Spatial Arrangement of the Drug Ibuprofen in a Model Membrane in the Presence of Lipid Rafts.

Many pharmaceutical drugs are known to interact with lipid membranes through nonspecific molecular interactions, which affect their therapeutic effect. Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID) and one of the most commonly prescribed. In the presence of cholesterol, lipid bilayers can separate into nanoscale liquid-disordered and liquid-ordered structures, the latter known as lipid rafts. Here, we study spin-labeled ibuprofen (ibuprofen-SL) in the model membrane consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol in the molar ratio of (0.5-0.5xchol)/(0.5-0.5xchol)/xchol. Electron paramagnetic resonance (EPR) spectroscopy is employed, along with its pulsed version of double electron-electron resonance (DEER, also known as PELDOR). The data obtained indicate lateral lipid-mediated clustering of ibuprofen-SL molecules with a local surface density noticeably larger than that expected for random lateral distribution. In the absence of cholesterol, the data can be interpreted as indicating alternating clustering in two opposing leaflets of the bilayer. In the presence of cholesterol, for xchol ≥ 20 mol %, the results show that ibuprofen-SL molecules have a quasi-regular lateral distribution, with a "superlattice" parameter of ∼3.0 nm. This regularity can be explained by the entrapment of ibuprofen-SL molecules by lipid rafts known to exist in this system with the additional assumption that lipid rafts have a nanoscale substructure.

Independent Mutation of Two Bridging Carboxylate Ligands Stabilizes Alternate Conformers of the Photosynthetic O2-Evolving Mn4CaO5 Cluster in Photosystem II.

The O2-evolving Mn4CaO5 cluster in photosystem II is ligated by six carboxylate residues. One of these is D170 of the D1 subunit. This carboxylate bridges between one Mn ion (Mn4) and the Ca ion. A second carboxylate ligand is D342 of the D1 subunit. This carboxylate bridges between two Mn ions (Mn1 and Mn2). D170 and D342 are located on opposite sides of the Mn4CaO5 cluster. Recently, it was shown that the D170E mutation perturbs both the intricate networks of H-bonds that surround the Mn4CaO5 cluster and the equilibrium between different conformers of the cluster in two of its lower oxidation states, S1 and S2, while still supporting O2 evolution at approximately 50% the rate of the wild type. In this study, we show that the D342E mutation produces much the same alterations to the cluster's FTIR and EPR spectra as D170E, while still supporting O2 evolution at approximately 20% the rate of the wild type. Furthermore, the double mutation, D170E + D342E, behaves similarly to the two single mutations. We conclude that D342E alters the equilibrium between different conformers of the cluster in its S1 and S2 states in the same manner as D170E and perturbs the H-bond networks in a similar fashion. This is the second identification of a Mn4CaO5 metal ligand whose mutation influences the equilibrium between the different conformers of the S1 and S2 states without eliminating O2 evolution. This finding has implications for our understanding of the mechanism of O2 formation in terms of catalytically active/inactive conformations of the Mn4CaO5 cluster in its lower oxidation states.

HYSCORE and QM/MM Studies of Second Sphere Variants of the Type 1 Copper Site in Azurin: Influence of Mutations on the Hyperfine Couplings of Remote Nitrogens.

Secondary coordination sphere (SCS) interactions have been shown to play important roles in tuning reduction potentials and electron transfer (ET) properties of the Type 1 copper proteins, but the precise roles of these interactions are not fully understood. In this work, we examined the influence of F114P, F114N, and N47S mutations in the SCS on the electronic structure of the T1 copper center in azurin (Az) by studying the hyperfine couplings of (i) histidine remote Nε nitrogens and (ii) the amide Np using the two-dimensional (2D) pulsed electron paramagnetic resonance (EPR) technique HYSCORE (hyperfine sublevel correlation) combined with quantum mechanics/molecular mechanics (QM/MM) and DLPNO-CCSD calculations. Our data show that some components of hyperfine tensor and isotropic coupling in N47SAz and F114PAz (but not F114NAz) deviate by up to ∼±20% from WTAz, indicating that these mutations significantly influence the spin density distribution between the CuII site and coordinating ligands. Furthermore, our calculations support the assignment of Np to the backbone amide of residue 47 (both in Asn and Ser variants). Since the spin density distributions play an important role in tuning the covalency of the Cu-Scys bond of Type 1 copper center that has been shown to be crucial in controlling the reduction potentials, this study provides additional insights into the electron spin factor in tuning the reduction potentials and ET properties.

LETHAL TOXIN NEUTRALIZING ANTIBODY RESPONSE INDUCED FOLLOWING ORAL VACCINATION WITH A MICROENCAPSULATED BACILLUS ANTHRACIS STERNE STRAIN 34F2 VACCINE PROOF-OF-CONCEPT STUDY IN WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS).

Improved methods are needed to prevent wildlife deaths from anthrax. Caused by Bacillus anthracis, naturally occurring outbreaks of anthrax are frequent but unpredictable. The commercially available veterinary vaccine is labeled for subcutaneous injection and is impractical for large-scale wildlife vaccination programs; therefore, oral vaccination is the most realistic method to control and prevent these outbreaks. We reported the induction of an anthrax-specific lethal toxin (LeTx) neutralizing antibody response in mice following oral vaccination with alginate microcapsules containing B. anthracis Sterne strain 34F2 spores, coated with poly-L-lysine (PLL) and vitelline protein B (VpB). We continued evaluating our novel vaccine formulation through this proof-of-concept study in white-tailed deer (WTD; Odocoileus virginianus; n = 9). We orally vaccinated WTD via needle-free syringe with three formulations of the encapsulated vaccine: 1) PLL-VpB-coated microcapsules with 107-8 spores/ml (n = 5), 2) PLL-VpB-coated microcapsules with 109-10 spores/ml (n = 2), and 3) PLL-coated microcapsules with 109-10 spores/ml (n = 2). Although the limited sample sizes require continued experimentation, we observed an anthrax-specific antibody response in WTD serum following oral vaccination with PLL-coated microcapsules containing 109 spores/ ml. Furthermore, this antibody response neutralized anthrax LeTx in vitro, suggesting that continued development of this vaccine may allow for realistic wildlife anthrax vaccination programs.

Debye Temperature Evaluation for Secondary Battery Cathode of α-SnxFe1-xOOH Nanoparticles Derived from the 57Fe- and 119Sn-Mössbauer Spectra.

Debye temperatures of α-SnxFe1-xOOH nanoparticles (x = 0, 0.05, 0.10, 0.15 and 0.20, abbreviated as Sn100x NPs) prepared by hydrothermal reaction were estimated with 57Fe- and 119Sn-Mössbauer spectra measured by varying the temperature from 20 to 300 K. Electrical properties were studied by solid-state impedance spectroscopy (SS-IS). Together, the charge-discharge capacity of Li- and Na-ion batteries containing Sn100x NPs as a cathode were evaluated. 57Fe-Mössbauer spectra of Sn10, Sn15, and Sn20 measured at 300 K showed only one doublet due to the superparamagnetic doublet, while the doublet decomposed into a sextet due to goethite at the temperature below 50 K for Sn 10, 200 K for Sn15, and 100 K for Sn20. These results suggest that Sn10, Sn15 and Sn20 had smaller particles than Sn0. On the other hand, 20 K 119Sn-Mössbauer spectra of Sn15 were composed of a paramagnetic doublet with an isomer shift (δ) of 0.24 mm s-1 and quadrupole splitting (∆) of 3.52 mm s-1. These values were larger than those of Sn10 (δ: 0.08 mm s-1, ∆: 0.00 mm s-1) and Sn20 (δ: 0.10 mm s-1, ∆: 0.00 mm s-1), suggesting that the SnIV-O chemical bond is shorter and the distortion of octahedral SnO6 is larger in Sn15 than in Sn10 and Sn20 due to the increase in the covalency and polarization of the SnIV-O chemical bond. Debye temperatures determined from 57Fe-Mössbauer spectra measured at the low temperature were 210 K, 228 K, and 250 K for Sn10, Sn15, and Sn20, while that of α-Fe2O3 was 324 K. Similarly, the Debye temperature of 199, 251, and 269 K for Sn10, Sn15, and Sn20 were estimated from the temperature-dependent 119Sn-Mössbauer spectra, which were significantly smaller than that of BaSnO3 (=658 K) and SnO2 (=382 K). These results suggest that Fe and Sn are a weakly bound lattice in goethite NPs with low crystallinity. Modification of NPs and addition of Sn has a positive effect, resulting in an increase in DC conductivity of almost 5 orders of magnitude, from a σDC value of 9.37 × 10-7 (Ω cm)-1 for pure goethite Sn (Sn0) up to DC plateau for samples containing 0.15 and 0.20 Sn (Sn15 and Sn20) with a DC value of ~4 × 10-7 (Ω cm)-1 @423 K. This non-linear conductivity pattern and levelling at a higher Sn content suggests that structural modifications have a notable impact on electron transport, which is primarily governed by the thermally activated via three-dimensional hopping of small polarons (SPH). Measurements of SIB performance, including the Sn100x cathode under a current density of 50 mA g-1, showed initial capacities of 81 and 85 mAh g-1 for Sn0 and Sn15, which were larger than the others. The large initial capacities were measured at a current density of 5 mA g-1 found at 170 and 182 mAh g-1 for Sn15 and Sn20, respectively. It is concluded that tin-goethite NPs are an excellent material for a secondary battery cathode and that Sn15 is the best cathode among the studied Sn100x NPs.

Exploring the Effect of V2O5 and Nb2O5 Content on the Structural, Thermal, and Electrical Characteristics of Sodium Phosphate Glasses and Glass-Ceramics.

Na-V-P-Nb-based materials have gained substantial recognition as cathode materials in high-rate sodium-ion batteries due to their unique properties and compositions, comprising both alkali and transition metal ions, which allow them to exhibit a mixed ionic-polaronic conduction mechanism. In this study, the impact of introducing two transition metal oxides, V2O5 and Nb2O5, on the thermal, (micro)structural, and electrical properties of the 35Na2O-25V2O5-(40 - x)P2O5 - xNb2O5 system is examined. The starting glass shows the highest values of DC conductivity, σDC, reaching 1.45 × 10-8 Ω-1 cm-1 at 303 K, along with a glass transition temperature, Tg, of 371 °C. The incorporation of Nb2O5 influences both σDC and Tg, resulting in non-linear trends, with the lowest values observed for the glass with x = 20 mol%. Electron paramagnetic resonance measurements and vibrational spectroscopy results suggest that the observed non-monotonic trend in σDC arises from a diminishing contribution of polaronic conductivity due to the decrease in the relative number of V4+ ions and the introduction of Nb2O5, which disrupts the predominantly mixed vanadate-phosphate network within the starting glasses, consequently impeding polaronic transport. The mechanism of electrical transport is investigated using the model-free Summerfield scaling procedure, revealing the presence of mixed ionic-polaronic conductivity in glasses where x < 10 mol%, whereas for x ≥ 10 mol%, the ionic conductivity mechanism becomes prominent. To assess the impact of the V2O5 content on the electrical transport mechanism, a comparative analysis of two analogue series with varying V2O5 content (10 and 25 mol%) is conducted to evaluate the extent of its polaronic contribution.

Conformational Heterogeneity of ß-Barrel Membrane Proteins Observed In Situ Using Orthogonal Spin Labels and Pulsed ESR Spectroscopy.

Outer membrane proteins (OMPs) of Gram-negative bacteria are involved in many essential functions of the cell. They are tightly packed in the outer membrane, which is an asymmetric lipid bilayer. Electron spin resonance (ESR) spectroscopic techniques combined with site-directed spin labeling (SDSL) enable observation of structure and conformational dynamics of these proteins directly in their native environments. Here we depict a protocol for site-directed spin labeling of ß-barrel membrane proteins in isolated outer membranes and intact E. coli using nitroxide, triarylmethyl (trityl), and Gd3+-based spin tags. Furthermore, subsequent continuous wave (CW) and orthogonal pulsed electron-electron double resonance (PELDOR) measurements are described along with experimental setup at Q-band (34 GHz), the data analysis, and interpretation.

Directional TV algorithm for fast EPR imaging.

Precise radiation guided by oxygen images has demonstrated superiority over the traditional radiation methods. Electron paramagnetic resonance (EPR) imaging has proven to be the most advanced oxygen imaging modality. However, the main drawback of EPR imaging is the long scan time. For each projection, we usually need to collect the projection many times and then average them to achieve high signal-to-noise ratio (SNR). One approach to fast scan is to reduce the repeating time for each projection. While the projections would be noisy and thus the traditional commonly-use filtered backprojection (FBP) algorithm would not be capable of accurately reconstructing images. Optimization-based iterative algorithms may accurately reconstruct images from noisy projections for they may incorporate prior information into optimization models. Based on the total variation (TV) algorithms for EPR imaging, in this work, we propose a directional TV (DTV) algorithm to further improve the reconstruction accuracy. We construct the DTV constrained, data divergence minimization (DTVcDM) model, derive its Chambolle-Pock (CP) solving algorithm, validate the correctness of the whole algorithm, and perform evaluations via simulated and real data. The experimental results show that the DTV algorithm outperforms the existing TV and FBP algorithms in fast EPR imaging. Compared to the standard FBP algorithm, the proposed algorithm may achieve 10 times of acceleration.

Estimating the initial absorbed dose of radiation in dried figs using EPR spectroscopy.

Dried figs were studied by Electron Paramagnetic Resonance (EPR) spectroscopy for identification of radiation treatment and dosage assessment. Gamma-irradiated samples show a multicomponent "sugar-like" EPR spectrum with line width of 6-8 mT, centered at g = 2.004. The investigation of the influence of the instrumental parameters microwave power and modulation amplitude on the EPR signal show saturation effect at microwave power above 2 mW and over modulation at modulation amplitude above 0.4 mT. Determination of the stability of radiation induced signals shows, that identification of previous radiation treatment is possible for a long time period after irradiation even more than one year. Dose-response curves of gamma-irradiated samples exhibits a linear response up to about 4 kGy and the saturation of the EPR signal at higher doses. A Single Aliquot Additive dosing method used to estimate the initial absorbed dose in irradiated dried fig flesh shows initial dose 0.25 kGy for the sample irradiated by 5 kGy and 3.7 kGy for those irradiated using 10 kGy. Taking into account the signal decay after 150 days of storage, the dose defined as initial should be 4.65 kGy for the 5 kGy irradiated sample and 8 kGy for that irradiated using 10 kGy.

[Mechanistic Analysis of Oxidative Stress-related Diseases Using Nitroxyl Radicals and Electron Spin Resonance].

Excessive production of reactive oxygen species (ROS) causes oxidative stress and is involved in the development and progression of a wide variety of diseases. Therefore, techniques for measuring oxidative stress are indispensable for analysis of the mechanisms of various diseases. The method involving ESR and the durable nitroxyl radical (ESR/spin probe method) is useful for this purpose, because the ESR signal intensity of the spin probe changes on reacting with ROS and other unstable radicals. In this review, the author's research applying the ESR/spin probe method to clarify disease mechanisms in vivo and in vitro is presented. The ESR signal of the probe injected into animals may decay through a few mechanisms besides reaction with ROS; thus, interpretation of the results is complicated. As the first approach to solving this problem, a probe resistant to enzymatic reduction by introducing a bulky group adjacent to the nitroxy group was created. The second approach was the use of a hydroxylamine probe which dominantly oxidized to nitroxyl radicals by reacting with superoxide anion radicals and oxidants. Using acyl-protected hydroxyl amine, it was demonstrated that sepsis model mice are under oxidative stress due to ROS production by activated phagocytes. On the other hand, it was shown in vitro that the UV-induced radical reaction of ketoprofen also occurs in lipid membranes, and that the reaction is related to ROS generation and membrane disruption. We believe that use of the ESR/spin probe method with ingenuity will clarify the mechanisms of various diseases.

Characterization of ferredoxins involved in electron transfer pathways for nitrogen fixation implicates differences in electronic structure in tuning 2[4Fe4S] Fd activity.

Ferredoxins (Fds) are small proteins which shuttle electrons to pathways like biological nitrogen fixation. Physical properties tune the reactivity of Fds with different pathways, but knowledge on how these properties can be manipulated to engineer new electron transfer pathways is lacking. Recently, we showed that an evolved strain of Rhodopseudomonas palustris uses a new electron transfer pathway for nitrogen fixation. This pathway involves a variant of the primary Fd of nitrogen fixation in R. palustris, Fer1, in which threonine at position 11 is substituted for isoleucine (Fer1T11I). To understand why this substitution in Fer1 enables more efficient electron transfer, we used in vivo and in vitro methods to characterize Fer1 and Fer1T11I. Electrochemical characterization revealed both Fer1 and Fer1T11I have similar redox transitions (-480 mV and - 550 mV), indicating the reduction potential was unaffected despite the proximity of T11 to an iron­sulfur (FeS) cluster of Fer1. Additionally, disruption of hydrogen bonding around an FeS cluster in Fer1 by substituting threonine with alanine (T11A) or valine (T11V) did not increase nitrogenase activity, indicating that disruption of hydrogen bonding does not explain the difference in activity observed for Fer1T11I. Electron paramagnetic resonance spectroscopy studies revealed key differences in the electronic structure of Fer1 and Fer1T11I, which indicate changes to the high spin states and/or spin-spin coupling between the FeS clusters of Fer1. Our data implicates these electronic structure differences in facilitating electron flow and sets a foundation for further investigations to understand the connection between these properties and intermolecular electron transfer.

Structural Flexibility of Tau in Its Interaction with Microtubules as Viewed by Site-Directed Spin Labeling EPR Spectroscopy.

Tau is a microtubule-associated protein that belongs to the Intrinsically Disordered Proteins (IDPs) family. IDPs or Intrinsically Disordered Regions (IDRs) play key roles in protein interaction networks and their dysfunctions are often related to severe diseases. Defined by their lack of stable secondary and tertiary structures in physiological conditions while being functional, these proteins use their inherent structural flexibility to adapt to and interact with various binding partners. Knowledges on the structural dynamics of IDPs and their different conformers are crucial to finely decipher fundamental biological processes controlled by mechanisms such as conformational adaptations or switches, induced fit, or conformational selection events. Different mechanisms of binding have been proposed: among them, the so-called folding-upon-binding in which the IDP adopts a certain conformation upon interacting with a partner protein, or the formation of a "fuzzy" complex in which the IDP partly keeps its dynamical character at the surface of its partner. The dynamical nature and physicochemical properties of unbound as well as bound IDPs make this class of proteins particularly difficult to characterize by classical bio-structural techniques and require specific approaches for the fine description of their inherent dynamics.Among other techniques, Site-Directed Spin Labeling combined with Electron Paramagnetic Resonance (SDSL-EPR) spectroscopy has gained much interest in this last decade for the study of IDPs. SDSL-EPR consists in grafting a paramagnetic label (mainly a nitroxide radical) at selected site(s) of the macromolecule under interest followed by its observation using and/or combining different EPR strategies. These nitroxide spin labels detected by continuous wave (cw) EPR spectroscopy are used as perfect reporters or "spy spins" of their local environment, being able to reveal structural transitions, folding/unfolding events, etc. Another approach is based on the measurement of inter-label distance distributions in the 1.5-8.0 nm range using pulsed dipolar EPR experiments, such as Double Electron-Electron Resonance (DEER) spectroscopy. The technique is then particularly well suited to study the behavior of Tau in its interaction with its physiological partner: microtubules (MTs). In this chapter we provide a detailed experimental protocol for the labeling of Tau protein and its EPR study while interacting with preformed (Paclitaxel-stabilized) MTs, or using Tau as MT inducer. We show how the choice of nitroxide label can be crucial to obtain functional information on Tau/tubulin complexes.

Electron Paramagnetic Resonance (EPR) Biodosimetry with Human Teeth: A Crucial Technique for Acute and Chronic Exposure Assessment.

ABSTRACT: Radiation exposure is a primary concern in emergency response scenarios and long-term health assessments. Accurate quantification of radiation doses is critical for informed decision-making and patient care. This paper reviews the dose reconstruction technique using both X- and Q-bands, with tooth enamel as a reliable dosimeter. Tooth enamel, due to its exceptional resistance to alteration over time, offers a unique opportunity for assessing both acute and chronic radiation exposures. This review delves into the principles underlying enamel dosimetry, the mechanism of radiation interactions, and dose retention in tooth enamel. We explore state-of-the-art analytical methods, such as electron paramagnetic resonance (EPR) spectroscopy, that accurately estimate low and high doses in acute and chronic exposure. Furthermore, we discuss the applicability of tooth enamel dosimetry in various scenarios, ranging from historical radiological incidents to recent nuclear events or radiological incidents. The ability to reconstruct radiation doses from dental enamel provides a valuable tool for epidemiological studies, validating the assessment of health risks associated with chronic exposures and aiding in the early detection and management of acute radiation incidents. This paper underscores the significance of tooth enamel as an essential medium for radiation dose reconstruction and its broader implications for enhancing radiation protection, emergency response, and public health preparedness. Incorporating enamel EPR dosimetry into standard protocols has the potential to transform the field of radiation assessment, ensuring more accurate and timely evaluations of radiation exposure and its associated risks.

Highly efficient visible-light driven dye degradation via 0D BiVO4 nanoparticles/2D BiOCl nanosheets p-n heterojunctions.

The construction of hybrid heterojunction photocatalysts is an effective strategy to improve the utilization of photogenerated carriers and photocatalytic activity. To enhance the separation distance of photogenerated carriers and accelerate the effective separation at the heterojunction of the interface, a unique 0D-2D hierarchical nanostructured p-n heterojunction was successfully fabricated in this work. BiOCl (BOC) nanosheets (p-type) were in situ grown on BiVO4 (BVO) nanoparticles (n-type) using the microemulsion-calcination method for highly efficient visible-light-driven organic dye degradation. Compared with pure BVO (the degradation rate of rhodamine B (RhB): about 32.0% in 55 min, the mineralization rate: 24.9% in 120 min), the RhB degradation rate can reach about 99.5% in 55 min and the mineralization rate of 62.1% in 120 min by utilizing BVO/25%BOC heterojunction photocatalyst under visible light irradiation. Various characterizations demonstrate that the formation of BVO/BOC p-n heterojunction greatly facilitates photogenerated carriers separation efficiency. Meanwhile, the results of the scavenging experiments and electron spin resonance tests indicate that ·O2- and h+ are the prominent active species for Rh B degradation. In addition, possible degradation pathways for Rh B were proposed using LC-MS tests. This work proves that building low dimensional p-n heterojunction photocatalysts is a promising strategy for developing photocatalysts with high efficiency.

Radical S-Adenosyl-l-Methionine Enzyme PylB: A C-Centered Radical to Convert l-Lysine into (3R)-3-Methyl-d-Ornithine.

PylB is a radical S-adenosyl-l-methionine (SAM) enzyme predicted to convert l-lysine into (3R)-3-methyl-d-ornithine, a precursor in the biosynthesis of the 22nd proteogenic amino acid pyrrolysine. This protein highly resembles that of the radical SAM tyrosine and tryptophan lyases, which activate their substrate by abstracting a H atom from the amino-nitrogen position. Here, combining in vitro assays, analytical methods, electron paramagnetic resonance spectroscopy, and theoretical methods, we demonstrated that instead, PylB activates its substrate by abstracting a H atom from the Cγ position of l-lysine to afford the radical-based ß-scission. Strikingly, we also showed that PylB catalyzes the reverse reaction, converting (3R)-3-methyl-d-ornithine into l-lysine and using catalytic amounts of the 5'-deoxyadenosyl radical. Finally, we identified significant in vitro production of 5'-thioadenosine, an unexpected shunt product that we propose to result from the quenching of the 5'-deoxyadenosyl radical species by the nearby [Fe4S4] cluster.

Application of electron paramagnetic resonance spectroscopy to examine free radicals formed in indapamide and torasemide storage under UV irradiation and at the higher temperatures which appear under light exposition.

Free radical formation in two diuretics: indapamide and torasemide was examined during UV irradiation and storage at higher temperatures using X-band (9.3 GHz) electron paramagnetic resonance spectroscopy (EPR). The aim of this study was to investigate the possibility of storing indapamide and torasemide under UV irradiation and at higher temperatures, which may occur during exposure to light. The diuretic samples were exposed to UVA irradiation for 15, 30 and 45 minutes, and stored at temperatures of 40 °C and 50 °C by 30 minutes. The EPR spectra were analyzed to determine the amplitudes (A), linewidths (ΔBpp), and integral intensities (I) and g factors. The concentrations of free radical (N) in the diuretic samples were also determined. The influence of microwave power on amplitudes, linewidths and the asymmetry parameter were evaluated. The result showed that the tested indapamide and torasemide samples exhibited high free radical concentrations in the range of 1018-1019 spin/g after UV irradiation and heat treatment. Therefore, due to the significant free radical formation indapamide and torasemide should not be stored under UV light and at temperatures of 40 °C and 50 °C. The complex character of free radical systems in the diuretic samples was proved as evidenced by the changes of the asymmetry parameters of the EPR lines with increasing microwave power. Fast spin-lattice relaxation processes were observed in all tested diuretic samples, regardless of the storage conditions. Electron paramagnetic resonance spectroscopy is proposed as a useful method in pharmacy to determine the appropriate storage conditions for diuretics.

Cleaving DNA with DNA: Cooperative Tuning of Structure and Reactivity Driven by Copper Ions.

A copper-dependent self-cleaving DNA (DNAzyme or deoyxyribozyme) previously isolated by in vitro selection has been analyzed by a combination of Molecular Dynamics (MD) simulations and advanced Electron Paramagnetic Resonance (Electron Spin Resonance) EPR/ESR spectroscopy, providing insights on the structural and mechanistic features of the cleavage reaction. The modeled 46-nucleotide deoxyribozyme in MD simulations forms duplex and triplex sub-structures that flank a highly conserved catalytic core. The DNA self-cleaving construct can also form a bimolecular complex that has a distinct substrate and enzyme domains. The highly dynamic structure combined with an oxidative site-specific cleavage of the substrate are two key-aspects to elucidate. By combining EPR/ESR spectroscopy with selectively isotopically labeled nucleotides it has been possible to overcome the major drawback related to the "metal-soup" scenario, also known as "super-stoichiometric" ratios of cofactors versus substrate, conventionally required for the DNA cleavage reaction within those nucleic acids-based enzymes. The focus on the endogenous paramagnetic center (Cu2+) here described paves the way for analysis on mixtures where several different cofactors are involved. Furthermore, the insertion of cleavage reaction within more complex architectures is now a realistic perspective towards the applicability of EPR/ESR spectroscopic studies.

Hydrophilic Reduction-Resistant Spin Labels of Pyrrolidine and Pyrroline Series from 3,4-Bis-hydroxymethyl-2,2,5,5-tetraethylpyrrolidine-1-oxyl.

Highly resistant to reduction nitroxides open new opportunities for structural studies of biological macromolecules in their native environment inside living cells and for functional imaging of pH and thiols, enzymatic activity and redox status in living animals. 3,4-Disubstituted nitroxides of 2,2,5,5-tetraethylpyrrolidine and pyrroline series with a functional group for binding to biomolecules and a polar moiety for higher solubility in water and for more rigid attachment via additional coordination to polar sites were designed and synthesized. The EPR spectra, lipophilicities, kinetics of the reduction in ascorbate-containing systems and the decay rates in liver homogenates were measured. The EPR spectra of all 3,4-disubstituted pyrrolidine nitroxides showed additional large splitting on methylene hydrogens of the ethyl groups, while the spectra of similar pyrroline nitroxides were represented with a simple triplet with narrow lines and hyperfine structure of the nitrogen manifolds resolved in oxygen-free conditions. Both pyrrolidine and pyrroline nitroxides demonstrated low rates of reduction with ascorbate, pyrrolidines being a bit more stable than similar pyrrolines. The decay of positively charged nitroxides in the rat liver homogenate was faster than that of neutral and negatively charged radicals, with lipophilicity, rate of reduction with ascorbate and the ring type playing minor role. The EPR spectra of N,N-dimethyl-3,4-bis-(aminomethyl)-2,2,5,5-tetraethylpyrrolidine-1-oxyl showed dependence on pH with pKa = 3, ΔaN = 0.055 mT and ΔaH = 0.075 mT.

ENDOR Spectroscopy Reveals the "Free" 5'-Deoxyadenosyl Radical in a Radical SAM Enzyme Active Site Actually is Chaperoned by Close Interaction with the Methionine-Bound [4Fe-4S]2+ Cluster.

1/2H and 13C hyperfine coupling constants to 5'-deoxyadenosyl (5'-dAdo•) radical trapped within the active site of the radical S-adenosyl-l-methionine (SAM) enzyme, pyruvate formate lyase-activating enzyme (PFL-AE), both in the absence of substrate and the presence of a reactive peptide-model of the PFL substrate, are completely characteristic of a classical organic free radical whose unpaired electron is localized in the 2pπ orbital of the sp2 C5'-carbon (J. Am. Chem. Soc. 2019, 141, 12139-12146). However, prior electron-nuclear double resonance (ENDOR) measurements had indicated that this 5'-dAdo• free radical is never truly "free": tight van der Waals contact with its target partners and active-site residues guide it in carrying out the exquisitely precise, regioselective reactions that are hallmarks of RS enzymes. Here, our understanding of how the active site chaperones 5'-dAdo• is extended through the finding that this apparently unexceptional organic free radical has an anomalous g-tensor and exhibits significant 57Fe, 13C, 15N, and 2H hyperfine couplings to the adjacent, isotopically labeled, methionine-bound [4Fe-4S]2+ cluster cogenerated with 5'-dAdo• during homolytic cleavage of cluster-bound SAM. The origin of the 57Fe couplings through nonbonded radical-cluster contact is illuminated by a formal exchange-coupling model and broken symmetry-density functional theory computations. Incorporation of ENDOR-derived distances from C5'(dAdo•) to labeled-methionine as structural constraints yields a model for active-site positioning of 5'-dAdo• with a short, nonbonded C5'-Fe distance (∼3 Å). This distance involves substantial motion of 5'-dAdo• toward the unique Fe of the [4Fe-4S]2+ cluster upon S-C(5') bond-cleavage, plausibly an initial step toward formation of the Fe-C5' bond of the organometallic complex, Ω, the central intermediate in catalysis by radical-SAM enzymes.

Extending the Range of Distances Accessible by 19F Electron-Nuclear Double Resonance in Proteins Using High-Spin Gd(III) Labels.

Fluorine electron-nuclear double resonance (19F ENDOR) has recently emerged as a valuable tool in structural biology for distance determination between F atoms and a paramagnetic center, either intrinsic or conjugated to a biomolecule via spin labeling. Such measurements allow access to distances too short to be measured by double electron-electron resonance (DEER). To further extend the accessible distance range, we exploit the high-spin properties of Gd(III) and focus on transitions other than the central transition (|-1/2⟩ ↔ |+1/2⟩), that become more populated at high magnetic fields and low temperatures. This increases the spectral resolution up to ca. 7 times, thus raising the long-distance limit of 19F ENDOR almost 2-fold. We first demonstrate this on a model fluorine-containing Gd(III) complex with a well-resolved 19F spectrum in conventional central transition measurements and show quantitative agreement between the experimental spectra and theoretical predictions. We then validate our approach on two proteins labeled with 19F and Gd(III), in which the Gd-F distance is too long to produce a well-resolved 19F ENDOR doublet when measured at the central transition. By focusing on the |-5/2⟩ ↔ |-3/2⟩ and |-7/2⟩ ↔ |-5/2⟩ EPR transitions, a resolution enhancement of 4.5- and 7-fold was obtained, respectively. We also present data analysis strategies to handle contributions of different electron spin manifolds to the ENDOR spectrum. Our new extended 19F ENDOR approach may be applicable to Gd-F distances as large as 20 Å, widening the current ENDOR distance window.