Curvature-Induced Electron Spin Catalysis with Carbon Spheres.

Here, we report curvature-induced electron spin catalysis by using solid carbon spheres as catalysts, which were synthesized using positive curvature molecular hexabromocyclopentadiene as a precursor molecule, following a radical coupling mechanism. The curvature spin of carbon is regarded as an overlapping state of σ- and π-radical, which is identified by the inverse Laplace transform of pulse-electron paramagnetic resonance. The growth mechanism of carbon spheres abiding by Kroto's model, is supported by the density functional theory study of thermodynamics and kinetics calculations. The solid carbon spheres present excellent catalytic behaviour of oxidation coupling of amines to form corresponding imines with the conversion of >99%, selectivity of 98.7%, and yield of 97.7%, which is attributed to the predominantly curvature-induced electron spin catalysis of carbon, supported by the calculation of oxygen adsorption energy. This work proposes a view of curvature-induced spin catalysis of carbon, which opens up a research direction for curvature-induced electron spin catalysis.

Approaches to determination of the mechanism of the Rieske monooxygenase salicylate 5-hydroxylase.

Rieske oxygenases catalyze an exceptionally broad range of discrete types of reactions despite the utilization of a highly conserved quaternary structure and metal cofactor complement. Oxygen activation within this family occurs at a mononuclear FeII site, which is located approximately 12 Å from a one-electron reduced Rieske-type iron-sulfur cluster. Electron transfer from the Rieske cluster to the mononuclear iron site occurs during O2 activation and product formation. A key question is whether all Rieske oxygenase reactions involve the same type of activated oxygen species. This question has been explored using the Rieske oxygenase salicylate 5-hydroxylase, which catalyzes both aromatic hydroxylation of salicylate and aromatic methyl hydroxylation when a methyl substituent is placed in the normal position of aromatic ring hydroxylation. We show here that the combined application of kinetic, biophysical, computational, and isotope effect methods reveals a uniform mechanism for initial O2 activation and substrate attack for both types of reactivity. However, the mechanism diverges during the later phases of the reactions in response to the electronic nature and geometry of the substrates as well as the lifetime of intermediates. Similar factors may be encountered broadly in the Rieske oxygenase family.

Unveiling the mechanism of cysteamine dioxygenase: A combined HPLC-MS assay and metal-substitution approach.

Mammalian cysteamine dioxygenase (ADO), a mononuclear non-heme Fe(II) enzyme with three histidine ligands, plays a key role in cysteamine catabolism and regulation of the N-degron signaling pathway. Despite its importance, the catalytic mechanism of ADO remains elusive. Here, we describe an HPLC-MS assay for characterizing thiol dioxygenase catalytic activities and a metal-substitution approach for mechanistic investigation using human ADO as a model. Two proposed mechanisms for ADO differ in oxygen activation: one involving a high-valent ferryl-oxo intermediate. We hypothesized that substituting iron with a metal that has a disfavored tendency to form high-valent states would discriminate between mechanisms. This chapter details the expression, purification, preparation, and characterization of cobalt-substituted ADO. The new HPLC-MS assay precisely measures enzymatic activity, revealing retained reactivity in the cobalt-substituted enzyme. The results obtained favor the concurrent dioxygen transfer mechanism in ADO. This combined approach provides a powerful tool for studying other non-heme iron thiol oxidizing enzymes.

Insights into electron transfer and bifurcation of the Synechocystis sp. PCC6803 hydrogenase reductase module.

The NAD+-reducing soluble [NiFe] hydrogenase (SH) is the key enzyme for production and consumption of molecular hydrogen (H2) in Synechocystis sp. PCC6803. In this study, we focused on the reductase module of the SynSH and investigated the structural and functional aspects of its subunits, particularly the so far elusive role of HoxE. We demonstrated the importance of HoxE for enzyme functionality, suggesting a regulatory role in maintaining enzyme activity and electron supply. Spectroscopic analysis confirmed that HoxE and HoxF each contain one [2Fe2S] cluster with an almost identical electronic structure. Structure predictions, alongside experimental evidence for ferredoxin interactions, revealed a remarkable similarity between SynSH and bifurcating hydrogenases, suggesting a related functional mechanism. Our study unveiled the subunit arrangement and cofactor composition essential for biological electron transfer. These findings enhance our understanding of NAD+-reducing [NiFe] hydrogenases in terms of their physiological function and structural requirements for biotechnologically relevant modifications.

Oxidative Stability of Fish Oil-Loaded Nanocapsules Produced by Electrospraying Using Kafirin or Zein Proteins as Wall Materials.

The encapsulation of fish oil by monoaxial electrospraying using kafirin or zein proteins as hydrophobic wall materials was investigated. Kafirin resulted in spherical fish oil-loaded nanocapsules (>50% of capsules below 1 µm), whereas zein led to fish oil-loaded nanocapsules with non-spherical morphology (>80% of capsules below 1 µm). Both hydrophobic encapsulating materials interacted with fish oil, successfully entrapping the oil within the protein matrix as indicated by Fourier-transform infrared spectroscopy (FTIR) and Raman spectroscopy results. FTIR also suggested hydrogen bonding between fish oil and the proteins. Trapped radicals in the encapsulation matrix that were detected by electron paramagnetic resonance (EPR), indicated oxidation during electrospraying and storage. Results from isothermal (140 °C) differential scanning calorimetry (DSC) denoted that the encapsulation of fish oil by electrospraying using both kafirin or zein as wall materials protected fish oil from oxidation. In particular, the zein-based nanocapsules were 3.3 times more oxidatively stable than the kafirin-based nanocapsules, which correlates with the higher oil encapsulation efficiency found for zein-based capsules. Thus, this study shows that kafirin might be considered a hydrophobic wall material for the encapsulation of fish oil by electrospraying, although it prevented lipid oxidation to a lower extent when compared to zein.

An Ultrasensitive Molecular Detector for Direct Sensing of Spin Currents at Room Temperature.

The experimental analysis of pure spin currents at interfaces is one major goal in the field of magnonics and spintronics. Complementary to the established Spin-Hall effect using the spin-to-charge conversion in heavy metals for information processing, we present a novel approach based on spin pumping detection by an interfacial, resonantly excited molecular paramagnet adsorbed to the surface of the spin current generating magnet. Here, we show that the sensitivity of this electron paramagnetic resonance (EPR) detector can be enhanced by orders of magnitude through intramolecular transfer of spin polarization at room temperature. Our proof-of-principle sample consists of octahedral-shaped ferrimagnetic Fe3O4 nanoparticles covered by oleic acid (OA) which has two paramagnetic centers, S1 and S2. S1 arises from the chemisorption of OA and is located directly at the interface to Fe3O4. S2 originates from radical formation at the center of the molecule close to the double bond of oleic acid and is not influenced by chemisorption. Using ferromagnetic resonance (FMR) excitation of the Fe3O4 nanoparticles to pump spins into S1, a population inversion of the spin-split levels of S2 is formed, vastly enhancing the detection sensitivity on the atomic scale.

Analysis of the Intensity of Nitric Oxide Production in Different Parts of the Spinal Cord after Modeling Combined Cerebral and Spinal Injury.

Using the method of electron paramagnetic resonance spectroscopy, we showed that NO production decreases by 60% (p<0.05) in the region located rostral to the spinal cord injury 7 days after combined injury to the brain and spinal cord. At the same time, NO production did not change in the site of spinal cord injury and caudal to the injury. The intensity of NO production in similar parts of the spinal cord in intact animals remained unchanged.

Probing skin photoallergens in reconstructed human epidermis: An EPR spin trapping investigation.

Photoallergic contact dermatitis is a skin disease caused by combined exposure to photoreactive chemicals and sunlight. Exposure to allergens and the risk of skin sensitization is an essential regulatory issue within the industry. Yet, only few non-validated assays for photoallergy assessment exist as the pathogenesis is not fully deciphered. Improving such assays and/or developing new ones require an understanding of the chemical mechanisms involved. The first key event in the photosensitization process, namely chemical binding of the photoallergen to endogenous proteins, is thought to proceed via photo-mediated radicals arising from the photoallergen. Moreover, the mechanism of action of these radicals if formed in the epidermis is not known and far from being unraveled. We present here an original proof-of-concept methodology to probe radical generation from allergens in contact with photoexposed skin, using electron paramagnetic resonance and spin trapping in a reconstructed human epidermis model mimicking real-life exposure scenarios.

Evaluation of Nitric Oxide-Donating Properties of 11H-indeno[1,2-b]quinoxalin-11-one Oxime (IQ-1) by Electron Paramagnetic Resonance Spectroscopy.

IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) is a specific c-Jun N-terminal kinase (JNK) inhibitor with anticancer and neuro- and cardioprotective properties. Because aryloxime derivatives undergo cytochrome P450-catalyzed oxidation to nitric oxide (NO) and ketones in liver microsomes, NO formation may be an additional mechanism of IQ-1 pharmacological action. In the present study, electron paramagnetic resonance (EPR) of the Fe2+ complex with diethyldithiocarbamate (DETC) as a spin trap and hemoglobin (Hb) was used to detect NO formation from IQ-1 in the liver and blood of rats, respectively, after IQ-1 intraperitoneal administration (50 mg/kg). Introducing the spin trap and IQ-1 led to signal characteristics of the complex (DETC)2-Fe2+-NO in rat liver. Similarly, the introduction of the spin trap components and IQ-1 resulted in an increase in the Hb-NO signal for both the R- and the T-conformers in blood samples. The density functional theory (DFT) calculations were in accordance with the experimental data and indicated that the NO formation of IQ-1 through the action of superoxide anion radical is thermodynamically favorable. We conclude that the administration of IQ-1 releases NO during its oxidoreductive bioconversion in vivo.

Hexagonal Boron Nitride Based Photonic Quantum Technologies.

Hexagonal boron nitride is rapidly gaining interest as a platform for photonic quantum technologies, due to its two-dimensional nature and its ability to host defects deep within its large band gap that may act as room-temperature single-photon emitters. In this review paper we provide an overview of (1) the structure, properties, growth and transfer of hexagonal boron nitride; (2) the creationof colour centres in hexagonal boron nitride and assignment of defects by comparison with ab initio calculations for applications in photonic quantum technologies; and (3) heterostructure devices for the electrical tuning and charge control of colour centres that form the basis for photonic quantum technology devices. The aim of this review is to provide readers a summary of progress in both defect engineering and device fabrication in hexagonal boron nitride based photonic quantum technologies.

An electron paramagnetic resonance time-course study of oxidative stress in the plasma of electronic cigarette exposed rats.

The long-term consequences of electronic cigarette (Ecig) use in humans are not yet known, but it is known that Ecig aerosols contain many toxic compounds of concern. We have recently shown that Ecig exposure impairs middle cerebral artery (MCA) endothelial function and that it takes 3 days for MCA reactivity to return to normal. However, the sources contributing to impairment of the endothelium were not investigated. We hypothesized that the increased levels of oxidative stress markers in the blood are correlated with impaired MCA reactivity. We used electron paramagnetic resonance (EPR) spectroscopy to examine plasma from 4-month-old male Sprague-Dawley rats that were exposed to either air (n = 5) or 1 h Ecig exposure, after which blood samples were collected at varying times after exposure (i.e., 1-4, 24, 48 and 72 h postexposure, n = 4 or 5 in each time group). The EPR analyses were performed using the redox-sensitive hydroxylamine spin probe 1-hydroxy-3-carboxymethyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) to measure the level of reactive oxidant species in the plasma samples. We found that EPR signal intensity from the CM• radical was significantly increased in plasma at 1-4, 24 and 48 h (P < 0.05, respectively) and returned to control (air) levels by 72 h. When evaluating the EPR results with MCA reactivity, we found a significant negative correlation (Pearson's P = 0.0027). These data indicate that impaired cerebrovascular reactivity resulting from vaping is associated with the oxidative stress level (measured by EPR from plasma) and indicate that a single 1 h vaping session can negatively influence vascular health for up to 3 days after vaping. HIGHLIGHTS: What is the central question of this study? Does the time course of oxidative stress triggered by electronic cigarette exposure follow the cerebral vascular dysfunction? What is the main finding and its importance? Electron paramagnetic resonance analysis shows that the oxidative stress induced after a single 1 h exposure to electronic cigarette aerosol takes ≤72 h to return to normal, which mirrors the time course for vascular dysfunction in the middle cerebral artery that we have reported previously.

Light-Induced Charge Separation in Photosystem I from Different Biological Species Characterized by Multifrequency Electron Paramagnetic Resonance Spectroscopy.

Photosystem I (PSI) serves as a model system for studying fundamental processes such as electron transfer (ET) and energy conversion, which are not only central to photosynthesis but also have broader implications for bioenergy production and biomimetic device design. In this study, we employed electron paramagnetic resonance (EPR) spectroscopy to investigate key light-induced charge separation steps in PSI isolated from several green algal and cyanobacterial species. Following photoexcitation, rapid sequential ET occurs through either of two quasi-symmetric branches of donor/acceptor cofactors embedded within the protein core, termed the A and B branches. Using high-frequency (130 GHz) time-resolved EPR (TR-EPR) and deuteration techniques to enhance spectral resolution, we observed that at low temperatures prokaryotic PSI exhibits reversible ET in the A branch and irreversible ET in the B branch, while PSI from eukaryotic counterparts displays either reversible ET in both branches or exclusively in the B branch. Furthermore, we observed a notable correlation between low-temperature charge separation to the terminal [4Fe-4S] clusters of PSI, termed FA and FB, as reflected in the measured FA/FB ratio. These findings enhance our understanding of the mechanistic diversity of PSI's ET across different species and underscore the importance of experimental design in resolving these differences. Though further research is necessary to elucidate the underlying mechanisms and the evolutionary significance of these variations in PSI charge separation, this study sets the stage for future investigations into the complex interplay between protein structure, ET pathways, and the environmental adaptations of photosynthetic organisms.

Characterization of dielectric properties and their impact on MAS-DNP NMR applications.

The dielectric properties of materials play a crucial role in the propagation and absorption of microwave beams employed in Magic Angle Spinning - Dynamic Nuclear Polarization (MAS-DNP) NMR experiments. Despite ongoing optimization efforts in sample preparation, routine MAS-DNP NMR applications often fall short of theoretical sensitivity limits. Offering a different perspective, we report the refractive indices and extinction coefficients of diverse materials used in MAS-DNP NMR experiments, spanning a frequency range from 70 to 960 GHz. Knowledge of their dielectric properties enables the accurate simulation of electron nutation frequencies, thereby guiding the design of more efficient hardware and sample preparation of biological or material samples. This is illustrated experimentally for four different rotor materials (sapphire, yttria-stabilized zirconia (YSZ), aluminum nitride (AlN), and SiAlON ceramics) used for DNP at 395 GHz/1H 600 MHz. Finally, electromagnetic simulations and state-of-the-art MAS-DNP numerical simulations provide a rational explanation for the observed magnetic field dependence of the enhancement when using nitroxide biradicals, offering insights that will improve MAS-DNP NMR at high magnetic fields.

Field-domain rapid-scan EPR at 240GHz for studies of protein functional dynamics at room temperature.

We present field-domain rapid-scan (RS) electron paramagnetic resonance (EPR) at 8.6T and 240GHz. To enable this technique, we upgraded a home-built EPR spectrometer with an FPGA-enabled digitizer and real-time processing software. The software leverages the Hilbert transform to recover the in-phase (I) and quadrature (Q) channels, and therefore the raw absorptive and dispersive signals, χ' and χ'', from their combined magnitude (I2+Q2). Averaging a magnitude is simpler than real-time coherent averaging and has the added benefit of permitting long-timescale signal averaging (up to at least 2.5×106 scans) because it eliminates the effects of source-receiver phase drift. Our rapid-scan (RS) EPR provides a signal-to-noise ratio that is approximately twice that of continuous wave (CW) EPR under the same experimental conditions, after scaling by the square root of acquisition time. We apply our RS EPR as an extension of the recently reported time-resolved Gd-Gd EPR (TiGGER) [Maity et al., 2023], which is able to monitor inter-residue distance changes during the photocycle of a photoresponsive protein through changes in the Gd-Gd dipolar couplings. RS, opposed to CW, returns field-swept spectra as a function of time with 10ms time resolution, and thus, adds a second dimension to the static field transients recorded by TiGGER. We were able to use RS TiGGER to track time-dependent and temperature-dependent kinetics of AsLOV2, a light-activated phototropin domain found in oats. The results presented here combine the benefits of RS EPR with the improved spectral resolution and sensitivity of Gd chelates at high magnetic fields. In the future, field-domain RS EPR at high magnetic fields may enable studies of other real-time kinetic processes with time resolutions that are otherwise difficult to access in the solution state.

Xerogel mesoporous materials based on ultrastable Blatter radicals for efficient sorption of nitric oxide.

The reduction of hazardous nitric oxide emissions remains a significant ecological challenge. Despite the variety of possibilities, sorbents able to capture low concentrations of NO from flue gas with high selectivity are still in demand. In this work a new type of mesoporous xerogel material highly loaded with ultrastable Blatter radicals (BTR, >60 % by mass) that act as selective NO sorption sites is developed. Electron Paramagnetic Resonance (EPR) spectroscopy evidences reversible NO sorption in nanometer-scale pores of BTR-based xerogels and indicates the high NO capacity of such radical-rich sorbent. Efficient NO capture from model flue gas mixture is also evidenced in experiments with a fixed bed reactor. Such advanced properties of new materials as selectivity, strong binding with NO and an ability for mild regeneration via thermodesorption promote them for future ecological applications.

Inhibitory effects of cold plasma-activated water on the generation of advanced glycation end products and methylimidazoles in cookies and mechanistic evaluation using electron paramagnetic resonance.

The inhibitory effects of cold plasma-activated water (PAW) on the formation of AGEs and methylimidazoles in cookies was examined. The results showed that different PAW (parameters: 50 W-50 s, 50 W-100 s, 50 W-150 s, 100 W-50 s, 100 W-100 s, and 100 W-150 s) reduced the contents of AGEs and methylimidazoles, in which the maximum inhibition rates were 47.38% and 40.17% for free and bound AGEs and 44.16% and 40.31% for free and bound methylimidazoles, respectively. Moreover, the mechanisms associated with the elimination of carbonyl intermediates and free radicals was determined by electron paramagnetic resonance (EPR) and high performance liquid chromatography-ultraviolet/visible absorption detector (HPLC-UV/Vis). The results showed the quenching of total free radicals, alkyl free radicals, and HO· by PAW, leading to the suppression of glyoxal and methylglyoxal intermediates. These findings support PAW as a promising agent to enhance the safety of cookies.

Mössbauer and EPR detection of iron trafficking kinetics and possibly labile iron pools in whole Saccharomyces cerevisiae cells.

The kinetics of iron trafficking in whole respiring Saccharomyces cerevisiae cells were investigated using Mössbauer and EPR spectroscopies. The Mössbauer-active isotope 57Fe was added to cells growing under iron-limited conditions; cells were analyzed at different times post iron addition. Spectroscopic changes suggested that the added 57Fe initially entered the labile iron pool, and then distributed to vacuoles and mitochondria. The first spectroscopic feature observed, ∼ 3 min after adding 57Fe plus a 5 to 15 min processing dead time, was a quadrupole doublet typical of nonheme high-spin FeII. This feature likely arose from labile FeII pools in the cell. At later times (15-150 min), magnetic features due to S = 5/2 FeIII developed; these likely arose from FeIII in vacuoles. Corresponding EPR spectra were dominated by a g = 4.3 signal from the S = 5/2 FeIII ions that increased in intensity over time. Developing at a similar rate was a quadrupole doublet typical of S = 0 [Fe4S4]2+ clusters and low-spin FeII hemes; such centers are mainly in mitochondria, cytosol, and nuclei. Development of these features was simulated using a published mathematical model, and simulations compared qualitatively well with observations. In the five sets of experiments presented, all spectroscopic features developed within the doubling time of the cells, implying that the detected iron trafficking species are physiologically relevant. These spectroscopy-based experiments allow the endogenous labile iron pool within growing cells to be detected without damaging or altering the pool, as definitely occurs using chelator-probe detection and possibly occurs using chromatographic separations.

TDP43 and huntingtin Exon-1 undergo a conformationally specific interaction that strongly alters the fibril formation of both proteins.

Protein aggregation is a common feature of many neurodegenerative diseases. In Huntington's disease, mutant huntingtin is the primary aggregating protein, but the aggregation of other proteins, such as TDP43, is likely to further contribute to toxicity. Moreover, mutant huntingtin is also a risk factor for TDP pathology in ALS. Despite this co-pathology of huntingtin and TDP43, it remains unknown whether these amyloidogenic proteins directly interact with each other. Using a combination of biophysical methods, we show that the aggregation-prone regions of both proteins, huntingtin exon-1 (Httex1) and the TDP43 low complexity domain (TDP43-LCD), interact in a conformationally specific manner. This interaction significantly slows Httex1 aggregation, while it accelerates TDP43-LCD aggregation. A key intermediate responsible for both effects is a complex formed by liquid TDP43-LCD condensates and Httex1 fibrils. This complex shields seeding competent surfaces of Httex1 fibrils from Httex1 monomers, which are excluded from the condensates. In contrast, TDP43-LCD condensates undergo an accelerated liquid-to-solid transition upon exposure to Httex1 fibrils. Cellular studies show co-aggregation of untagged Httex1 with TDP43. This interaction causes mislocalization of TDP43, which has been linked to TDP43 toxicity. The protection from Httex1 aggregation in lieu of TDP43-LCD aggregation is interesting, as it mirrors what has been found in disease models, namely that TDP43 can protect from huntingtin toxicity, while mutant huntingtin can promote TDP43 pathology. These results suggest that direct protein interaction could, at least in part, be responsible for the linked pathologies of both proteins.

Intermediate steps in the formation of neuronal SNARE complexes.

Neuronal exocytosis requires the assembly of three SNARE proteins, syntaxin and SNAP25 on the plasma membrane and synaptobrevin on the vesicle membrane. However, the precise steps in this process and the points at which assembly and fusion are controlled by regulatory proteins are unclear. In the present work, we examine the kinetics and intermediate states during SNARE assembly in vitro using a combination of time resolved fluorescence and EPR spectroscopy. We show that syntaxin rapidly forms a dimer prior to forming the kinetically stable 2:1 syntaxin:SNAP25 complex and that the 2:1 complex is not diminished by the presence of excess SNAP25. Moreover, the 2:1 complex is temperature-dependent with a reduced concentration at 37 °C. The two segments of SNAP25 behave differently. The N-terminal SN1 segment of SNAP25 exhibits a pronounced increase in backbone ordering from the N- to the C-terminus that is not seen in the C-terminal SNAP25 segment SN2. Both the SN1 and SN2 segments of SNAP25 will assemble with syntaxin; however, while the association of the SN1 segment with syntaxin produces a stable 2:2 (SN1:syntaxin) complex, the complex formed between SN2 and syntaxin is largely disordered. Synaptobrevin fails to bind syntaxin alone but will associate with syntaxin in the presence of either the SN1 or SN2 segments; however, the synaptobrevin:syntaxin:SN2 complex remains disordered. Taken together, these data suggest that synaptobrevin and syntaxin do not assemble in the absence of SNAP25 and that the SN2 segment of SNAP25 is the last to enter the SNARE complex.

Investigations on the EPR g factors and local structures for the orthorhombic and tetragonal Cu2+ centers in Pb[Zr0.54Ti0.46]O3.

The defect models of the orthorhombical and tetragonal Cu2+ centers in Pb[Zr0.54Ti0.46]O3 are attributed to Cu2+ ions occupying the sixfold coordinated octahedral Ti4+ site with and without charge compensation, respectively. The electron paramagnetic resonance (EPR) g factors gi (i = x, y, z) of the Cu2+ centers in Pb[Zr0.54Ti0.46]O3 are theoretically studied by using the perturbation formulas of a 3d9 ion under orthorhombically and tetragonally elongated octahedra. Based on the calculation, the impurity off-center displacements are about 0.253 and 0.162 Å for the orthorhombical and tetragonal Cu2+ centers, respectively. Meanwhile, the planar Cu2+-O2- bonds are found to experience the relative variation ΔR (≈0.102 Å) along the a- and b-axes for the orthorhombical Cu2+ center due to the Jahn-Teller (JT) effect. The theoretical EPR g factors based on the above local structures agree well with the observed values.