Improved resolution of 3-mercaptopropionate dioxygenase active site provided by ENDOR spectroscopy offers insight into catalytic mechanism.

3-mercaptopropionate (3MPA) dioxygenase (MDO) is a mononuclear nonheme iron enzyme that catalyzes the O2-dependent oxidation of thiol-bearing substrates to yield the corresponding sulfinic acid. MDO is a member of the cysteine dioxygenase family of small molecule thiol dioxygenases and thus shares a conserved sequence of active site residues (Serine-155, Histidine-157, and Tyrosine-159), collectively referred to as the SHY-motif. It has been demonstrated that these amino acids directly interact with the mononuclear Fe-site, influencing steady-state catalysis, catalytic efficiency, O2-binding, and substrate coordination. However, the underlying mechanism by which this is accomplished is poorly understood. Here, pulsed electron paramagnetic resonance spectroscopy [1H Mims electron nuclear double resonance spectroscopy] is applied to validate density functional theory computational models for the MDO Fe-site simultaneously coordinated by substrate and nitric oxide (NO), (3MPA/NO)-MDO. The enhanced resolution provided by electron nuclear double resonance spectroscopy allows for direct observation of Fe-bound substrate conformations and H-bond donation from Tyr159 to the Fe-bound NO ligand. Further inclusion of SHY-motif residues within the validated model reveals a distinct channel restricting movement of the Fe-bound NO-ligand. It has been argued that the iron-nitrosyl emulates the structure of potential Fe(III)-superoxide intermediates within the MDO catalytic cycle. While the merit of this assumption remains unconfirmed, the model reported here offers a framework to evaluate oxygen binding at the substrate-bound Fe-site and possible reaction mechanisms. It also underscores the significance of hydrogen bonding interactions within the enzymatic active site.

Robotic-assisted endoscopic onlay repair (R-ENDOR) for concomitant ventral hernias and diastasis recti: initial results and surgical technique.

BACKGROUND: The surgical management of midline ventral hernias complicated by concomitant diastasis recti presents a significant clinical challenge. The Endoscopic Onlay Repair (ENDOR) offers a minimally invasive solution, effectively addressing both conditions. This study focuses on describing the adaptation of ENDOR to a robotic platform, termed R-ENDOR, aiming to report initial outcomes along with other established robotic surgical approaches. METHODS: This retrospective case series study included consecutive adult patients who underwent R-ENDOR approach from October 2018 to April 2023, performed by a single surgeon. A comprehensive description of the surgical technique is included. Patient demographics, operative, and hernia-specific characteristics, as well as clinical outcomes are described. RESULTS: A total of 15 patients undergoing R-ENDOR for ventral hernia repair with diastasis recti plication were included. The median age was 59 years (IQR 42-63), with 60% (n = 9) female patients. The majority (86%, n = 13) had an ASA score of ≤ 2, and the median BMI was 24 kg/m2, with 20% (n = 3) classified as obese. Median hernia size was 2 cm (IQR 2-2.25), with a median diastasis length of 19 cm (IQR 15-21.5) and width of 4 cm (IQR 3-6). The median operative time was 129 min (IQR 113-166). Most repairs (93%, n = 14) were reinforced with mesh, predominantly self-fixating (73.3%, n = 11). Eighty percent of patients (n = 12) were discharged on the same day, with a median follow-up of 153 days (IQR 55-309). Notable complications included clinically significant seromas in 20% of patients (n = 3), long-term hypoesthesia in 40% (n = 6), and readmission in one patient (6.6%) for surgical site infection (SSI) requiring IV antibiotic therapy. CONCLUSION: Midline ventral hernias associated to diastasis recti can be managed robotically by ENDOR with safe and consistent 90-day outcomes in a carefully selected group of patients.

Statistical analysis of ENDOR spectra.

Electron-nuclear double resonance (ENDOR) measures the hyperfine interaction of magnetic nuclei with paramagnetic centers and is hence a powerful tool for spectroscopic investigations extending from biophysics to material science. Progress in microwave technology and the recent availability of commercial electron paramagnetic resonance (EPR) spectrometers up to an electron Larmor frequency of 263 GHz now open the opportunity for a more quantitative spectral analysis. Using representative spectra of a prototype amino acid radical in a biologically relevant enzyme, the [Formula: see text] in Escherichia coli ribonucleotide reductase, we developed a statistical model for ENDOR data and conducted statistical inference on the spectra including uncertainty estimation and hypothesis testing. Our approach in conjunction with 1H/2H isotopic labeling of [Formula: see text] in the protein unambiguously established new unexpected spectral contributions. Density functional theory (DFT) calculations and ENDOR spectral simulations indicated that these features result from the beta-methylene hyperfine coupling and are caused by a distribution of molecular conformations, likely important for the biological function of this essential radical. The results demonstrate that model-based statistical analysis in combination with state-of-the-art spectroscopy accesses information hitherto beyond standard approaches.

Pulse EPR Methods in the Angstrom to Nanometre Scale Shed Light on the Conformational Flexibility of a Fluoride Riboswitch.

Riboswitches control gene regulation upon external stimuli such as environmental factors or ligand binding. The fluoride sensing riboswitch from Thermotoga petrophila is a complex regulatory RNA proposed to be involved in resistance to F- cytotoxicity. The details of structure and dynamics underpinning the regulatory mechanism are currently debated. Here we demonstrate that a combination of pulsed electron paramagnetic resonance (ESR/EPR) spectroscopies, detecting distances in the angstrom to nanometre range, can probe distinct regions of conformational flexibility in this riboswitch. PELDOR (pulsed electron-electron double resonance) revealed a similar preorganisation of the sensing domain in three forms, i.e. the free aptamer, the Mg2+-bound apo, and the F--bound holo form. 19F ENDOR (electron-nuclear double resonance) was used to investigate the active site structure of the F--bound holo form. Distance distributions without a priori structural information were compared with in silico modelling of spin label conformations based on the crystal structure. While PELDOR, probing the periphery of the RNA fold, revealed conformational flexibility of the RNA backbone, ENDOR indicated low structural heterogeneity at the ligand binding site. Overall, the combination of PELDOR and ENDOR with sub-angstrom precision gave insight into structural organisation and flexibility of a riboswitch, not easily attainable by other biophysical techniques.

Structure and Internal Dynamics of Short RNA Duplexes Determined by a Combination of Pulsed EPR Methods and MD Simulations.

We used EPR spectroscopy to characterize the structure of RNA duplexes and their internal twist, stretch and bending motions. We prepared eight 20-base-pair-long RNA duplexes containing the rigid spin-label Çm, a cytidine analogue, at two positions and acquired orientation-selective PELDOR/DEER data. By using different frequency bands (X-, Q-, G-band), detailed information about the distance and orientation of the labels was obtained and provided insights into the global conformational dynamics of the RNA duplex. We used 19F Mims ENDOR experiments on three singly Çm- and singly fluorine-labeled RNA duplexes to determine the exact position of the Çm spin label in the helix. In a quantitative comparison to MD simulations of RNA with and without Çm spin labels, we found that state-of-the-art force fields with explicit parameterization of the spin label were able to describe the conformational ensemble present in our experiments. The MD simulations further confirmed that the Çm spin labels are excellent mimics of cytidine inducing only small local changes in the RNA structure. Çm spin labels are thus ideally suited for high-precision EPR experiments to probe the structure and, in conjunction with MD simulations, motions of RNA.

Final Stages in the Biosynthesis of the [FeFe]-Hydrogenase Active Site.

The paper aims to elucidate the final stages in the biosynthesis of the [2Fe]H active site of the [FeFe]-hydrogenases. The recently hypothesized intermediate [Fe2(SCH2NH2)2(CN)2(CO)4]2- ([1]2-) was prepared by a multistep route from [Fe2(S2)(CN)(CO)5]-. The following synthetic intermediates were characterized in order: [Fe2(SCH2NHFmoc)2(CNBEt3)(CO)5]-, [Fe2(SCH2NHFmoc)2(CN)-(CO)5]-, and [Fe2(SCH2NHFmoc)2(CN)2(CO)4]2-, where Fmoc is fluorenylmethoxycarbonyl). Derivatives of these anions include [K(18-crown-6)]+, PPh4 + and PPN+ salts as well as the 13CD2-isotopologues. These Fe2 species exist as a mixture of two isomers attributed to diequatorial (ee) and axial-equatorial (ae) stereochemistry at sulfur. In vitro experiments demonstrate that [1]2- maturates HydA1 in the presence of HydF and a cocktail of reagents. HydA1 can also be maturated using a highly simplified cocktail, omitting HydF and other proteins. This result is consistent with HydA1 participating in the maturation process and refines the roles of HydF.

Structure and Flexibility of Copper-Modified DNA G-Quadruplexes Investigated by 19 F ENDOR Experiments at 34†GHz.

DNA G-quadruplexes (GQs) are of great interest due to their involvement in crucial biological processes such as telomerase maintenance and gene expression. Furthermore, they are reported as catalytically active DNAzymes and building blocks in bio-nanotechnology. GQs exhibit remarkable structural diversity and conformational heterogeneity, necessitating precise and reliable tools to unravel their structure-function relationships. Here, we present insights into the structure and conformational flexibility of a unimolecular GQ with high spatial resolution via electron-nuclear double resonance (ENDOR) experiments combined with Cu(II) and fluorine labeling. These findings showcase the successful application of the 19 F-ENDOR methodology at 34 GHz, overcoming the limitations posed by the complexity and scarcity of higher-frequency spectrometers. Importantly, our approach retains both sensitivity and orientational resolution. This integrated study not only enhances our understanding of GQs but also expands the methodological toolbox for studying other macromolecules.

Two closed ATP- and ADP-dependent conformations in yeast Hsp90 chaperone detected by Mn(II) EPR spectroscopic techniques.

Hsp90 plays a central role in cell homeostasis by assisting folding and maturation of a large variety of clients. It is a homo-dimer, which functions via hydrolysis of ATP-coupled to conformational changes. Hsp90's conformational cycle in the absence of cochaperones is currently postulated as apo-Hsp90 being an ensemble of "open"/"closed" conformations. Upon ATP binding, Hsp90 adopts an active ATP-bound closed conformation where the N-terminal domains, which comprise the ATP binding site, are in close contact. However, there is no consensus regarding the conformation of the ADP-bound Hsp90, which is considered important for client release. In this work, we tracked the conformational states of yeast Hsp90 at various stages of ATP hydrolysis in frozen solutions employing electron paramagnetic resonance (EPR) techniques, particularly double electron-electron resonance (DEER) distance measurements. Using rigid Gd(III) spin labels, we found the C domains to be dimerized with same distance distribution at all hydrolysis states. Then, we substituted the ATPase Mg(II) cofactor with paramagnetic Mn(II) and followed the hydrolysis state using hyperfine spectroscopy and measured the inter-N-domain distance distributions via Mn(II)-Mn(II) DEER. The point character of the Mn(II) spin label allowed us resolve 2 different closed states: The ATP-bound (prehydrolysis) characterized by a distance distribution having a maximum of 4.3 nm, which broadened and shortened, shifting the mean to 3.8 nm at the ADP-bound state (posthydrolysis). This provides experimental evidence to a second closed conformational state of Hsp90 in solution, referred to as "compact." Finally, the so-called high-energy state, trapped by addition of vanadate, was found structurally similar to the posthydrolysis state.

Atomic-scale evidence for highly selective electrocatalytic N-N coupling on metallic MoS2.

Molybdenum sulfide (MoS2) is the most widely studied transition-metal dichalcogenide (TMDs) and phase engineering can markedly improve its electrocatalytic activity. However, the selectivity toward desired products remains poorly explored, limiting its application in complex chemical reactions. Here we report how phase engineering of MoS2 significantly improves the selectivity for nitrite reduction to nitrous oxide, a critical process in biological denitrification, using continuous-wave and pulsed electron paramagnetic resonance spectroscopy. We reveal that metallic 1T-MoS2 has a protonation site with a pKa of ∼5.5, where the proton is located ∼3.26 Šfrom redox-active Mo site. This protonation site is unique to 1T-MoS2 and induces sequential proton-electron transfer which inhibits ammonium formation while promoting nitrous oxide production, as confirmed by the pH-dependent selectivity and deuterium kinetic isotope effect. This is atomic-scale evidence of phase-dependent selectivity on MoS2, expanding the application of TMDs to selective electrocatalysis.

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.

GdIII -19 F Distance Measurements for Proteins in Cells by Electron-Nuclear Double Resonance.

Studies of protein structure and dynamics are usually carried out in dilute buffer solutions, conditions that differ significantly from the crowded environment in the cell. The double electron-electron resonance (DEER) technique can track proteins' conformations in the cell by providing distance distributions between two attached spin labels. This technique, however, cannot access distances below 1.8 nm. Here, we show that GdIII -19 F Mims electron-nuclear double resonance (ENDOR) measurements can cover part of this short range. Low temperature solution and in-cell ENDOR measurements, complemented with room temperature solution and in-cell GdIII -19 F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin-labeled with rigid GdIII tags. The proteins were delivered into human cells via electroporation. The solution and in-cell derived GdIII -19 F distances were essentially identical and lie in the 1-1.5 nm range revealing that both, GB1 and Ub, retained their overall structure in the GdIII and 19 F regions in the cell.

Primary donor triplet states of Photosystem I and II studied by Q-band pulse ENDOR spectroscopy.

The photoexcited triplet state of the "primary donors" in the two photosystems of oxygenic photosynthesis has been investigated by means of electron-nuclear double resonance (ENDOR) at Q-band (34 GHz). The data obtained represent the first set of 1H hyperfine coupling tensors of the 3P700 triplet state in PSI and expand the existing data set for 3P680. We achieved an extensive assignment of the observed electron-nuclear hyperfine coupling constants (hfcs) corresponding to the methine α-protons and the methyl group ß-protons of the chlorophyll (Chl) macrocycle. The data clearly confirm that in both photosystems the primary donor triplet is located on one specific monomeric Chl at cryogenic temperature. In comparison to previous transient ENDOR and pulse ENDOR experiments at standard X-band (9-10 GHz), the pulse Q-band ENDOR spectra demonstrate both improved signal-to-noise ratio and increased resolution. The observed ENDOR spectra for 3P700 and 3P680 differ in terms of the intensity loss of lines from specific methyl group protons, which is explained by hindered methyl group rotation produced by binding site effects. Contact analysis of the methyl groups in the PSI crystal structure in combination with the ENDOR analysis of 3P700 suggests that the triplet is located on the Chl a' (PA) in PSI. The results also provide additional evidence for the localization of 3P680 on the accessory ChlD1 in PSII.

Sequential Hydrogen Tunneling in o-Tolylmethylene.

o-Tolylmethylene 1 is a metastable triplet carbene that rearranges to o-xylylene 2 even at temperatures as low as 2.7 K via [1,4] H atom tunneling. Electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopical techniques were used to identify two conformers of 1 (anti and syn) in noble gas matrices and in frozen organic solutions. Conformer-specific kinetic measurements revealed that the rate constants for the rearrangements of the anti and syn conformers of 1 are very similar. However, the orbital alignment in the syn conformer is less favorable for the hydrogen transfer reaction than the orbital configuration in the anti conformer. Our spectroscopic and quantum chemical investigations indicate that anti 1 and syn 1 rapidly interconvert via efficient quantum tunneling forming a rotational pre-equilibrium. The subsequent second tunneling reaction, the [1,4] H migration from anti 1 to 2, is rate-limiting for the formation of 2. We here present an efficient strategy for the study of such tunneling equilibria.

Measurement of Angstrom to Nanometer Molecular Distances with 19 F Nuclear Spins by EPR/ENDOR Spectroscopy.

Spectroscopic and biophysical methods for structural determination at atomic resolution are fundamental in studies of biological function. Here we introduce an approach to measure molecular distances in bio-macromolecules using 19 F nuclear spins and nitroxide radicals in combination with high-frequency (94 GHz/3.4 T) electron-nuclear double resonance (ENDOR). The small size and large gyromagnetic ratio of the 19 F label enables to access distances up to about 1.5 nm with an accuracy of 0.1-1 Å. The experiment is not limited by the size of the bio-macromolecule. Performance is illustrated on synthesized fluorinated model compounds as well as spin-labelled RNA duplexes. The results demonstrate that our simple but strategic spin-labelling procedure combined with state-of-the-art spectroscopy accesses a distance range crucial to elucidate active sites of nucleic acids or proteins in the solution state.

Flavodoxin with an air-stable flavin semiquinone in a green sulfur bacterium.

Flavodoxins are small proteins with a non-covalently bound FMN that can accept two electrons and accordingly adopt three redox states: oxidized (quinone), one-electron reduced (semiquinone), and two-electron reduced (quinol). In iron-deficient cyanobacteria and algae, flavodoxin can substitute for ferredoxin as the electron carrier in the photosynthetic electron transport chain. Here, we demonstrate a similar function for flavodoxin from the green sulfur bacterium Chlorobium phaeovibrioides (cp-Fld). The expression of the cp-Fld gene, found in a close proximity with the genes for other proteins associated with iron transport and storage, increased in a low-iron medium. cp-Fld produced in Escherichia coli exhibited the optical, ERP, and electron-nuclear double resonance spectra that were similar to those of known flavodoxins. However, unlike all other flavodoxins, cp-Fld exhibited unprecedented stability of FMN semiquinone to oxidation by air and difference in midpoint redox potentials for the quinone-semiquinone and semiquinone-quinol couples (- 110 and - 530 mV, respectively). cp-Fld could be reduced by pyruvate:ferredoxin oxidoreductase found in the membrane-free extract of Chl. phaeovibrioides cells and photo-reduced by the photosynthetic reaction center found in membrane vesicles from these cells. The green sulfur bacterium Chl. phaeovibrioides appears thus to be a new type of the photosynthetic organisms that can use flavodoxin as an alternative electron carrier to cope with iron deficiency.

Changing the site energy of per-614 in the Peridinin-chlorophyll a-protein does not alter its capability of chlorophyll triplet quenching.

The peridinin-chlorophyll-a protein (PCP) is a water-soluble light harvesting protein of the dinoflagellate Amphidinium carterae, employing peridinin (Per) as the main carotenoid to fulfil light harvesting and photo-protective functions. Per molecules bound to the protein experience specific molecular surroundings which lead to different electronic and spectral properties. In the refolded N89 L variant PCP (N89 L-RFPCP) a significant part of the intensity on the long wavelength side of the absorption spectrum is shifted to shorter wavelengths due to a significant change in the Per-614 site energy. Since Per-614 has been shown to be the main chlorophyll (Chl) triplet quencher in the protein, and the relative geometry of pigments is not affected by the mutation as verified by X-ray crystallography, this variant is ideally suited to study the dependence of the triplet-triplet energy transfer (TTET) mechanism on the pigment site energy. By using a combination of Optically Detected Magnetic Resonance (ODMR), pulse Electron Paramagnetic Resonance (EPR) and Electron Nuclear DOuble Resonance (ENDOR) we found that PCP maintains the efficient Per-614-to-Chl-a TTET despite the change of Per-614 local energy. This shows the robustness of the photoprotective site, which is very important for the protection of the system.

Thermally Activated Delayed Fluorescence in a Y3 N@C80 Endohedral Fullerene: Time-Resolved Luminescence and EPR Studies.

The endohedral fullerene Y3 N@C80 exhibits luminescence with reasonable quantum yield and extraordinary long lifetime. By variable-temperature steady-state and time-resolved luminescence spectroscopy, it is demonstrated that above 60 K the Y3 N@C80 exhibits thermally activated delayed fluorescence with maximum emission at 120 K and a negligible prompt fluorescence. Below 60 K, a phosphorescence with a lifetime of 192±1 ms is observed. Spin distribution and dynamics in the triplet excited state is investigated with X- and W-band EPR and ENDOR spectroscopies and DFT computations. Finally, electroluminescence of the Y3 N@C80 /PFO film is demonstrated opening the possibility for red-emitting fullerene-based organic light-emitting diodes (OLEDs).

Surface Sensing of Quantum Dots by Electron Spins.

The nanoscale design of quantum dots (QDs) requires advanced analytical techniques. However, those that are commonly used do not have sufficient sensitivity or spatial resolution. Here, we use magnetic resonance techniques combined with paramagnetic Mn impurities in PbS QDs for sensitive probing of the QD surface and environment. In particular, we reveal inequivalent proton spin relaxations of the capping ligands and solvent molecules, strengths and anisotropies of the Mn nuclear spin interactions, and Mn nuclei distances with ∼1 Šsensitivity. These findings demonstrate the potential of magnetically doped QDs as sensitive magnetic nanoprobes and the use of electron spins for surface sensing.

Proton Matrix ENDOR Studies on Ca2+-depleted and Sr2+-substituted Manganese Cluster in Photosystem II.

Proton matrix ENDOR spectra were measured for Ca(2+)-depleted and Sr(2+)-substituted photosystem II (PSII) membrane samples from spinach and core complexes from Thermosynechococcus vulcanus in the S2 state. The ENDOR spectra obtained were similar for untreated PSII from T. vulcanus and spinach, as well as for Ca(2+)-containing and Sr(2+)-substituted PSII, indicating that the proton arrangements around the manganese cluster in cyanobacterial and higher plant PSII and Ca(2+)-containing and Sr(2+)-substituted PSII are similar in the S2 state, in agreement with the similarity of the crystal structure of both Ca(2+)-containing and Sr(2+)-substituted PSII in the S1 state. Nevertheless, slightly different hyperfine separations were found between Ca(2+)-containing and Sr(2+)-substituted PSII because of modifications of the water protons ligating to the Sr(2+) ion. Importantly, Ca(2+) depletion caused the loss of ENDOR signals with a 1.36-MHz separation because of the loss of the water proton W4 connecting Ca(2+) and YZ directly. With respect to the crystal structure and the functions of Ca(2+) in oxygen evolution, it was concluded that the roles of Ca(2+) and Sr(2+) involve the maintenance of the hydrogen bond network near the Ca(2+) site and electron transfer pathway to the manganese cluster.

Advanced paramagnetic resonance spectroscopies of iron-sulfur proteins: Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM).

The advanced electron paramagnetic resonance (EPR) techniques, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies, provide unique insights into the structure, coordination chemistry, and biochemical mechanism of nature's widely distributed iron-sulfur cluster (FeS) proteins. This review describes the ENDOR and ESEEM techniques and then provides a series of case studies on their application to a wide variety of FeS proteins including ferredoxins, nitrogenase, and radical SAM enzymes. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.