Combined Electron Paramagnetic Resonance Research Articles

Molecular insights into the role of heme in the transcriptional regulatory system AppA/PpsR

Heme has been shown to have a crucial role in the signal transduction mechanism of the facultative photoheterotrophic bacterium Rhodobacter sphaeroides. It interacts with the transcriptional regulatory complex AppA/PpsR, in which AppA and PpsR function as the antirepressor and repressor, respectively, of photosynthesis gene expression. The mechanism, however, of this interaction remains incompletely understood. In this study, we combined electron paramagnetic resonance (EPR) spectroscopy and Förster resonance energy transfer (FRET) to demonstrate the ligation of heme in PpsR with a proposed cysteine residue. We show that heme binding in AppA affects the fluorescent properties of the dark-adapted state of the protein, suggesting a less constrained flavin environment compared with the absence of heme and the light-adapted state. We performed ultrafast transient absorption measurements in order to reveal potential differences in the dynamic processes in the full-length AppA and its heme-binding domain alone. Comparison of the CO-binding dynamics demonstrates a more open heme pocket in the holo-protein, qualitatively similar to what has been observed in the CO sensor RcoM-2, and suggests a communication path between the blue-light-using flavin (BLUF) and sensing containing heme instead of cobalamin (SCHIC) domains of AppA. We have also examined quantitatively the affinity of PpsR to bind to individual DNA fragments of the puc promoter using fluorescence anisotropy assays. We conclude that oligomerization of PpsR is initially triggered by binding of one of the two DNA fragments and observe a ∼10-fold increase in the dissociation constant Kd for DNA binding upon heme binding to PpsR. Our study provides significant new insight at the molecular level on the regulatory role of heme that modulates the complex transcriptional regulation in R. sphaeroides and supports the two levels of heme signaling, via its binding to AppA and PpsR and via the sensing of gases like oxygen.

Maximizing the applicability of continuous wave (CW) Electron Paramagnetic Resonance (EPR): what more can we do after a century?

Electron Paramagnetic Resonance/Electron Spin Resonance (EPR/ESR) spectroscopy is a powerful experimental approach solving challenging problems that are inaccessible to other experimental techniques. While pulsed EPR techniques and their applications have been the central focus of the field nowadays, methodology development and application of continuous wave (CW) EPR have never been undervalued. Although the importance of EPR in research has been recognized in many areas as indicated by the increasing number of EPR research articles published every year, the growth in the number of EPR research teams seems to not match the former, likely caused by the relatively high cost of modern pulsed EPR spectrometers. In this perspective, we aim to discuss the new areas and new directions of the application of CW EPR in combination with spin labeling that are slightly different from the classic/traditional applications of the CW EPR technique. Based on our experience, we show example applications of CW EPR on the interfaces of protein-confinement materials, protein-inorganic crystal lattices, protein-nanoparticles, and protein-polymers. Various structure and dynamics information can be resolved from the data analysis of CW EPR, leading to enhanced understanding of protein biophysics upon interaction with the synthetic nanostructures as well as possible guidance of hybrid materials development based on the combination of proteins and nanostructures. We also discussed the possibilities of even broadening the application spectrum of EPR spectroscopy. The relatively high affordability of CW EPR spectrometers (as compared to the pulsed ones) also facilitates the growth in EPR research groups worldwide.

Intermolecular interactions of tetracyanoethylene (TCNE) and fumaronitrile (FN) with minor amines: A combined UV-Vis and EPR study.

In this paper, the nature of interactions between two cyanocarbons-tetracyanoethylene (TCNE) and fumaronitrile (FN)-and a series of four secondary amines possessing a general formula C4HxN (x = 5-11) is thoroughly scrutinized. For all of the TCNE-amine pairs, tricyanovinylation (TCV) reaction is observed; however, only for pyrrole, it is accompanied with a visible charge-transfer (CT) complex formation-no such chemical individuals, characteristic for TCNE, have been noticed for aliphatic and alicyclic amines. On the contrary, FN forms such complexes with all the amines studied. Interestingly, a rather unexpected reaction of FN with alicyclic amines has been observed. The recorded electron paramagnetic resonance (EPR) spectra indicate the presence of both TCNE●- and FN●- radicals in the analyzed samples, assigned to a complete charge (electron) transfer process within the CT complexes, whose efficiency can be additionally enhanced by photoirradiation. The origination of the former radical, whose presence is observed also in the TCNE-diethylamine mixture, is as well proposed to result indirectly from the TCV reaction, occurring for this system. Finally, the superhyperfine structure of EPR spectra, indicating the existence of some secondary interactions of the radicals with surrounding compounds, is discussed. Formation of CT complexes and tricyanovinylates has been investigated and characterized with UV-Vis spectroscopy, while the presence of (cyano)radicals in the analyzed mixtures has been evidenced by (photoinduced) EPR measurements. Interpretation of the experimental results is also supplemented with computer simulations including density functional theory calculations.

Crystallographic snapshots of a B12-dependent radical SAM methyltransferase

By catalysing the microbial formation of methane, methyl-coenzyme M reductase has a central role in the global levels of this greenhouse gas1,2. The activity of methyl-coenzyme M reductase is profoundly affected by several unique post-translational modifications3–6, such as a unique C-methylation reaction catalysed by methanogenesis marker protein 10 (Mmp10), a radical S-adenosyl-l-methionine (SAM) enzyme7,8. Here we report the spectroscopic investigation and atomic resolution structure of Mmp10 from Methanosarcina acetivorans, a unique B12 (cobalamin)-dependent radical SAM enzyme9. The structure of Mmp10 reveals a unique enzyme architecture with four metallic centres and critical structural features involved in the control of catalysis. In addition, the structure of the enzyme–substrate complex offers a glimpse into a B12-dependent radical SAM enzyme in a precatalytic state. By combining electron paramagnetic resonance spectroscopy, structural biology and biochemistry, our study illuminates the mechanism by which the emerging superfamily of B12-dependent radical SAM enzymes catalyse chemically challenging alkylation reactions and identifies distinctive active site rearrangements to provide a structural rationale for the dual use of the SAM cofactor for radical and nucleophilic chemistry.

Electrocatalytic Activity of Polyaniline in Magnesium-Sulfur Batteries.

Rechargeable magnesium-sulfur (Mg-S) batteries offer the potential for inexpensive energy storage alternatives to other metal-ion batteries for the grid scale and household applications. Despite all economic and resource advantages, Mg-S battery chemistry suffers from a complicated reaction mechanism and extremely slow reaction kinetics. To improve the kinetics, we improvise a new electrode architecture where a conductive polymer is used along with a carbon network. This report will bring an important insight of electrocatalytic activity of polyaniline, on the basis of free-radical coupling and is a completely new concept in Mg-S battery chemistry. By the combined electron spin resonance spectroscopy, X-ray photoelectron spectroscopy, and fluorescence lifetime measurements, we perceived that the polyaniline anchors the S3•- species from the electrolyte/catholyte through a free-radical-coupling process and thus promotes the reduction to end-discharged products, via a chemical adduct. The concept of free-radical catalysis in Mg/S batteries will open a new knowledge to enhance the active material utilization in the Mg-S batteries.

Electron paramagnetic resonance and spin trapping to detect free radicals from allergenic hydroperoxides in contact with the skin: From the molecule to the tissue.

A major research topic consists of revealing the contribution of radical-mediated reactions in dermatological diseases related to xenobiotic-induced stress to succeed risk-assessment procedures protecting producers and consumers. Allergic contact dermatitis is the clinically relevant consequence of skin sensitization, one of the most critical occupational and environmental health issues related to xenobiotics exposure. The first key event identified for the skin sensitization process to a chemical is its aptitude to react with epidermal proteins and form antigenic structures that will further trigger the immune response. Many chemical sensitizers are suspected to react through mechanisms involving radical intermediates. This review focuses on the recent progress we have accomplished over the last few years studying radical intermediates derived from skin-sensitizing chemicals by electron paramagnetic resonance in combination with the spin-trapping technique. Our work is carried out "from the molecule", performing studies in solution, "to the tissue", by the development of a methodology on a reconstructed human epidermis model, very close in terms of histology and metabolic/enzymatic activity to real human epidermis, that can be used as suitable biological tissue model. The benefits are to test chemicals under conditions close to human use and real-life sensitization exposures and benefit from the three-dimensional (3D) microenvironment.

Singlet oxygen-dominated electrocatalytic oxidation treatment for the high-salinity quaternary ammonium compound wastewater with Ti/(RuxIry)O2 anode

The widespread application of quaternary ammonium compounds (QAC) has posed a serious hazard to the environment and human being, and high concentration of Cl− in QAC wastewater may further increase the difficulty of pollutants elimination. In this study, such a QAC wastewater under high salinity conditions was chosen as the target, the prepared Ti/(RuxIry)O2 anode exhibited favorable catalytic performance for the oxidation and mineralization of QAC under high salinity conditions. Increasing the Ru/Ir ratio of Ti-based electrode coating also slightly promoted the inner catalytic capacity. The combination of electron paramagnetic resonance (EPR) and quenching experiments indicates that 1O2 served as a main reactive specie in the Ti/(RuxIry)O2 electrooxidation system. The increase of pH could decrease the removal efficiency of QAC for the reduced 1O2 yield, and the rise of Cl− concentration could favor the QAC oxidation, and Cl− was a better electrolyte to promote the oxidation of organic contaminants when compared to Na2SO4 or Na2CO3. Additionally, the conversion pathway of the model pollutant was tentatively investigated, the results demonstrated that there were almost no halogenated final products residual by electrocatalytic oxidation with Ti/(RuxIry)O2 anode. This study not only elucidate the reaction mechanism of Ti/(RuxIry)O2 anode electrocatalytic oxidation of high salinity QAC wastewater, but also may provide an efficacious and eco-friendly method for the treatment of high salinity QAC wastewater.

Emergent perpendicular magnetic anisotropy at the interface of an oxide heterostructure

Controlling the magnetic anisotropy in magnetic oxides is critical for the development of oxide spintronics. Here we report on the finding of an emergent interfacial magnetism, with unique perpendicular magnetic anisotropy (PMA), at the interface of a half-doped manganite ${\mathrm{La}}_{1/2}{\mathrm{Sr}}_{1/2}\mathrm{Mn}{\mathrm{O}}_{3}$ film on $\mathrm{LaAl}{\mathrm{O}}_{3}$ substrate, through combined electron spin resonance measurements and density functional theory calculations. The interfacial magnetism can be explained by the $3{d}_{3{z}^{2}\ensuremath{-}{r}^{2}}$ orbital-mediated ferromagnetic exchange interaction between Mn ions near the interface, which is enhanced by the compressive strain from the substrate. Our results provide a useful PMA system for possible spintronic applications, as well as atomistic-level insight to the magnetic anisotropy in strained manganite oxide interfaces.

Uptake of Cell-Penetrating Peptide RL2 by Human Lung Cancer Cells: Monitoring by Electron Paramagnetic Resonance and Confocal Laser Scanning Microscopy.

RL2 is a recombinant analogue of a human κ-casein fragment, capable of penetrating cells and inducing apoptosis of cancer cells with no toxicity to normal cells. The exact mechanism of RL2 penetration into cells remains unknown. In this study, we investigated the mechanism of RL2 penetration into human lung cancer A549 cells by a combination of electron paramagnetic resonance (EPR) spectroscopy and confocal laser scanning microscopy. EPR spectra of A549 cells incubated with RL2 (sRL2) spin-labeled by a highly stable 3-carboxy-2,2,5,5-tetraethylpyrrolidine-1-oxyl radical were found to contain three components, with their contributions changing with time. The combined EPR and confocal-microscopy data allowed us to assign these three forms of sRL2 to the spin-labeled protein sticking to the membrane of the cell and endosomes, to the spin-labeled protein in the cell interior, and to spin labeled short peptides formed in the cell because of protein digestion. EPR spectroscopy enabled us to follow the kinetics of transformations between different forms of the spin-labeled protein at a minimal spin concentration (3–16 μM) in the cell. The prospects of applications of spin-labeled cell-penetrating peptides to EPR imaging, DNP, and magnetic resonance imaging are discussed, as is possible research on an intrinsically disordered protein in the cell by pulsed dipolar EPR spectroscopy.

First Chronological Constraints for the High Terraces of the Upper Ebro Catchment

The Cenozoic sedimentary basins in the Iberian Peninsula show a change from long-term basin infill to incision, a transition that indicates a period of major drainage reorganization that culminated in the throughflow of the networks to the Atlantic and Mediterranean oceans. Both the cause of the transition from aggradation to degradation and the linkages to tectonic, climatic, and geomorphic events hinge on the chronology of the fluvial network incision and excavation of the basin’s sedimentary fills. In this paper, we describe the first chronologic data on the highest fluvial terraces of the upper area of the Ebro River, one of the largest fluvial systems in the Iberian Peninsula, to determine the onset of incision and excavation in the basin. For this purpose, we combine electron spin resonance (ESR) and paleomagnetism methods to date strath terraces found at 140, 90, and 85 m above the current river level. Our results show ages of ca. 1.2 and 1.5 Ma for the uppermost river terraces in the upper Ebro catchment, constraining the minimum age of the entrenchment of the upper Ebro River.

Investigation onto the performance and mechanism of visible light photodegradation of methyl orange catalyzed by M/CeO2 (M=Pt, Ag, Au)

In this paper, M/CeO2 (M=Pt, Ag, Au) photocatalysts were prepared and used for visible light degradation of methyl orange (MO) with H2O2 as a sacrificial reagent. Utilizing X-ray diffraction (XRD), UV-vis diffuse reflectance spectroscopy (DRS), transmission electron microscopy (TEM), N2 physical adsorption (BET), X-ray photoelectron spectroscopy (XPS), photocurrent characterization and electrochemical impedance spectroscopy, etc., the structure, morphology, chemical state, and optical properties of the catalyst were investigated. The effects of the amount of catalyst, the initial concentration of methyl orange, the pH value of the solution and the number of reuses on the photocatalytic degradation efficiency of MO were studied. The results showed that under optimized reaction condition of 15 mg 2Pt/CeO2 catalyst, the initial concentration of MO was 20 mg/L, pH=5, the reaction time was 195 min and the amount of H2O2 was 0.2 mL, the degradation percentage of MO reached 75.8%. The main active species of photodegradation are hydroxyl radicals and superoxide radicals, which was determined by free radical capture experiments and combined electron spin resonance (ESR). The photocatalytic mechanism based on work function difference and electron transfer was also discussed in detail.

Reaction, structure and spectroscopic properties of bis(cyano) cobalt(III) porphyrin complexes

Cyanocobalamin and analogues have drawn much attention for the promising applications in photovoltaic and photocatalytic systems. In this study, two low spin bis(cyano) cobalt(III) porphyrin complexes [K(222)][Co[Formula: see text](TPP)(CN)2] and [K(222)][Co[Formula: see text](TMP)(CN)2] (222 = 4,7,13,16,21,24-hexaoxo-1,10-diazabicyclo[8.8.8]hexacosane, TPP = meso-tetraphenylporphyrin dianion, TMP = meso-tetramesitylporphyrin dianion), which were isolated from the reactions between [Co[Formula: see text](Porph)] (Porph = Porphyrin) and [K(222)(CN)], are characterized by a single crystal X-ray diffraction, FT-IR and UV-vis spectroscopies. Combined UV-vis and electron paramagnetic resonance (EPR) investigations have been conducted to understand the reaction mechanisms. The work gives new insights into the reactivities and spectroscopic properties of cyano cobalt macrocyclic complexes.

Fast spin flips for entangled atoms

Spin Physics The integration of pump-probe spectroscopy with scanning tunneling microscopy (STM) tools has allowed studies of spin relaxation in atoms on the nanosecond time scale. However, following the free evolution of a pair of entangled spins requires faster initial excitation than can be delivered with microwave pulses. Veldman et al. sequentially combined electron spin resonance–STM and direct-current pump-probe techniques to instantaneously flip spins while preserving spin coherence. They used this method to follow free, coherent flip-flop evolution of two coupled spin-1/2 atoms (hydrogenated titanium atom dimers) on a magnesium oxide surface. Science , abg8223, this issue p. [964][1] [1]: /lookup/doi/10.1126/science.abg8223

Terahertz Magneto-Optical Excitations of the sd-Hybrid States of Lithium Nitridocobaltate Li2(Li1–xCox)N

We report the results of the experimental and theoretical study of the magnetic anisotropy of single crystals of the Co-doped lithium nitride Li2(Li1-xCox)N with x = 0.005, 0.01, and 0.02. It was shown recently that doping of the Li3N crystalline matrix with 3d transition metal (TM) ions yields superior magnetic properties comparable with the strongly anisotropic single-molecule magnetism of rare-earth complexes. Our combined electron spin resonance (ESR) and THz spectroscopic investigations of Li2(Li1-xCox)N in a very broad frequency range up to 1.7 THz and in magnetic fields up to 16 T enable an accurate determination of the energies of the spin levels of the ground state multiplet Ŝ = 1 of the paramagnetic Co(I) ion. In particular, we find a very large zero field splitting (ZFS) of almost 1 THz (∼4 meV or 33 cm-1) between the ground-state singlet and the first excited doublet state. On the computational side, ab initio many-body quantum chemistry calculations reveal a ZFS gap consistent with the experimental value. Such a large ZFS energy yields a very strong single-ion magnetic anisotropy of easy-plane type resembling that of rare-earth ions. Its microscopic origin is the unusual linear coordination of the Co(I) ions in Li2(Li1-xCox)N with two nitrogen ligands. Our calculations also evidence a strong 3d-4s hybridization of the electronic shells resulting in significant electron spin density at the 59Co nuclei, which may be responsible for the experimentally observed extraordinary large hyperfine structure of the ESR signals. Altogether, our experimental spectroscopic and computational results enable comprehensive insights into the remarkable properties of the Li2[Li1-x(TM)x]N magnets on the microscopic level.

Fluorescent and Water Dispersible Single‐Chain Nanoparticles: Core–Shell Structured Compartmentation

AbstractSingle‐chain nanoparticles (SCNPs) are highly versatile structures resembling proteins, able to function as catalysts or biomedical delivery systems. Based on their synthesis by single‐chain collapse into nanoparticular systems, their internal structure is complex, resulting in nanosized domains preformed during the crosslinking process. In this study we present proof of such nanocompartments within SCNPs via a combination of electron paramagnetic resonance (EPR) and fluorescence spectroscopy. A novel strategy to encapsulate labels within these water dispersible SCNPs with hydrodynamic radii of ≈5 nm is presented, based on amphiphilic polymers with additional covalently bound labels, attached via the copper catalyzed azide/alkyne “click” reaction (CuAAC). A detailed profile of the interior of the SCNPs and the labels’ microenvironment was obtained via electron paramagnetic resonance (EPR) experiments, followed by an assessment of their photophysical properties.

Fluorescent and Water Dispersible Single-Chain Nanoparticles: Core-Shell Structured Compartmentation.

Single‐chain nanoparticles (SCNPs) are highly versatile structures resembling proteins, able to function as catalysts or biomedical delivery systems. Based on their synthesis by single‐chain collapse into nanoparticular systems, their internal structure is complex, resulting in nanosized domains preformed during the crosslinking process. In this study we present proof of such nanocompartments within SCNPs via a combination of electron paramagnetic resonance (EPR) and fluorescence spectroscopy. A novel strategy to encapsulate labels within these water dispersible SCNPs with hydrodynamic radii of ≈5 nm is presented, based on amphiphilic polymers with additional covalently bound labels, attached via the copper catalyzed azide/alkyne “click” reaction (CuAAC). A detailed profile of the interior of the SCNPs and the labels’ microenvironment was obtained via electron paramagnetic resonance (EPR) experiments, followed by an assessment of their photophysical properties.

Preparation and spectroscopic characterization of lyophilized Mo nitrogenase

Mo nitrogenase is the primary source of biologically fixed nitrogen, making this system highly interesting for developing new, energy efficient ways of ammonia production. Although heavily investigated, studies of the active site of this enzyme have generally been limited to spectroscopic methods that are compatible with the presence of water and relatively low protein concentrations. One method of overcoming this limitation is through lyophilization, which allows for measurements to be performed on solvent free, high concentration samples. This method also has the potential for allowing efficient protein storage and solvent exchange. To investigate the viability of this preparatory method with Mo nitrogenase, we employ a combination of electron paramagnetic resonance, Mo and Fe K-edge X-ray absorption spectroscopy, and acetylene reduction assays. Our results show that while some small distortions in the metallocofactors occur, oxidation and spin states are maintained through the lyophilization process and that reconstitution of either lyophilized protein component into buffer restores acetylene reducing activity.Graphic abstract

Direct oxidation of methane to oxygenates on supported single Cu atom catalyst

Catalytically converting CH4 to chemicals and fuels is of paramount importance but remains a major challenge to simultaneously obtain high activity and selectivity. Here, we report Cu1/ZSM-5 single atom catalyst is highly active (C1 oxygenates productivity of 4800 μmol⋅gcat−1 at 50 °C and 12,000 μmol⋅gcat-1 at 70 °C within 30 min) and selective (C1 oxygenates selectivity of 99 % at 50 °C) for direct CH4 oxidation, comparable to most of those state-of-the-art noble metal catalysts. The combined DFT calculation, electronic microscope, X-ray absorption and electron paramagnetic resonance results confirm that each isolated Cu atom stabilized by four O moieties on the ZSM-5 support possesses uniform Cu1-O4 entity as active site and preferentially activates CH4 instead of CH3OH that is advantageous for highly selective C1 oxygenates production, especially for methanol. Our molecular-level finding on the atomic structure of Cu active site paves the way to design better catalysts for methane conversion.

Reactive oxygen species formation at Pt nanoparticles revisited by electron paramagnetic resonance and electrochemical analysis

A combined electrochemical and electron paramagnetic resonance (EPR) procedure for the study of oxygen reduction reaction (ORR) intermediates generated at Pt nanoparticles (NPs) is validated in this work. Using spin-trap EPR complemented by electrochemical analysis, we show that we can detect and identify the free radicals that are produced during the ORR through trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) which are otherwise difficult to detect. Experiments with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as spin trap show no evidence of DEPMPO-OOH and indicate that only OH radicals are trapped during the ORR. The results of this study serve as a functional proof-of-concept for further research on the identification of radical ORR intermediates in solution. We propose that our procedure can be used for a more rigorous quantification of free radicals involved in other electrochemical reactions.

Development of Cu2+-Based Distance Methods and Force Field Parameters for the Determination of PNA Conformations and Dynamics by EPR and MD Simulations.

Peptide nucleic acids (PNAs) are a promising group of synthetic analogues of DNA and RNA that offer several distinct advantages over the naturally occurring nucleic acids for applications in biosensing, drug delivery, and nanoelectronics. Because of its structural differences from DNA/RNA, methods to analyze and assess the structure, conformations, and dynamics are needed. In this work, we develop synergistic techniques for the study of the PNA conformation. We use CuQ2, a Cu2+ complex with 8-hydroxyquinoline (HQ), as an alternative base pair and as a spin label in electron paramagnetic resonance (EPR) distance methods. We use molecular dynamics (MD) simulations with newly developed force field parameters for the spin labels to interpret the distance constraints determined by EPR. We complement these methods by UV-vis and circular dichroism measurements and assess the efficacy of the Cu2+ label on a PNA duplex whose backbone is based on aminoethylglycine and a duplex with a hydroxymethyl backbone modification. We show that the Cu2+ label functions efficiently within the standard PNA and the hydroxymethyl-modified PNA and that the MD parameters may be used to accurately reproduce our EPR findings. Through the combination of EPR and MD, we gain new insights into the PNA structure and conformations as well as into the mechanism of orientational selectivity in Cu2+ EPR at X-band. These results present for the first time a rigid Cu2+ spin label used for EPR distance measurements in PNA and the accompanying MD force fields for the spin label. Our studies also reveal that the spin labels have a low impact on the structure of the PNA duplexes. The combined MD and EPR approach represents an important new tool for the characterization of the PNA duplex structure and provides valuable information to aid in the rational application of PNA at large.