans, 1 (B, C), 2 (A), 4 (D); magnetic field values were corrected against an offset using a Bruker weak pitch sample.

respectively. They show the rhombic EPR signature of the lpH Mo(V) species characterized by a g-tensor with principal values g1,2,3 = (2.0009, 1.9847, 1.9638) further split by hyperfine interaction originating from a strongly coupled I = 1/2 nucleus with principal values A1,2,3 ≈ (1.20, 0.89, 0.87) mT [i.e., A1,2,3 ≈ (33.6, 24.7, 23.9) MHz], consistent with literature values.9,10,16 In contrast, 98Mo- and 15N/98Mo-enriched NarGH samples prepared at pH 8.5 and 10 (hereafter referred to as hpH-14N and hpH-15N, respectively) show the typical hpH resonances with g1,2,3 ≈ (1.9869, 1.9800, 1.9607) and no resolved hyperfine structure (Figure 2C,D). Remarkably, the absence of I = 5/2 95,97 Mo isotopes in these samples allows to clearly resolve a weak contribution of the lpH species (mostly visible in the lowfield edge of the spectra shown in Figure 2C,D), which otherwise would have been hardly detectable because of its overlap with the signal from the 95,97Mo nuclei representing ∼25% of the natural abundance.9 Therefore, both lpH and hpH species contribute to the measured spectra.15,16 This holds true for all spectra shown in Figure 2. In particular, simulating the amplitude ratio between the two hyperfine lines at g3 of the lpH Mo(V) signal in samples prepared at acidic pH allows to estimate the contribution of the hpH species in these samples. This conclusion is further supported by the 14,15N HYSCORE experiments shown hereafter. The difficulty to isolate the contribution from each species when changing the pH of the D


Article

Inorganic Chemistry

Figure 3. 14N HYSCORE spectra of the Mo(V) species in the lpH-14N (A) and hpH-14N (B) samples prepared at pH 6.0 and 8.5, respectively. (C, D) The best simulations of HYSCORE cross-features shown in (A, B), respectively. Simulations were obtained using the parameters given in Table 1 for nuclei 14NI and 14NII by summing the contribution of the two Mo(V) species in the following proportions: 80% of 14NI + 20% 14NII (C), 15% of 14 NI + 85% 14NII (D). The signals on the diagonal are due to incomplete echo inversion by the mixing π pulse in the HYSCORE experiment and are therefore not reproduced in the simulations. Experimental parameters: time between first and second pulses τ = 136 ns, microwave frequency 9.690 84 GHz (A) and 9.693 69 GHz (B), and magnetic field, 348.9 mT (A) and 349.3 mT (B).

MHz also appears on the diagonal. These give rise to correlation peaks between frequencies ∼5.5 and 2.4 MHz (cross-peaks 2A in Figure 3B), 5.5 and 1.9 MHz (cross-peaks 2C in Figure 3B), and 5.5 and 0.6 MHz (cross-peaks 2B in Figure 3B). These observations suggest the spectral contribution of at least two nitrogen nuclei. The additive relationship satisfied by the three former frequencies (0.6 + 1.9 MHz is close to 2.4 MHz), the correlation pattern as well as the typical increase in the HYSCORE modulation depth measured in the timedomain spectrum (not shown) reflect the peculiar situation where the hyperfine coupling constant of the nitrogen responsible for these peaks is nearly equal to twice the nuclear Larmor frequency (aiso ≈ 2νI, cancellation condition). Consequently, the nuclear Zeeman and hyperfine splitting effectively cancel each other in one electron spin manifold Ms so that, in this manifold, the nuclear spin Hamiltonian reduces to the purely quadrupole Hamiltonian. Therefore, the three sharp lines detected in the hpH-14N HYSCORE spectrum are assigned to the three pure nuclear quadrupole resonance frequencies originating from a single 14N nucleus, hereafter referred to as NII. These frequencies are related by the equations

solvent implies that care must be taken when analyzing Mo(V) species in Nar preparations with naturally abundant molybdenum isotopes, for example, in EPR-monitored redox titration experiments. Spectral simulations (shown as red dotted lines in Figure 2) were performed to quantify the relative proportion of the two species in each sample. The results are given in the legend of Figure 2 and confirm that the variation of the relative proportion of the two Mo(V) species against pH cannot be accounted for by a simple acid−base equilibrium.15,16 14 N HYSCORE Spectroscopy of Mo(V) in 98MoEnriched NarGH. To detect weak 14N magnetic interactions with the Mo(V) ion that are unresolved in the EPR spectra shown in previous section, HYSCORE measurements were performed on the same samples. Representative 14 N HYSCORE spectra of the lpH-14N and hpH-14N samples are shown in Figure 3A,B, respectively. At pH 6.0, the spectrum is dominated by two intense offdiagonal cross-peaks (1A in Figure 3A) in the (+,+) quadrant, correlating nuclear transition frequencies at 2.7 and 4.0 MHz. Additional signals are detected, including two diagonal peaks at 0.6 and 2.3 MHz in the (+,+) quadrant as well as two pairs of cross-peaks correlating nuclear frequencies at 2.4 and 5.4 MHz and visible in both the (+,+) and (+,−) quadrants (referred to as 2A in Figure 3A). All these features remain in the HYSCORE spectrum of the hpH-14N sample prepared at pH 8.5, albeit with different relative amplitudes (Figure 3B). While crosspeaks 1A exhibit largely decreased amplitudes in the hpH-14N sample, the HYSCORE spectrum dominantly displays three sharp diagonal peaks at frequencies ∼0.6, 1.9 and 2.4 MHz. A broad line with lower amplitude having a maximum at ∼5.5

ν+ = κ(3 + η), ν− = κ(3 − η), ν0 = 2κη

(1a)

where κ is the quadrupolar coupling constant, and η is the asymmetry parameter of the electric field gradient on the nucleus.48 This situation of intermediate coupling is also characterized by the absence of single quantum peak in the second electron spin manifold Ms and by a broadening of the double quantum (dq) peak. This line has maximum intensity at a frequency approximated by E


Article

Inorganic Chemistry

Figure 4. 15N HYSCORE spectra of the Mo(V) species in the lpH-15N (A) and hpH-15N (B) samples prepared at pH 6.2 and 10, respectively. (C, D) The best simulations of spectra shown in (A, B), respectively. Simulations were obtained using the parameters given in Table 1 for 15NI and 15NII by summing the contribution of the two Mo(V) species in the following proportions: 60% 15NI + 40% 15NII (A), 20% 15NI + 80% 15NII (B). As the number of weakly coupled 15N nuclei to the two Mo(V) forms that are responsible for the dominant diagonal peak at the free 15N Larmor frequency is not known, an additional independent contribution from a lpH-like Mo(V) spin system having a single coupled 15N nucleus with (aiso, T) = (0, 0.25) MHz was added in simulations. Its spectral contribution was adjusted to fit with the experimental peak amplitude and did not affect the parameters found for NI and NII. This procedure was used to improve visual agreement between the experimental and simulated spectra. Experimental parameters: time between first and second pulses τ = 200 ns, microwave frequency 9.701 11 GHz (A), 9.675 52 GHz (B), and magnetic field, 349.6 mT (A) and 348.9 mT (B).

νdq ± = 2(νef ±2 + c)0.5

with the ratio between the effective nuclear frequency in each manifold νef± = |νI ± aiso/2| and the quadrupole coupling constant κ satisfying νef ±/κ > 1. In this case, only a single double quantum transition line without pronounced orientation dependence from each electron spin manifold is expected to show up in a frozen solution spectrum. Therefore, cross-peaks 1A are assigned to the two double quantum transitions νdq+ = 4.0 MHz and νdq− = 2.7 MHz from a 14N nucleus, hereafter referred to as NI. Application of eq 2 then leads to aiso(14NI) = 1.1 MHz and c = 1.5 MHz2, that is, a quadrupole coupling constant κ(NI) in the interval [0.6−0.7] (assuming 0 ≤ η ≤ 1). The circular contour line shape of cross peaks 1A indicates that the hyperfine interaction tensor is weakly anisotropic. Finally, taking into account the variation of the amplitude of the HYSCORE peaks against pH, we conclude that NI and NII nuclei are coupled to the lpH and hpH Mo(V) species, respectively. The detection of both NI and NII in lpH-14N and hpH-14N samples is consistent with the EPR data shown above indicating a contribution of both Mo(V) species in these samples. 15 N HYSCORE Spectroscopy of Mo(V) in 15N/ 98Mo Doubly Enriched NarGH. To substantiate and simplify the analysis of the nitrogen environment of the lpH and hpH forms, HYSCORE spectra were recorded in samples containing 15 N/98Mo doubly enriched NarGH prepared at pH 6.2 or 10. Indeed, I = 1/2 15N nuclei lack nuclear quadrupole interactions, so the 15N HYSCORE modulation is solely induced by the

(2)

where νef ± = |νI ± A/2|

(3)

c = κ 2(3 + η2)

(4)

A is the secular part of the hyperfine coupling tensor determined mainly from its isotropic part aiso in the case of a small anisotropic hyperfine tensor. In the HYSCORE spectrum shown in Figure 3B, cross peaks 2A, 2B, and 2C correlate 5.5 MHz with ν+ = 2.4 MHz, ν− = 1.9 MHz, and ν0 = 0.6 MHz in the two quadrants. Such an observation provides an unambiguous assignment of the 5.5 MHz nuclear frequency to the double quantum transition frequency from the hyperfine manifold, where the nuclear Zeeman and the hyperfine interactions are additive. Therefore, application of eqs 1 provides a first estimate of the quadrupolar parameters of NII, that is, κ(NII) = 0.71 MHz and η(NII) = 0.40. From eq 2, the assignment of νdq± = 5.5 MHz leads to the value A(14NII) ≈ 2.7 MHz. Although this estimated A value slightly deviates from the 2νI value (i.e., |A − 2νI| = 0.55 MHz), this deviation does not exceed 4κ/3 ≈ 0.95 MHz, which is the limiting value for the validity of the cancellation condition. Beside the HYSCORE features ascribed to NII, the observation of only two narrow cross peaks 1A correlating 14 N nuclear transition frequencies belonging to opposite electron spin manifolds indicates a 14N hyperfine interaction F


Article

Inorganic Chemistry

naturally abundant nitrogen isotopes. The hyperfine and quadrupolar parameters used for these simulations are given in bold in Table 1. The optimal proportions of both Mo(V)

anisotropic portion of the hyperfine interaction. This provides the off-diagonal elements in the spin Hamiltonian required to mix nuclear spin states and to drive both the allowed and semi forbidden EPR transitions. This anisotropic portion corresponds to the nonsecular term (B) of the modulation depth parameter, k (eq 7 in Supporting Information). In stark contrast to EPR spectra, 15N-enrichment of NarGH strongly affects the HYSCORE pattern, as illustrated when comparing the HYSCORE spectra of the lpH-15N sample and of the lpH-14N sample in Figures 4A and 3A, respectively. The former contains a prominent peak 0 on the diagonal at the 15N Zeeman frequency (νI(15N) = 1.48 MHz), which mostly arises from 15N nuclei around the Mo(V) that weakly interact with the unpaired electron. In addition, two cross peaks 1′ positioned symmetrically with respect to this diagonal peak 0 are clearly resolved in the (+,+) quadrant, with maxima at frequencies 0.7 and 2.3 MHz. They arise from a 15N nucleus with predominant isotropic hyperfine coupling, and we estimate the hyperfine coupling constant aiso(15N) ≈ 1.6 MHz from the span between cross-peaks 1′. By scaling 15N to 14N values by | A(15N)/A(14N)| = |gn(15N)/gn(14N)| ≈ 1.4, this translates into aiso(14N) ≈ 1.1 MHz, in excellent agreement with the value aiso(14NI) = 1.1 MHz determined above. Therefore, it can be safely concluded that the cross-peaks 1A observed in unlabeled NarGH are related with the appearance of cross peaks 1′ in the 15 N-labeled protein, so that cross-peaks 1A and 1′ arise from the same nitrogen, namely, NI. An additional pair of cross peaks 2′ is well-resolved in the (+,−) quadrant of the spectrum shown in Figure 4A, correlating frequencies ∼0.6 to ∼3.3 MHz. They must therefore arise from a more strongly coupled 15N nucleus than NI. Beside the strong diagonal peak 0, cross peaks 2′ are the dominant features of the HYSCORE spectrum recorded on the hpH-15N sample and shown in Figure 4B. Additional crosspeaks of weaker intensities appear at similar frequencies in the (+,+) quadrant, indicating that features 2′ arise from a single 15 N nucleus in the intermediate coupling regime, with an estimated A(15N) = 3.8 MHz corresponding to A(14N) = 2.7 MHz, in agreement with the value estimated for 14NII in the analysis shown above. Therefore, correlations 2′ arise from NII, which is coupled to the hpH Mo(V) species and experiences the cancellation condition. Translated into 15N and given the modest anisotropy of the hyperfine interaction, this indicates that the matching condition (4νI = 2aiso + T in the case of an axial hyperfine tensor) is fulfilled for 15NII.49 As a consequence, the lower of the two frequencies (να ≈ 0.6 MHz) no longer depends on the angle θ between the dipolar hyperfine interaction axis and the direction of the external magnetic field but solely on the dipolar term T, and it is given by να = 3T/4. A small rhombicity of the hyperfine coupling influences the width but not the position of the να peak. Hence, according to this analysis, a value of T(15NII) ≈ 0.8 MHz (corresponding to T(14NII) ≈ 0.6 MHz when rescaled to 14N nucleus) is obtained. Eventually, the relative intensities of cross-peaks 1′ and 2′ in Figure 4A,B are consistent with our assignment of NI and NII arising from the lpH and hpH Mo(V) species, respectively. Numerical Simulations of 14,15N HYSCORE Spectra. The procedure we used to simulate the Mo(V) HYSCORE spectra is detailed in the Supporting Information. The best simulations are shown as contour plots in Figure 4C,D, for 98 Mo/15N doubly labeled NarGH, and in Figure 3C,D, for HYSCORE spectra measured on 98Mo-enriched NarGH with

Table 1. Principal Valuesa of the g and A Tensors of 1H, 14N, and 15N Nuclei, and 14N Nuclear Quadrupole Parameters of the lpH and hpH Mo(V) Species lpH Mo(V)

gexp gcalc 15 NI exp 14

NI exp

14

NI calc

1

Hexp Hcalc gexp 15 NII exp 1

hpH Mo(V)

14

1

NII exp

Hexp

g1, g2, g3 = 2.0009, 1.9847, 1.9638 g1, g2, g3 = 2.0023, 1.9849, 1.9612 (aiso, T) (MHz) = (±1.3, ±0.35) or (∓1.4, ±0.35)b (aiso, T) (MHz) = (±0.9, ±0.30) or (∓1.0, ±0.30)b (|K| (MHz), η) = (0.69, 0.44)c A1, A2, A3 (MHz) = 0.27, 0.31, 0.54 (|K| (MHz), η) = (0.80, 0.35) A1, A2, A3 (MHz) = 34, 25, 25 A1, A2, A3 (MHz) = 33.4, 21.6, 20.4 g1, g2, g3 = 1.9869, 1.9800, 1.9605 (aiso, T) (MHz) = (±3.1, ±0.75) or (∓3.4, ±0.75)d (aiso, T) (MHz) = (±2.2, ±0.60) or (∓2.4, ±0.60)d (|K| (MHz), η) = (0.66, 0.40)e A1, A2, A3 (MHz) = 8.4, 8.4, 8.4f

a

Calculated values for the lpH species are issued from DFT calculations on computational model 1A, whereas experimental values were determined by simulating EPR and HYSCORE spectra. The large uncertainty on the Euler angles is due to the weak orientation selectivity of X-band HYSCORE experiments on the Mo(V) species in Nar. Estimated errors on the experimental values given in Table 1 are: gexp, ±0.0005, aiso(14,15N), ±0.1 MHz, T(14,15N), ±0.05 MHz, |K|, ±0.02 MHz, η, ±0.02, 1Hexp Ai, ±3 MHz). Hyperfine parameters used for simulating HYSCORE spectra are indicated in bold. bNo influence of Euler angles in the simulations of HYSCORE spectra. cEuler angles (100° ± 20°, 100° ± 80°, 170° ± 30°). dOnly a weak influence of the Euler angle in the simulations of HYSCORE spectra was found, leading to β = 20° ± 10°. eEuler angles (170° ± 150°, 90° ± 30°, 90° ± 10° or 270° ± 10°). fFrom ref 14.

species that were freely adjusted to fit the relative amplitudes of the NI and NII HYSCORE signals are in fairly good agreement with those obtained from EPR analysis of the corresponding spectra recorded on the same samples (see legend of Figures 2−4). Notably, the positions of the NI and NII cross-peaks in the 14N HYSCORE spectra are well-simulated by using similar nqi parameters for both nuclei (Table 1). Given the high sensitivity of these parameters to the chemical environment of a nucleus, we conclude that NI and NII arise from the same kind of nitrogen atom in the structure. DFT Calculations to Address the Structure of the lpH and hpH Mo(V) Species. How the EPR-detectable Mo(V) species relate to the differing crystal structures of the Mo active site in NarGHI or in NarGH remains debated.1,2,8,15 To tackle this issue and assign the detected nitrogen nuclei, we performed DFT calculations on geometry-optimized molecular models of the active site based on atomic coordinates from the NarGH crystal structure. The g-values of the hpH and lpH Mo(V) species in Nar are characteristic of a MoS4O2 coordination sphere, which is shared by all enzymes of the MobisPGD superfamily harboring a serine or an aspartate as direct ligand to the metal ion.2,15 In addition, the presence of a relatively large and exchangeable proton hyperfine coupling to the hpH and lpH Mo(V) species with aiso(1H) ≈ 27.7 and 8.4 MHz, G


Article

Inorganic Chemistry respectively, strongly constrains the possible structures of the Moco.9,14 Indeed, to account for such couplings, the most likely candidates are the exogenous oxygen-containing ligand or the carboxylic acid side chain of the Asp222 ligand. First, we calculated the g- and 1H-hfi tensors for several Mo(V) computational models shown in Figure 5, and these two distinct magnetic parameters were both considered to select the best model.

Figure 6. Correlation diagram of the experimental (red symbols) and calculated (black symbols) g values of the Mo(V) species discussed in the present work and plot against giso = 1/3 × (g1 + g2 + g3). (■) g1, (●) g2, (▲) g3 values. For models 1−5, SSSS angles are −20°, −20°, 5°, 10°, 0°, respectively. These values correspond to the conformation leading to the best agreement with experimental values that minimize 3 Δg = ∑i = 1 |g i calc − g iexp|for each model. The SSSS dihedral angle has been fixed to 0° for model 1A.

the hydroxo ligand is oriented in such a manner that it makes a strong hydrogen bond with Asp222. Regarding the hpH Mo(V) species and in contrast to model 1 for the lpH species, it appears that none of these simple models satisfactorily accounts for the local structure of the hpH species (Tables S1−S5). For model 3, the predicted principal values of the proton A-tensor are much weaker than their experimental counterparts, but it gives g values that are the closest to the experimental ones. Therefore, it cannot be firmly discarded, remaining a possible model for the hpH species. Vertebrate sulfite oxidases also exhibit two pH-dependent Mo(V) EPR signatures with features qualitatively similar to those of the lpH and hpH Mo(V) species in Nar.13,50 Indeed, the lpH Mo(V) species in chicken liver sulfite oxidase (cSO) is characterized by a g tensor with principal values g1,2,3 = (2.007, 1.974, 1.968) further split by a strong hyperfine interaction originating from an exchangeable proton with principal values A1,2,3 ≈ (42.7, 19.5, 17.3) MHz, assigned to a Mo-OH group.13,51 As observed in Nar, no splitting is resolved on the EPR spectrum of the hpH Mo(V) signal typically observed at pH > 8 and characterized by the g principal components g1,2,3 ≈ (1.990, 1.966, 1.954). However, direct evidence for the presence of an exchangeable proton coupled to the hpH species in cSO and assigned to a Mo-OH(n) species has been provided using variable-frequency pulsed EPR measurements. 52,53 Indeed, these techniques can detect weak interactions between Mo(V) and exchangeable 1,2H nuclei. Therefore, it has been proposed that both the lpH and hpH Mo(V) species in cSO have an equatorial hydroxo ligand coordinated to the metal ion. These conclusions are also supported by experiments on samples prepared in H217Oenriched buffer.24,40,54 DFT calculations performed on small molecular models indicated that differences in the magnitude of the experimentally determined 1H and 17O hyperfine coupling constants between both species are most likely due to a distinct orientation of the hydroxo ligand.24,40 Thus, we performed a similar systematic variation of the OMoOH dihedral angle on

Figure 5. Schematic structures of small computational models 1−5. The SSSS dihedral angle is defined as SaSbScSd. P and Q refer to the proximal and distal pyranopterins, respectively, according to their distance to the proximal FS0 [4Fe-4S] cluster.46

Asp222 was considered either in monodentate (models 1−3, 5) or bidentate (model 4) coordination, and in protonated (models 2−4) or deprotonated (models 1 and 5) state. For models with monodentate Asp222, the sixth position was occupied by an exogenous ligand, namely, a hydroxo (models 1 and 2), an oxo (model 3), or a water molecule (model 5). Only models with at least one exchangeable proton were considered. The SSSS dihedral angle formed by the two pairs of dithiolene sulfur donors (originating from the pyranopterins) was kept fixed to avoid nonphysiological conformations during geometrical optimizations. However, to study its effect on the calculated g and A(1H) values, a variation of this angle from −30° to + 30° was performed for each model. Indeed, examination of the available crystal structures of Mo-bisPGD enzymes indicates that this variation corresponds to the domain effectively covered. Corresponding calculated g and A(1H) values of all computational models are reported in Supporting Information (Tables S1−S5), and the set of g values leading to the closest agreement with experimental values is plotted for each model in Figure 6 against the isotropic g value. Considering both the g and A tensors, model 1 with a monodentate deprotonated Asp222 and a hydroxo ligand best fits with experimental data on lpH species (Figure 6). The best agreement is obtained for an SSSS dihedral angle of −20°, leading to g = 2.001, 1.984, 1.962 and A1,2,3(1HOH) = 32.4, 20.0, 17.5 MHz (Table S1). As illustrated in Figure 6, these g values are remarkably close to the experimental ones given the simplicity of the model. In the geometry-optimized model 1, H


Article

Inorganic Chemistry

larger model is called 1A thereafter, and a schematic representation of the geometry-optimized structure is shown in Figure 8.

model 1, varying it over the whole possible range with a 20° step, and we calculated the g-tensor principal values and the isotropic hfi constant of the hydroxo proton for each angle value. Beside the close agreement obtained for the g and isotropic hfi constants of the lpH species when this angle is close to 0°, the calculated parameters significantly deviate from those experimentally determined for the hpH Mo(V) species for all orientations (Figure 7). In contrast to cSO, a simple rotation of the OH group about the MoO bond alone is not sufficient to explain the hpH/lpH transition in Nar.

Figure 8. Schematic representation of the geometry-optimized computational model 1A obtained using the X-ray crystal coordinates from the soluble NarGH form (PDB 1R27).

The SSSS dihedral angle was fixed to either 0° or −15°, as it was not possible to reach the convergence criteria for model 1A with a SSSS angle of −20°. As the former angle yielded calculated g-values closer to experimental ones than the latter (see Table S6 in the Supporting Information), only the model with 0° is considered in the following. The calculated values of the spin Hamiltonian parameters for model 1A are given in Table 1, and the g-values are plotted in the correlation diagram shown in Figure 6. As observed for model 1, the g and A(1HOH) values calculated with model 1A are in good agreement with those of the lpH Mo(V) species with differences not exceeding 25 × 10−4 (Table 1). Calculated hfi parameters from nitrogen atoms included in model 1A are provided in Table 2. The Table 2. DFT-Calculated 14N hfi and nqi Parameters of Nitrogena Nuclei Included in Model 1A Figure 7. Effect of the variation of the dihedral angle θOH on the Mo(V) g-values (A) and on the proton isotropic hfi constant aiso(1HOH) (B). θOH is the OMoOH dihedral angle. θOH = 0° corresponds to the OH being coplanar with Mo-O(Asp). Parameters are calculated using model 1 and compared with those experimentally determined for the lpH (continuous lines) and hpH (dotted lines) Mo(V) species. The vertical dark shaded area shows the range of θOH for which the experimental and calculated g-values and hfi constants are in good agreement for lpH. The three calculated principal values g1, g2, and g3 of the Mo(V) g-tensor are indicated in (A) by black squares, red circles and blue triangles, respectively, whereas the 1H isotropic hfi constants are shown as black squares in (B).

nitrogen atom

A1; A2; A3 [MHz]

|κ| [MHz]

η

Asn52 Nδ Gly579 Np pyranopterin P N5 pyranopterin P N10 pyranopterin Q N5 pyranopterin Q N10 NI-lpH Mo(V) NII-hpH Mo(V) Asn Nδ Gly Np

0.27; 0.31; 0.54 0.05; −0.20; −0.20 0.90; 1.01; 1.29 0.10; 0.11; 0.18 0.71; 0.73; 0.89 −0.01; −0.05; −0.05 ±1.3; ±1.3; ±0.4 ±3.0; ±3.0; ±1.2

0.799 0.919 1.305 1.191 1.287 1.198 0.69 0.66 0.63 0.80−0.87

0.348 0.162 0.157 0.096 0.167 0.087 0.44 0.40 0.39 0.37−0.42

a

Pyranopterin nitrogen atoms are numbered as shown in Figure 8. Only nitrogen atoms carrying significant spin density are reported. Corresponding experimental values for NI and NII are also recalled. Nqi parameters measured by nuclear quadrupole resonance for Asn56 and polyglycine57 are given in the last two rows.

Further, to identify the origin of NI and NII, we included nitrogen atoms in the vicinity of the molybdenum while maintaining a coordination of the metal ion based on model 1. Examination of the Moco surrounding in the available X-ray crystal structures indicates that, beyond those belonging to the pyranopterin moieties, the two closest nitrogen atoms to the metal are Asn52-Nδ (d = 3.8−3.9 Å) and the Gly579-N (d = 3.7−3.8 Å), being therefore primary candidates. A larger computational model extended from model 1 was developed that includes the entire tetrahydropyranopterins, the Asn52, and the amide bond between Val578 and Gly579 residues. This

results indicate that nitrogen atoms from both pyranopterin moieties (i.e., N5 & N10 according to numbering given in Figure 8) and the protein environment (i.e., Asn52-Nδ and, to a lesser extent, the amide bond between Val578 and Gly579) can accommodate enough electron spin density to give rise to the experimentally measured nonzero isotropic hyperfine coupling constant (aiso ≈ 1 MHz for 14NI). However, the high sensitivity of weak 14N hfi parameters to small changes in I


Article

Inorganic Chemistry

Figure 9. Experimental (A) and simulated (B) HYSCORE spectra of the Mo(V) in as prepared 98Mo-enriched and selectively 15N-labeled NarGH (obtained using uniformly 15N-enriched Asn) prepared at pH 6.2. The relative amplitudes of the correlation peaks are well-simulated assuming that the spectrum results from the contribution of 25% of the hpH Mo(V) species and 75% of the lpH one, in agreement with the cw EPR data (see Figure S1 in the Supporting Information). Experimental parameters: time between first and second pulses τ = 136 ns; microwave frequency 9.698 74 GHz; magnetic field, 349.2 mT; the peak on the diagonal at 0.6 MHz arises from the 14NII single quantum transition ν0 and is due to a residual contribution of 14N in the sample. The signal on the antidiagonal around 4.5 MHz in the (+,−) quadrant is due to incomplete excitation of the spins by the mixing π pulse in the HYSCORE experiment.

sample at pH 6.2 exhibits a Mo(V) cw EPR signature wellsimulated by adding 75% of lpH to 25% of hpH Mo(V) species (see Figure S1 in the Supporting Information). A corresponding representative HYSCORE spectrum of this sample is shown in Figure 9A. It displays cross features identical to those produced by NI and NII in the HYSCORE spectrum measured on uniformly 15N-labeled NarGH prepared at the same pH value (Figure 4A). Further, the lack of signal at the 15N Zeeman frequency is consistent with the crystallographic data showing a single asparagine residue in the Moco surrounding, that is, Asn52. Moreover, the relative amplitudes of the detected HYSCORE correlation peaks are well-simulated assuming relative proportions of the Mo(V) species identical to those determined by simulation of the corresponding cw EPR spectrum (Figure 9B). These observations allow to unambiguously assign both NI and NII to the Asn52-Nδ terminal nitrogen in lpH and hpH species, as predicted from DFT calculations on model 1A. Structure of the Low-pH and High-pH Mo(V) Species. The present work provides the first structural model of the molybdenum active site in NarG that accounts for the magnetic parameters of the low pH Mo(V) determined from EPR and HYSCORE analyses. In this model, the first coordination sphere of the metal ion is provided by four sulfur atoms originating from the two pyranopterin moieties, a hydroxo ligand and an oxygen atom from the monodentate deprotonated Asp222 side chain, as observed in the NarGH structure.6 It is noteworthy that, using a similar approach, it was not yet possible to definitively account for the magnetic parameters of the high-pH Mo(V) species, although model 3 remains a possible candidate. In particular, (i) the bidentate coordination of Asp222 and the lack of oxo/hydroxo ligand as resolved in the NarGHI structure5 (model 4) leads to an unrealistically high gtensor anisotropy (Figure 6 and Table S4), (ii) the magnitude of the exchangeable proton hyperfine coupling to the hpH form is too large to arise from a protonated aspartate ligand, (iii) using small computational models, variations of the OMoOH dihedral angle previously proposed to be mainly responsible for the transition between the high- and low-pH Mo(V) species in cSO24,40 could not account alone for the same phenomenon in Nar (Figure 7). Such a situation is most likely due to the important role of the more distant environment in tuning the Moco physicochemical properties, which is not taken into

the electronic structure of the investigated models prevents using them as unique criteria to ascertain the identity of NI and NII. Rather, the nqi parameters, which are sensitive to the electric field gradient created by the local environment (bonds and charges) near the quadrupole nucleus, should be more reliable to identify the origin of interacting nuclei, provided that the computational model is large enough to adequately simulate the electric field gradient surrounding the atoms of interest. In the combined ESEEM and DFT study of 33S interactions to the blocked Mo(V) species generated in sulfite oxidases upon reduction of the enzyme by Na(33)SO3, the 33S nqi parameters were indeed the most determining factors to assign the interacting sulfur nucleus to a coordinated equatorial sulfite rather than to sulfate using either a truncated42 or a much larger (>250 atoms) computational model.55 Accordingly, significantly different κ values were calculated for the 14N nuclei included in model 1A (Table 2). In particular, pyranopterin nitrogen nuclei have calculated κ values in the range of [1.19−1.31], which is significantly higher than that of the two other nitrogen nuclei. Best agreement between the calculated and the experimentally determined nqi parameters is found for the terminal nitrogen (Nδ) of Asn52 in model 1A, indicating that the latter most likely corresponds to NI and NII (Table 1). This conclusion is further supported by direct comparison of the experimentally determined NI and NII nqi parameters with nuclear quadrupole resonance (NQR) data available from the literature.56,57 As illustrated in Table 2, this comparison shows that experimental κ values of NI and NII are very close to that determined for Asn Nδ by NQR (κ = 0.66 and 0.69 vs 0.63 MHz, respectively), and closer than those measured for peptide nitrogens in polyglycines. In the geometry-optimized structure, Asn52 Nδ atom makes a strong hydrogen bond with the oxygen of the hydroxo ligand of Mo (dN‑H = 1.74 Å), therefore allowing a significant transfer of spin density from the metal ion onto this nucleus. Importantly, this situation qualitatively accounts for the nonzero isotropic part of the hfi for NI and NII inferred from HYSCORE studies (Table 1). EPR and HYSCORE Spectroscopy of Mo(V) in 98MoEnriched and Selectively 15N-Asn-Labeled NarGH. To validate the identity of the nitrogen atom responsible for NI and NII, samples of purified 98Mo-enriched and 15N-Asn-labeled NarGH were prepared and studied by EPR. The as-purified J


Article

Inorganic Chemistry

suggesting a possible role of its side chain in water release after the two-electron reduction of the substrate.59 Asn52 is also structurally equivalent to Trp116 in the DMSO reductase from R. capsulatus, which appears hydrogen-bonded to the DMSO oxygen atom via its Nε in the structure of the DMSO reductase with the DMSO substrate bound to the active site (pdb code: 4DMR).62 This led to the suggestion that this residue participates in nitrate stabilization in the vicinity of the molybdenum cofactor in Nar during catalysis.6 As the lpH Mo(V) species is known to bind various anions, resolving the spectral contribution of Asn52 by HYSCORE spectroscopy opens new perspectives and the possibility to use it as a probe of the structure of the active site in the presence of these anions, including the substrate nitrate.

account in the small models used here (∼60 atoms for models 1−5). Although found in a tricyclic form in the vast majority of the three-dimensional (3D) crystal structures of the Mo/WbisPGD enzymes, bicyclic dihydropterins have been resolved in the complete NarGHI structure6 or in those of the evolutionary closest relatives ethylbenzene dehydrogenase58 and perchlorate reductase.59 Careful examination of these structures reveal that the immediate environment of the metal is only slightly affected by the opening of the pyrane ring. In particular, the SSSS dihedral angle, which strongly affects the Mo(V) g-values, is not significantly modified. Therefore, we anticipate that, rather than the geometry of the pyranopterin, the protein environment around the metal ion most likely tunes the lpH/hpH transition. Experiments aimed at evaluating the involvement of nearby residues in this process as well as theoretical studies using larger models are currently being performed in our laboratories to address these issues, keeping in mind that crystallographic structures may not reflect the conformation of the active protein in solution or may display altered conformation around the metal atoms due to structural heterogeneities and/or X-ray photoreduction.8 Detailed analysis of the HYSCORE spectra of the Mo(V) species prepared at acidic or basic pH indicates that the differences in the nuclear transition frequencies of NI and NII are mostly related to a change of nitrogen hyperfine coupling parameters (Table 1). On the basis of the unit spin 15N atomic hfi constant A = 2535.4 MHz,60 aiso(15N) values of 1.4 and 3.4 MHz for NI and NII correspond to 2s spin density populations ∼0.6 × 10−3 and ∼1.3 × 10−3 onto the interacting nitrogen, respectively; that is, the spin density onto the Asn52 Nδ increases by ∼0.7 × 10−3 from the lpH to the hpH species. This, together with the concomitant twofold increase of the anisotropic hfi, is qualitatively consistent with the interacting nucleus being closer to the Mo in the hpH form as compared to the lpH one. In light of our model, this could be structurally explained by subtle variations of the orientation and/or length of the H bond between the hydroxo ligand and the Asn52 terminal NH2 group. Such possibilities are currently being tested by DFT calculations. Detection and analysis of hyperfine interactions with additional nuclei such as 1,2H and 17O will certainly provide further constraints to identify and further distinguish the structure of the two Mo(V) species in Nar, as in the case of sulfite oxidizing enzymes.24 Role of Asn52 in the Catalytic Mechanism of NarGHI. Current mechanistic hypotheses for NarGHI catalytic cycle predict that the molybdenum hydroxo ligand could act as the labile group that is replaced by an oxygen atom from nitrate.61 The herein-proposed hydrogen bond between Asn52 and the molybdenum hydroxo ligand in the EPR-detected Mo(V) species anticipates an important role for this residue during catalysis. Asn52 is strictly conserved within members of the Nar/nitrite oxidoreductase/ethylbenzene dehydrogenase/ (per)chlorate reductase/selenite reductase/sterol C25 dehydrogenase/DMS dehydrogenase family, which all have an aspartate as Mo ligand and form a distinct clade on the phylogenetic tree of Mo/W-bisPGD enzymes.3 This Asn residue is situated on a highly conserved loop at the MobisPGD cofactor and FS0 cluster interface, and is adjacent to one of the FeS0 ligating Cys residues (e.g., Cys53 in Ec numbering). In the crystal structure of the perchlorate reductase solved in a reduced state with a substrate analogue bound to the active site (pdb code: 5CHC), the equivalent Asn35 stabilizes a water molecule with the Asp ligand,

■

CONCLUDING REMARKS An increasing number of structural and functional studies on mononuclear molybdenum enzymes underline the importance of the protein environment beside the first coordination sphere of the molybdenum cofactor for fine-tuning enzyme reactivity and molybdenum cofactor redox properties. In this study, by combining the use of multiple isotope enrichment strategies (98Mo- and uniform or selective 15N-enrichment) together with HYSCORE spectroscopy and DFT modeling, we propose a structural model for the lpH Mo(V) species in Nar discovered almost four decades ago by seminal studies of Bray and coworkers. Using such an approach, we demonstrate that details of the enzyme structural rearrangements around the Moco with pH can be precisely monitored. Further, we unveil the peculiar position of the conserved Asn52 to the Mo active site and its participation to H-bond network in both the lpH and hpH species. This work provides an immediate foundation for identifying the structures of the lpH-type Mo(V) intermediates generated upon addition of substrate or other anions (e.g., nitrite, chloride, fluoride) in Nar and in other molybdenum enzymes, including their catalytically compromised mutants, using 14,15N nuclei as magnetic probes.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b03129. EPR theory, procedure used for simulating HYSCORE spectra, calculated principal values of the g- and protonhyperfine coupling tensors for computational models 1− 5 upon variation of the SSSS dihedral angle. Comparison of g, hfi, and nqi parameters for model 1A with SSSS angle of 0° and −15°. Experimental and simulated Mo(V) spectra in as-purified 98Mo-enriched and selectively 15N-Asn-labeled NarGH (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 00-33-491-16-4557. Fax: 00-33-491-16-4097. ORCID

Pierre Ceccaldi: 0000-0001-8156-5035 Stéphane Grimaldi: 0000-0002-9559-6112 K


Article

Inorganic Chemistry Present Address

(11) Godfrey, C.; Greenwood, C.; Thomson, A. J.; Bray, R. C.; George, G. N. Electron-paramagnetic-resonance spectroscopy studies on the dissimilatory nitrate reductase from Pseudomonas-aeruginosa. Biochem. J. 1984, 224, 601−608. (12) Field, S. J.; Thornton, N. P.; Anderson, L. J.; Gates, A. J.; Reilly, A.; Jepson, B. J.; Richardson, D. J.; George, S. J.; Cheesman, M. R.; Butt, J. N. Reductive activation of nitrate reductases. Dalton Trans. 2005, 3580−6. (13) Lamy, M. T.; Gutteridge, S.; Bary, R. C. Electron-paramagneticresonance parameters of molybdenum(V) in sulfite oxidase from chicken liver. Biochem. J. 1980, 185, 397−403. (14) George, G. N.; Bray, R. C.; Morpeth, F. F.; Boxer, D. H. Complexes with halide and other anions of the molybdenum centre of nitrate reductase from Escherichia coli. Biochem. J. 1985, 227, 925−31. (15) Grimaldi, S.; Biaso, F.; Burlat, B.; Guigliarelli, B. In Molybdenum and Tungsten Enzymes: Spectroscopic and Theoretical Investigations; Hille, R., Kirk, M. L., Schulzke, C., Eds.; The Royal Society of Chemistry, 2016; pp 68−120. (16) Ceccaldi, P.; Rendon, J.; Leger, C.; Toci, R.; Guigliarelli, B.; Magalon, A.; Grimaldi, S.; Fourmond, V. Reductive activation of E. coli respiratory nitrate reductase. Biochim. Biophys. Acta, Bioenerg. 2015, 1847, 1055−63. (17) Magalon, A.; Asso, M.; Guigliarelli, B.; Rothery, R. A.; Bertrand, P.; Giordano, G.; Blasco, F. Molybdenum cofactor properties and [FeS] cluster coordination in Escherichia coli nitrate reductase A: investigation by site-directed mutagenesis of the conserved his-50 residue in the NarG subunit. Biochemistry 1998, 37, 7363−70. (18) Rothery, R. A.; Bertero, M. G.; Spreter, T.; Bouromand, N.; Strynadka, N. C. J.; Weiner, J. H. Protein Crystallography Reveals a Role for the FS0 Cluster of Escherichia coli Nitrate Reductase A (NarGHI) in Enzyme Maturation. J. Biol. Chem. 2010, 285, 8801− 8807. (19) Rothery, R. A.; Weiner, J. H. Shifting the metallocentric molybdoenzyme paradigm: the importance of pyranopterin coordination. JBIC, J. Biol. Inorg. Chem. 2015, 20, 349−372. (20) Wu, S. Y.; Rothery, R. A.; Weiner, J. H. Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase. J. Biol. Chem. 2015, 290, 25164−25173. (21) Jacques, J. G. J.; Fourmond, V.; Arnoux, P.; Sabaty, M.; Etienne, E.; Grosse, S.; Biaso, F.; Bertrand, P.; Pignol, D.; Leger, C.; Guigliarelli, B.; Burlat, B. Reductive activation in periplasmic nitrate reductase involves chemical modifications of the Mo-cofactor beyond the first coordination sphere of the metal ion. Biochim. Biophys. Acta, Bioenerg. 2014, 1837, 277−286. (22) Duval, S.; Santini, J. M.; Lemaire, D.; Chaspoul, F.; Russell, M. J.; Grimaldi, S.; Nitschke, W.; Schoepp-Cothenet, B. The H-bond network surrounding the pyranopterins modulates redox cooperativity in the molybdenum-bisPGD cofactor in arsenite oxidase. Biochim. Biophys. Acta, Bioenerg. 2016, 1857, 1353−62. (23) Blaney, S. M.; Pearsall, D. L.; Nieter Burgmayer, S. J.; Moore, E. M.; Sauk-Schubert, C. Redox reactions of the pyranopterin system of the molybdenum cofactor. JBIC, J. Biol. Inorg. Chem. 2004, 9, 59−66. (24) Klein, E. L.; Astashkin, A. V.; Raitsimring, A. M.; Enemark, J. H. Applications of pulsed EPR spectroscopy to structural studies of sulfite oxidizing enzymes. Coord. Chem. Rev. 2013, 257, 110−118. (25) Lorigan, G. A.; Britt, R. D.; Kim, J. H.; Hille, R. Electron-spin echo envelope modulation spectroscopy of the molybdenum center of xanthine-oxidase. Biochim. Biophys. Acta, Bioenerg. 1994, 1185, 284− 294. (26) Hanson, G. R.; Lane, I. In Metals in Biology-Applications of HighResolution EPR to Metalloenzymes; Berliner, G. H. a. L., Ed.; SpringerVerlag: New York, 2010; Chapter 7, pp 169−199. (27) Baba, T.; Ara, T.; Hasegawa, M.; Takai, Y.; Okumura, Y.; Baba, M.; Datsenko, K. A.; Tomita, M.; Wanner, B. L.; Mori, H. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2006, 2, 2006−0008. (28) Datsenko, K. A.; Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 6640−5.

§

Department of Chemistry, Boston University, 590 Commonwealth Avenue, 02215 Boston, MA, USA. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors are grateful to G. Gerbaud and E. Etienne for the maintenance of the EPR instrumentation used in this work, to B. Burlat and B. Schoepp-Cothenet for fruitful discussions, to L. Sylvi for her help in sample preparation, and to M. El Kousseifi for his work at the early stages of the present spectroscopic study. This work has been performed thanks to the support of the A*MIDEX project (No. ANR-11-IDEX-0001-02) funded by the “Investissements d’Avenir” French Government program, managed by the French National Research Agency (ANR), but also by the French national research agency (ANR, MC2 project, Grant No. 11-BSV5-005-01, https://anrmc2. wordpress.com, and MOLYERE project, Grant No. 16-CE290010-01), the CNRS for the “Émergence CO2” project from the “Mission interdisciplinarité”, and the national French EPR network (RENARD, IR3443, http://renard.univ-lille1.fr). Calculations were performed using computing resources from the “Centre Régional de Compétences en Modélisation Moléculaire” (Marseille). J. Rendon was supported by a CNRS− Institut de Chimie/Région Provence Alpes Côte d′Azur Ph.D. fellowship. The authors are part of the French Bioinorganic Chemistry group (http://frenchbic.cnrs.fr).

■

REFERENCES

(1) Hille, R.; Hall, J.; Basu, P. The mononuclear molybdenum enzymes. Chem. Rev. 2014, 114, 3963−4038. (2) Grimaldi, S.; Schoepp-Cothenet, B.; Ceccaldi, P.; Guigliarelli, B.; Magalon, A. The prokaryotic Mo/W-bisPGD enzymes family: a catalytic workhorse in bioenergetic. Biochim. Biophys. Acta, Bioenerg. 2013, 1827, 1048−1085. (3) Magalon, A.; Ceccaldi, P.; Schoepp-Cothenet, B. In Molybdenum and Tungsten Enzymes: Biochemistry; Hille, R., Schulzke, C., Kirk, M. L., Eds.; The Royal Society of Chemistry, 2016; pp 143−191. (4) Blasco, F.; Guigliarelli, B.; Magalon, A.; Asso, M.; Giordano, G.; Rothery, R. A. The coordination and function of the redox centres of the membrane-bound nitrate reductases. Cell. Mol. Life Sci. 2001, 58, 179−193. (5) Bertero, M. G.; Rothery, R. A.; Palak, M.; Hou, C.; Lim, D.; Blasco, F.; Weiner, J. H.; Strynadka, N. C. Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A. Nat. Struct. Biol. 2003, 10, 681−7. (6) Jormakka, M.; Richardson, D.; Byrne, B.; Iwata, S. Architecture of NarGH reveals a structural classification of Mo-bisMGD enzymes. Structure 2004, 12, 95−104. (7) George, G. N.; Turner, N. A.; Bray, R. C.; Morpeth, F. F.; Boxer, D. H.; Cramer, S. P. X-ray-absorption and electron-paramagneticresonance spectroscopic studies of the environment of molybdenum in high-pH and low-pH forms of Escherichia coli nitrate reductase. Biochem. J. 1989, 259, 693−700. (8) Pushie, M. J.; George, G. N. Spectroscopic studies of molybdenum and tungsten enzymes. Coord. Chem. Rev. 2011, 255, 1055−1084. (9) Vincent, S. P.; Bray, R. C. Electron-paramagnetic-resonance studies on nitrate reductase from Escherichia coli K12. Biochem. J. 1978, 171, 639−47. (10) Bray, R. C.; Vincent, S. P.; Lowe, D. J.; Clegg, R. A.; Garland, P. B. Electron-paramagnetic-resonance studies on the molybdenum of nitrate reductase from Escherichia coli K12. Biochem. J. 1976, 155, 201−3. L


Article

Inorganic Chemistry (29) Lanciano, P.; Vergnes, A.; Grimaldi, S.; Guigliarelli, B.; Magalon, A. Biogenesis of a respiratory complex is orchestrated by a single accessory protein. J. Biol. Chem. 2007, 282, 17468−74. (30) Guigliarelli, B.; Asso, M.; More, C.; Augier, V.; Blasco, F.; Pommier, J.; Giordano, G.; Bertrand, P. EPR and redox characterization of iron-sulfur centers in nitrate reductases A and Z from Escherichia coli: evidence for a high potential and low potential class and their relevance in the electron transfert mechanism. Eur. J. Biochem. 1992, 207, 61−68. (31) Grimaldi, S.; Arias Cartin, R.; Lanciano, P.; Lyubenova, S.; Endeward, B.; Prisner, T. F.; Magalon, A.; Guigliarelli, B. Direct evidence for nitrogen ligation to the high-stability semiquinone intermediate in Escherichia coli nitrate reductase A. J. Biol. Chem. 2010, 285, 179−187. (32) Arias-Cartin, R.; Lyubenova, S.; Ceccaldi, P.; Prisner, T.; Magalon, A.; Guigliarelli, B.; Grimaldi, S. HYSCORE evidence that endogenous mena- and ubisemiquinone bind at the same Q site (QD) of Escherichia coli nitrate reductase A. J. Am. Chem. Soc. 2010, 132, 5942−3. (33) Stoll, S.; Schweiger, A. EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J. Magn. Reson. 2006, 178, 42−55. (34) Stoll, S.; Britt, R. D. General and efficient simulation of pulse EPR spectra. Phys. Chem. Chem. Phys. 2009, 11, 6614−25. (35) Neese, F. The ORCA program system. Wiley Interdisciplinary Reviews-Computational Molecular Science 2012, 2, 73−78. (36) Buehl, M.; Kabrede, H. Geometries of transition-metal complexes from density-functional theory. J. Chem. Theory Comput. 2006, 2, 1282−1290. (37) Andrae, D.; Haussermann, U.; Dolg, M.; Stoll, H.; Preuss, H. Energy-adjusted ab initio pseudopotentials for the 2nd and 3rd row transition-elements. Theoretica Chimica Acta 1990, 77, 123−141. (38) Neese, F. An improvement of the resolution of the identity approximation for the formation of the Coulomb matrix. J. Comput. Chem. 2003, 24, 1740−1747. (39) Weigend, F. Accurate Coulomb-fitting basis sets for H to Rn. Phys. Chem. Chem. Phys. 2006, 8, 1057−1065. (40) Astashkin, A. V.; Klein, E. L.; Ganyushin, D.; Johnson-Winters, K.; Neese, F.; Kappler, U.; Enemark, J. H. Exchangeable oxygens in the vicinity of the molybdenum center of the high-pH form of sulfite oxidase and sulfite dehydrogenase. Phys. Chem. Chem. Phys. 2009, 11, 6733−6742. (41) Klein, E. L.; Astashkin, A. V.; Ganyushin, D.; Riplinger, C.; Johnson-Winters, K.; Neese, F.; Enemark, J. H. Direct Detection and Characterization of Chloride in the Active Site of the Low-pH Form of Sulfite Oxidase Using Electron Spin Echo Envelope Modulation Spectroscopy, Isotopic Labeling, and Density Functional Theory Calculations. Inorg. Chem. 2009, 48, 4743−4752. (42) Enemark, J. H.; Raitsimring, A. M.; Astashkin, A. V.; Klein, E. L. Implications for the mechanism of sulfite oxidizing enzymes from pulsed EPR spectroscopy and DFT calculations for ″difficult″ nuclei. Faraday Discuss. 2011, 148, 249−67. (43) Rajapakshe, A.; Astashkin, A. V.; Klein, E. L.; Reichmann, D.; Mendel, R. R.; Bittner, F.; Enemark, J. H. Structural Studies of the Molybdenum Center of Mitochondrial Amidoxime Reducing Component (mARC) by Pulsed EPR Spectroscopy and O-17Labeling. Biochemistry 2011, 50, 8813−8822. (44) Mtei, R. P.; Lyashenko, G.; Stein, B.; Rubie, N.; Hille, R.; Kirk, M. L. Spectroscopic and Electronic Structure Studies of a Dimethyl Sulfoxide Reductase Catalytic Intermediate: Implications for Electronand Atom-Transfer Reactivity. J. Am. Chem. Soc. 2011, 133, 9762− 9774. (45) Biaso, F.; Burlat, B.; Guigliarelli, B. DFT investigation of the molybdenum cofactor in periplasmic nitrate reductases: structure of the Mo(V) EPR-active species. Inorg. Chem. 2012, 51, 3409−19. (46) Rothery, R. A.; Workun, G. J.; Weiner, J. H. The prokaryotic complex iron-sulfur molybdoenzyme family. Biochim. Biophys. Acta, Biomembr. 2008, 1778, 1897−1929.

(47) Klamt, A.; Schuurmann, G. COSMO - A new approach to dielectric screening in solvents with explicit expressions for the screening energy and its gradient. J. Chem. Soc., Perkin Trans. 2 1993, 799−805. (48) Flanagan, H. L.; Singel, D. J. Analysis of 14N ESEEM patterns of randomly oriented solids. J. Chem. Phys. 1987, 87, 5606−5612. (49) Schweiger, A.; Jeschke, G. Principles of Pulse Electron Paramagnetic Resonance; Oxford University Press: New York, 2001. (50) Cohen, H. J.; Fridovich, I.; Rajagopalan, K. V. Hepatic sulfite oxidase. A functional role for molybdenum. J. Biol. Chem. 1971, 246, 374−82. (51) Astashkin, A. V.; Raitsimring, A. M.; Feng, C.; Johnson, J. L.; Rajagopalan, K. V.; Enemark, J. H. The Mo-OH proton of the low-pH form of sulfite oxidase: comparison of the hyperfine interactions obtained from pulsed ENDOR, CW-EPR and ESEEM measurements. Appl. Magn. Reson. 2002, 22, 421−430. (52) Raitsimring, A. M.; Pacheco, A.; Enemark, J. H. ESEEM investigations of the high pH and low pH forms of chicken liver sulfite oxidase. J. Am. Chem. Soc. 1998, 120, 11263−11278. (53) Astashkin, A. V.; Mader, M. L.; Pacheco, A.; Enemark, J. H.; Raitsimring, A. M. Direct detection of the proton-containing group coordinated to Mo(V) in the high pH form of chicken liver sulfite oxidase by refocused primary ESEEM spectroscopy: Structural and mechanistic implications. J. Am. Chem. Soc. 2000, 122, 5294−5302. (54) Cramer, S. P.; Johnson, J. L.; Rajagopalan, K. V.; Sorrell, T. N. Observation of O-17 effects on Mo-V EPR-spectra in sulfite oxidase, xanthine dehydrogenase, and MoO(SC6H5)4. Biochem. Biophys. Res. Commun. 1979, 91, 434−439. (55) Klein, E. L.; Raitsimring, A. M.; Astashkin, A. V.; Rajapakshe, A.; Johnson-Winters, K.; Arnold, A. R.; Potapov, A.; Goldfarb, D.; Enemark, J. H. Identity of the Exchangeable Sulfur-Containing Ligand at the Mo(V) Center of R160Q Human Sulfite Oxidase. Inorg. Chem. 2012, 51, 1408−1418. (56) Edmonds, D. T. Nuclear-Quadrupole Double-Resonance. Phys. Rep. 1977, 29, 233−290. (57) Rabbani, S. R.; Edmonds, D. T.; Gosling, P.; Palmer, M. H. Measurement of the N-14 quadrupole coupling-constants in Glycine, Diglycine, Triglycine, and Tetraglycine and a comparison with calculation. J. Magn. Reson. 1987, 72, 230−237. (58) Kloer, D. P.; Hagel, C.; Heider, J.; Schulz, G. E. Crystal structure of ethylbenzene dehydrogenase from Aromatoleum aromaticum. Structure 2006, 14, 1377−1388. (59) Youngblut, M. D.; Tsai, C. L.; Clark, I. C.; Carlson, H. K.; Maglaqui, A. P.; Gau-Pan, P. S.; Redford, S. A.; Wong, A.; Tainer, J. A.; Coates, J. D. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues. J. Biol. Chem. 2016, 291, 9190−9202. (60) Morton, J. R.; Preston, K. F. Atomic parameters for paramagnetic resonance data. J. Magn. Reson. 1978, 30, 577−582. (61) Correia, C.; Besson, S.; Brondino, C. D.; Gonzalez, P. J.; Fauque, G.; Lampreia, J.; Moura, I.; Moura, J. J. G. Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617. JBIC, J. Biol. Inorg. Chem. 2008, 13, 1321−1333. (62) McAlpine, A. S.; McEwan, A. G.; Bailey, S. The high resolution crystal structure of DMSO reductase in complex with DMSO. J. Mol. Biol. 1998, 275, 613−623.

M


and High-pH Mo(V) Species in Respiratory ... - ACS Publications

Recommend Documents