Heme Spin Distribution in the Substrate-Free and Inhibited Novel CYP116B5hd: A Multifrequency Hyperfine Sublevel Correlation (HYSCORE) Study.

The cytochrome P450 family consists of ubiquitous monooxygenases with the potential to perform a wide variety of catalytic applications. Among the members of this family, CYP116B5hd shows a very prominent resistance to peracid damage, a property that makes it a promising tool for fine chemical synthesis using the peroxide shunt. In this meticulous study, we use hyperfine spectroscopy with a multifrequency approach (X- and Q-band) to characterize in detail the electronic structure of the heme iron of CYP116B5hd in the resting state, which provides structural details about its active site. The hyperfine dipole-dipole interaction between the electron and proton nuclear spins allows for the locating of two different protons from the coordinated water and a beta proton from the cysteine axial ligand of heme iron with respect to the magnetic axes centered on the iron. Additionally, since new anti-cancer therapies target the inhibition of P450s, here we use the CYP116B5hd system-imidazole as a model for studying cytochrome P450 inhibition by an azo compound. The effects of the inhibition of protein by imidazole in the active-site geometry and electron spin distribution are presented. The binding of imidazole to CYP116B5hd results in an imidazole-nitrogen axial coordination and a low-spin heme FeIII. HYSCORE experiments were used to detect the hyperfine interactions. The combined interpretation of the gyromagnetic tensor and the hyperfine and quadrupole tensors of magnetic nuclei coupled to the iron electron spin allowed us to obtain a precise picture of the active-site geometry, including the orientation of the semi-occupied orbitals and magnetic axes, which coincide with the porphyrin N-Fe-N axes. The electronic structure of the iron does not seem to be affected by imidazole binding. Two different possible coordination geometries of the axial imidazole were observed. The angles between gx (coinciding with one of the N-Fe-N axes) and the projection of the imidazole plane on the heme were determined to be -60° and -25° for each of the two possibilities via measurement of the hyperfine structure of the axially coordinated 14N.

Cyanide replaces substrate in obligate-ordered addition of nitric oxide to the non-heme mononuclear iron AvMDO active site.

Thiol dioxygenases are a subset of non-heme mononuclear iron oxygenases that catalyze the O2-dependent oxidation of thiol-bearing substrates to yield sulfinic acid products. Cysteine dioxygenase (CDO) and 3-mercaptopropionic acid (3MPA) dioxygenase (MDO) are the most extensively characterized members of this enzyme family. As with many non-heme mononuclear iron oxidase/oxygenases, CDO and MDO exhibit an obligate-ordered addition of organic substrate before dioxygen. As this substrate-gated O2-reactivity extends to the oxygen-surrogate, nitric oxide (NO), EPR spectroscopy has long been used to interrogate the [substrate:NO:enzyme] ternary complex. In principle, these studies can be extrapolated to provide information about transient iron-oxo intermediates produced during catalytic turnover with dioxygen. In this work, we demonstrate that cyanide mimics the native thiol-substrate in ordered-addition experiments with MDO cloned from Azotobacter vinelandii (AvMDO). Following treatment of the catalytically active Fe(II)-AvMDO with excess cyanide, addition of NO yields a low-spin (S = 1/2) (CN/NO)-Fe-complex. Continuous wave and pulsed X-band EPR characterization of this complex produced in wild-type and H157N variant AvMDO reveal multiple nuclear hyperfine features diagnostic of interactions within the first- and outer-coordination sphere of the enzymatic Fe-site. Spectroscopically validated computational models indicate simultaneous coordination of two cyanide ligands replaces the bidentate (thiol and carboxylate) coordination of 3MPA allowing for NO-binding at the catalytically relevant O2-binding site. This promiscuous substrate-gated reactivity of AvMDO with NO provides an instructive counterpoint to the high substrate-specificity exhibited by mammalian CDO for L-cysteine.

Syntheses and Electrochemical and EPR Studies of Porphyrins Functionalized with Bulky Aromatic Amine Donors.

A series of nickel(II) porphyrins bearing one or two bulky nitrogen donors at the meso positions were prepared by using Ullmann methodology or more classical Buchwald-Hartwig amination reactions to create the new C-N bonds. For several new compounds, single crystals were obtained, and the X-ray structures were solved. The electrochemical data of these compounds are reported. For a few representative examples, spectroelectrochemical measurements were used to clarify the electron exchange process. In addition, a detailed electron paramagnetic resonance (EPR) study was performed to estimate the extent of delocalization of the generated radical cations. In particular, electron nuclear double resonance spectroscopy (ENDOR) was used to determine the coupling constants. DFT calculations were conducted to corroborate the EPR spectroscopic data.

Structure of 3-mercaptopropionic acid dioxygenase with a substrate analog reveals bidentate substrate binding at the iron center.

Thiol dioxygenases are a subset of nonheme iron oxygenases that catalyze the formation of sulfinic acids from sulfhydryl-containing substrates and dioxygen. Among this class, cysteine dioxygenases (CDOs) and 3-mercaptopropionic acid dioxygenases (3MDOs) are the best characterized, and the mode of substrate binding for CDOs is well understood. However, the manner in which 3-mercaptopropionic acid (3MPA) coordinates to the nonheme iron site in 3MDO remains a matter of debate. A model for bidentate 3MPA coordination at the 3MDO Fe-site has been proposed on the basis of computational docking, whereas steady-state kinetics and EPR spectroscopic measurements suggest a thiolate-only coordination of the substrate. To address this gap in knowledge, we determined the structure of Azobacter vinelandii 3MDO (Av3MDO) in complex with the substrate analog and competitive inhibitor, 3-hydroxypropionic acid (3HPA). The structure together with DFT computational modeling demonstrates that 3HPA and 3MPA associate with iron as chelate complexes with the substrate-carboxylate group forming an additional interaction with Arg168 and the thiol bound at the same position as in CDO. A chloride ligand was bound to iron in the coordination site assigned as the O2-binding site. Supporting HYSCORE spectroscopic experiments were performed on the (3MPA/NO)-bound Av3MDO iron nitrosyl (S = 3/2) site. In combination with spectroscopic simulations and optimized DFT models, this work provides an experimentally verified model of the Av3MDO enzyme-substrate complex, effectively resolving a debate in the literature regarding the preferred substrate-binding denticity. These results elegantly explain the observed 3MDO substrate specificity, but leave unanswered questions regarding the mechanism of substrate-gated reactivity with dioxygen.

Nature and Topology of Metal-Oxygen Binding Sites in Zeolite Materials: 17 O High-Resolution EPR Spectroscopy of Metal-Loaded ZSM-5.

Determining structural models is pivotal to the rational understanding and development of heterogeneous catalytic systems. A paradigmatic case is represented by open-shell metals supported on oxides, where the catalytic properties crucially depend on the nature of the metal-oxygen bonds and the extent of charge and spin transfer. Through a combination of selective 17 O isotopic enrichment and the unique properties of open-shell s-state monovalent Group 12 cations, we derive a site-specific topological description of active sites in an MFI zeolite. We show that just a few selected sites out of all possible are populated and that the relative occupancies depend on the specific properties of the metal, and we provide maps of charge and spin transfer at the metal-oxygen interface. This approach is not restricted to zeotype materials, rather it is applicable to any catalysts supported on oxygen-containing materials.

Snapshots of a Migrating H-Atom: Characterization of a Reactive Iron(III) Indenide Hydride and its Nearly Isoenergetic Ring-Protonated Iron(I) Isomer.

We report the characterization of an S= 1/2 iron π-complex, [Fe(η6 -IndH)(depe)]+ (Ind=Indenide (C9 H7- ), depe=1,2-bis(diethylphosphino)ethane), which results via C-H elimination from a transient FeIII hydride, [Fe(η3 :η2 -Ind)(depe)H]+ . Owing to weak M-H/C-H bonds, these species appear to undergo proton-coupled electron transfer (PCET) to release H2 through bimolecular recombination. Mechanistic information, gained from stoichiometric as well as computational studies, reveal the open-shell π-arene complex to have a BDFEC-H value of ≈50 kcal mol-1 , roughly equal to the BDFEFe-H of its FeIII -H precursor (ΔG°≈0 between them). Markedly, this reactivity differs from related Fe(η5 -Cp/Cp*) compounds, for which terminal FeIII -H cations are isolable and have been structurally characterized, highlighting the effect of a benzannulated ring (indene). Overall, this study provides a structural, thermochemical, and mechanistic foundation for the characterization of indenide/indene PCET precursors and outlines a valuable approach for the differentiation of a ring- versus a metal-bound H-atom by way of continuous-wave (CW) and pulse EPR (HYSCORE) spectroscopic measurements.

The effect of a C298D mutation in CaHydA [FeFe]-hydrogenase: Insights into the protein-metal cluster interaction by EPR and FTIR spectroscopic investigation.

A conserved cysteine located in the signature motif of the catalytic center (H-cluster) of [FeFe]-hydrogenases functions in proton transfer. This residue corresponds to C298 in Clostridium acetobutylicum CaHydA. Despite the chemical and structural difference, the mutant C298D retains fast catalytic activity, while replacement with any other amino acid causes significant activity loss. Given the proximity of C298 to the H-cluster, the effect of the C298D mutation on the catalytic center was studied by continuous wave (CW) and pulse electron paramagnetic resonance (EPR) and by Fourier transform infrared (FTIR) spectroscopies. Comparison of the C298D mutant with the wild type CaHydA by CW and pulse EPR showed that the electronic structure of the center is not altered. FTIR spectroscopy confirmed that absorption peak values observed in the mutant are virtually identical to those observed in the wild type, indicating that the H-cluster is not generally affected by the mutation. Significant differences were observed only in the inhibited state Hox-CO: the vibrational modes assigned to the COexo and Fed-CO in this state are shifted to lower values in C298D, suggesting different interaction of these ligands with the protein moiety when C298 is changed to D298. More relevant to the catalytic cycle, the redox equilibrium between the Hox and Hred states is modified by the mutation, causing a prevalence of the oxidized state. This work highlights how the interactions between the protein environment and the H-cluster, a dynamic closely interconnected system, can be engineered and studied in the perspective of designing bio-inspired catalysts and mimics.

Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non-diphosphate Inhibitors.

Isoprenoid biosynthesis is an important area for anti-infective drug development. One isoprenoid target is (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMBPP) reductase (IspH), which forms isopentenyl diphosphate and dimethylallyl diphosphate from HMBPP in a 2H+ /2e- reduction. IspH contains a 4 Fe-4 S cluster, and in this work, we first investigated how small molecules bound to the cluster by using HYSCORE and NRVS spectroscopies. The results of these, as well as other structural and spectroscopic investigations, led to the conclusion that, in most cases, ligands bound to IspH 4 Fe-4 S clusters by η1 coordination, forming tetrahedral geometries at the unique fourth Fe, ligand side chains preventing further ligand (e.g., H2 O, O2 ) binding. Based on these ideas, we used in silico methods to find drug-like inhibitors that might occupy the HMBPP substrate binding pocket and bind to Fe, leading to the discovery of a barbituric acid analogue with a Ki value of ≈500 nm against Pseudomonas aeruginosa IspH.

Combined Spectroscopic and Electrochemical Detection of a Ni(I) ⋠⋠⋠H-N Bonding Interaction with Relevance to Electrocatalytic H2 Production.

The [Ni(P(R) 2 N(R') 2 )2 ](2+) family of complexes are exceptionally active catalysts for proton reduction to H2 . In this manuscript, we explore the first protonation step of the proposed catalytic cycle by using a catalytically inactive Ni(I) complex possessing a sterically demanding variation of the ligand. Due to the paramagnetic nature of the Ni(I) oxidation state, the protonated Ni(I) intermediate has been characterized through a combination of cyclic voltammetry, electron nuclear double resonance (ENDOR) spectroscopy, and hyperfine sublevel correlation (HYSCORE) spectroscopy. Both the electrochemical and spectroscopic studies indicate that the Ni(I) complex is protonated at a pendant amine that is endo to Ni, which suggests the presence of an intramolecular Ni(I) ⋅⋅⋅HN bonding interaction. Using density functional theory, the hydrogen bond was found to involve three doubly-occupied, localized molecular orbitals: the 3dxz , 3d z 2, and 3dyz orbitals of nickel. These studies provide the first direct experimental evidence for this critical catalytic intermediate, and implications for catalytic H2 production are discussed.

Multi-frequency, multi-technique pulsed EPR investigation of the copper binding site of murine amyloid ß peptide.

Copper-amyloid peptides are proposed to be the cause of Alzheimer's disease, presumably by oxidative stress. However, mice do not produce amyloid plaques and thus do not suffer from Alzheimer's disease. Although much effort has been focused on the structural characterization of the copper- human amyloid peptides, little is known regarding the copper-binding mode in murine amyloid peptides. Thus, we investigated the structure of copper-murine amyloid peptides through multi-frequency, multi-technique pulsed EPR spectroscopy in conjunction with specific isotope labeling. Based on our pulsed EPR results, we found that Ala2, Glu3, His6, and His14 are directly coordinated with the copper ion in murine amyloid ß peptides at pH 8.5. This is the first detailed structural characterization of the copper-binding mode in murine amyloid ß peptides. This work may advance the knowledge required for developing inhibitors of Alzheimer's disease.

Molecular basis of [FeFe]-hydrogenase function: an insight into the complex interplay between protein and catalytic cofactor.

The precise electrochemical features of metal cofactors that convey the functions of redox enzymes are essentially determined by the specific interaction pattern between cofactor and enclosing protein environment. However, while biophysical techniques allow a detailed understanding of the features characterizing the cofactor itself, knowledge about the contribution of the protein part is much harder to obtain. [FeFe]-hydrogenases are an interesting class of enzymes that catalyze both, H2 oxidation and the reduction of protons to molecular hydrogen with significant efficiency. The active site of these proteins consists of an unusual prosthetic group (H-cluster) with six iron and six sulfur atoms. While H-cluster architecture and catalytic states during the different steps of H2 turnover have been thoroughly investigated during the last 20 years, possible functional contributions from the polypeptide framework were only assumed according to the level of conservancy and X-ray structure analyses. Due to the recent development of simpler and more efficient expression systems the role of single amino acids can now be experimentally investigated. This article summarizes, compares and categorizes the results of recent investigations based on site directed and random mutagenesis according to their informative value about structure function relationships in [FeFe]-hydrogenases. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.

CYP116B5hd, a self-sufficient P450 cytochrome: A dataset of its electronic and geometrical properties.

This paper documents the dataset obtained from the Electron Paramagnetic Resonance (EPR) study of the electronic properties of a self-sufficient cytochrome P450, CYP116B5hd, which possesses an interesting catalytic activity for synthetic purposes. In fact, when isolated, its heme domain can act as a peroxygenase on different substrates of biotechnological interest. Raw data shown in Famulari et al. (2022) and supplementary data in raw and processed forms (figures) are documented and available in this paper. Additionally, simulations of the experimental data together with simulation scripts based for EasySpin, a widespread MATLAB toolbox for EPR spectral simulations, are provided. The procedure for g-value analysis based on a crystal-field theory is also detailed here, offering an interesting tool for comparison of FeIII-heme P450 systems. Due to the catalytic interest of the protein, which has been recently discovered, and the correlation that has been reported between g-values and peroxidase function, both, CW-EPR and HYSCORE spectra and data set of the model CYPBM3hd are also provided. Finally, the materials and methods for enzyme production and purification, sample preparation and experimental and spectroscopic procedures a together with instrumental details are described in detail. The data files and simulation scripts can be found in: https://doi.org/10.5281/zenodo.6418626.

EPR characterization of the heme domain of a self-sufficient cytochrome P450 (CYP116B5).

CYP116B5 is a self-sufficient cytochrome P450 (CYP450) with interesting catalytic properties for synthetic purposes. When isolated, its heme domain can act as a peroxygenase on different substrates of biotechnological interest. Here, by means of continuous wave and advanced EPR techniques, the coordination environment of iron in the isolated CYP116B5 heme domain (CYP116b5hd) is characterized. The ligand-free protein shows the characteristic EPR spectrum of a low-spin (S = 1/2) FeIII-heme with [gz = 2.440 ± 0.005, gy = 2.25 ± 0.01, gx = 1.92 ± 0.01]. These g-values reflect an electronic ground state very similar to classical P450 monooxygenases rather than P450 peroxygenases. Binding of imidazole results in g-values very close to the ones reported for CYP152 peroxygenases. The detection of hyperfine interactions through HYperfine Sub-level CORrElation (HYSCORE) Spectroscopy experiments, shows that this is due to a nitrogen-mediated axial coordination. This work adds a piece of experimental evidence to the research, aimed at elucidating the features that distinguish the classical P450 enzymes from peroxygenases. It shows that the electronic environment of heme iron of CYP116B5 in the resting state is similar to the classical P450 monooxygenases. Therefore, it is not the critical factor that confers to CYP116B5hd its peroxygenase-like activity, suggesting a crucial role of the protein matrix.

High-Field (3.4 T) Electron Paramagnetic Resonance, 1H Electron-Nuclear Double Resonance, ESEEM, HYSCORE, and Relaxation Studies of Asphaltene Solubility Fractions of Bitumen for Structural Characterization of Intrinsic Carbon-Centered Radicals.

Petroleum asphaltenes are considered the most irritating components of various oil systems, complicating the extraction, transportation, and processing of hydrocarbons. Despite the fact that the paramagnetic properties of asphaltenes and their aggregates have been studied since the 1950s, there is still no clear understanding of the structure of stable paramagnetic centers in petroleum systems. The paper considers the possibilities of various electron paramagnetic resonance (EPR) techniques to study petroleum asphaltenes and their solubility fractions using a carbon-centered stable free radical (FR) as an intrinsic probe. The dilution of asphaltenes with deuterated toluene made it possible to refine the change in the structure at the initial stage of asphaltene disaggregation. From the measurements of samples of bitumen, a planar circumcoronene-like model of FR structure and FR-centered asphaltenes is proposed. The results show that EPR-based approaches can serve as sensitive numerical tools to follow asphaltenes' structure and their disaggregation.

A sensitivity leap for X-band EPR using a probehead with a cryogenic preamplifier.

Inspired by the considerable success of cryogenically cooled NMR cryoprobes, we present an upgraded X-band EPR probehead, equipped with a cryogenic low-noise preamplifier. Our setup suppresses source noise, can handle the high microwave powers typical in X-band pulsed EPR, and is compatible with the convenient resonator coupling and sample access found on commercially available spectrometers. Our approach allows standard pulsed and continuous-wave EPR experiments to be performed at X-band frequency with significantly increased sensitivity compared to the unmodified setup. The probehead demonstrates a voltage signal-to-noise ratio (SNR) enhancement by a factor close to 8× at a temperature of 6 K, and remains close to 2× at room temperature. By further suppressing room-temperature noise at the expense of reduced microwave power (and thus minimum π-pulse length), the factor of SNR improvement approaches 15 at 6 K, corresponding to an impressive 200-fold reduction in EPR measurement time. We reveal the full potential of this probehead by demonstrating such SNR improvements using a suite of typical hyperfine and dipolar spectroscopy experiments on exemplary samples.

Two-dimensional HYSCORE spectroscopy reveals a histidine imidazole as the axial ligand to Chl3A in the M688HPsaA genetic variant of Photosystem I.

Recent studies on Photosystem I (PS I) have shown that the six core chlorophyll a molecules are highly coupled, allowing for efficient creation and stabilization of the charge-separated state. One area of particular interest is the identity and function of the primary acceptor, A0, as the factors that influence its ultrafast processes and redox properties are not yet fully elucidated. It was recently shown that A0 exists as a dimer of the closely-spaced Chl2/Chl3 molecules wherein the reduced A0- state has an asymmetric distribution of electron spin density that favors Chl3. Previous experimental work in which this ligand was changed to a hard base (histidine, M688HPsaA) revealed severely impacted electron transfer processes at both the A0 and A1 acceptors; molecular dynamics simulations further suggested two distinct conformations of PS I in which the His residue coordinates and forms a hydrogen bond to the A0 and A1 cofactors, respectively. In this study, we have applied 2D HYSCORE spectroscopy in conjunction with molecular dynamics simulations and density functional theory calculations to the study of the M688HPsaA variant. Analysis of the hyperfine parameters demonstrates that the His imidazole serves as the axial ligand to the central Mg2+ ion in Chl3A in the M688HPsaA variant. Although the change in ligand identity does not alter delocalization of electron density over the Chl2/Chl3 dimer, a small shift in the asymmetry of delocalization, coupled with the electron withdrawing properties of the ligand, most likely accounts for the inhibition of forward electron transfer in the His-ligated conformation.

1,2H hyperfine spectroscopy and DFT modeling unveil the demethylmenasemiquinone binding mode to E. coli nitrate reductase A (NarGHI).

The quinol oxidation site QD in E. coli respiratory nitrate reductase A (EcNarGHI) reacts with the three isoprenoid quinones naturally synthesized by the bacterium, i.e. ubiquinones (UQ), menaquinones (MK) and demethylmenaquinones (DMK). The binding mode of the demethylmenasemiquinone (DMSK) intermediate to the EcNarGHI QD quinol oxidation site is analyzed in detail using 1,2H hyperfine (hf) spectroscopy in combination with H2O/D2O exchange experiments and DFT modeling, and compared to the menasemiquinone one bound to the QD site (MSKD) previously studied by us. DMSKD and MSKD are shown to bind in a similar and strongly asymmetric manner through a short (~1.7 Å) H-bond. The origin of the specific hf pattern resolved on the DMSKD field-swept EPR spectrum is unambiguously ascribed to slightly inequivalent contributions from two ß-methylene protons of the isoprenoid side chain. DFT calculations show that their large isotropic hf coupling constants (Aiso ~12 and 15 MHz) are consistent with both (i) a specific highly asymmetric binding mode of DMSKD and (ii) a near in-plane orientation of its isoprenyl chain at Cß relative to the aromatic ring, which differs by ~90° to that predicted for free or NarGHI-bound MSK. Our results provide new insights into how the conformation and the redox properties of different natural quinones are selectively fine-tuned by the protein environment at a single Q site. Such a fine-tuning most likely contributes to render NarGHI as an efficient and flexible respiratory enzyme to be used upon rapid variations of the Q-pool content.

Multiple drug binding modes in Mycobacterium tuberculosis CYP51B1.

The Mycobacterium tuberculosis (Mtb) genome encodes 20 different cytochrome P450 enzymes (CYPs), many of which serve essential biosynthetic roles. CYP51B1, the Mtb version of eukaryotic sterol demethylase, remains a potential therapeutic target. The binding of three drug fragments containing nitrogen heterocycles to CYP51B1 is studied here by continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) techniques to determine how each drug fragment binds to the heme active-site. All three drug fragments form a mixture of complexes, some of which retain the axial water ligand from the resting state. Hyperfine sublevel correlation spectroscopy (HYSCORE) and electron-nuclear double resonance spectroscopy (ENDOR) observe protons of the axial water and on the drug fragments that reveal drug binding modes. Binding in CYP51B1 is complicated by the presence of multiple binding modes that coexist in the same solution. These results aid our understanding of CYP-inhibitor interactions and will help guide future inhibitor design.

Determining the Electronic Structure of Paramagnetic Intermediates in membrane proteins: A high-resolution 2D 1H hyperfine sublevel correlation study of the redox-active tyrosines of photosystem II.

The photosynthetic reaction center, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in nature by using light energy to drive water oxidation. The four-electron water oxidation reaction occurs at the tetranuclear manganese­calcium-oxo (Mn4Ca-oxo) cluster that is present in the oxygen-evolving complex (OEC) of PSII. The water oxidation reaction is facilitated by proton-coupled electron transfer (PCET) at the redox-active tyrosine residue, YZ, in the OEC which is one of the two symmetric tyrosine residues, YZ and YD, in PSII. Although YZ and YD are chemically identical, their redox properties and reaction kinetics are very different. In the present study, we apply high-resolution two-dimensional (2D) 1H hyperfine sublevel correlation (HYSCORE) spectroscopy to determine the electronic structure of YZ and YD to understand better the functional tuning of PCET at each tyrosine. Most importantly, the 2D HYSCORE measurements that are described here are applicable for the study of paramagnetic cofactors in a wide variety of membrane-bound proteins.

Non-uniform HYSCORE: Measurement, processing and analysis with Hyscorean.

Non-uniform sampling (NUS) provides a considerable reduction of measurement time especially for multi-dimensional experiments. This comes at the cost of additional signal processing steps to reconstruct the complete signal from the experimental data points. Despite being routinely employed in NMR for many experiments, EPR applications have not benefited from NUS due to the lack of a straightforward implementation to perform NUS in common commercial spectrometers. In this work we present a novel method to perform NUS HYSCORE experiments on commercial Bruker EPR spectrometers, along with a benchmark of modern reconstruction methods, and new processing software tools for NUS HYSCORE signals. All of this comes in the form of a free-software package: Hyscorean. Experimental NUS spectra are measured and processed with this package using different reconstruction methods and compared to their uniform sampled counterparts, thereby showcasing the method's potential for EPR spectroscopy.