Analysis of the electron transfer pathway in small laccase by EPR and UV-vis spectroscopy coupled with redox titration

Bacterial small laccases(SLAC) are promising industrial biocatalysts due to their ability to oxidize a broad range of substrates with exceptional thermostability and tolerance for alkaline p H. Electron transfer between substrate, copper centers, and O2is one of the key steps in the catalytic turnover of SLAC. However, limited research has been conducted on the electron transfer pathway of SLAC and SLAC-catalyzed reactions, hindering further engineering of SLAC to produce tunable biocatalysts for novel applications. Herein, the combinational use of electron paramagnetic resonance(EPR) and ultraviolet-visible(UV-vis) spectroscopic methods coupled with redox titration were employed to monitor the electron transfer processes and obtain further insights into the electron transfer pathway in SLAC. The reduction potentials for type 1 copper(T1Cu), type 2 copper(T2Cu) and type 3copper(T3Cu) were determined to be 367 ± 2 mV, 378 ± 5 m V and 403 ± 2 mV,respectively. Moreover, the reduction potential of a selected substrate of SLAC, hydroquinone(HQ), was determined to be 288 mV using cyclic voltammetry(CV). In this way, an electron transfer pathway was identified based on the reduction potentials. Specifically,electrons are transferred from HQ to T1Cu, then to T2Cu and T3Cu, and finally to O2.Furthermore, superhyperfine splitting observed via EPR during redox titration indicated a modification in the covalency of T2Cu upon electron uptake, suggesting a conformational alteration in the protein environment surrounding the copper sites, which could potentially influence the reduction potential of the copper sites during catalytic processes. The results presented here not only provide a comprehensive method for analyzing the electron transfer pathway in metalloenzymes through reduction potential measurements, but also offer valuable insights for further engineering and directed evolution studies of SLAC in the aim for biotechnological and industrial applications.

A low-noise X-band microwave source with digital automatic frequency control for electron paramagnetic resonance spectroscopy

We report a new design of microwave source for X-band electron paramagnetic resonance spectrometer.The microwave source is equipped with a digital automatic frequency control circuit.The parameters of the digital automatic frequency control circuit can be flexibly configured for different experimental conditions,such as the input powers or the quality factors of the resonator.The configurability makes the microwave source universally compatible and greatly extends its application.To demonstrate the ability of adapting to various experimental conditions,the microwave source is tested by varying the input powers and the quality factors of the resonator.A satisfactory phase noise as low as-135 d Bc/Hz at 100-k Hz offset from the center frequency is achieved,due to the use of a phase-locked dielectric resonator oscillator and a direct digital synthesizer.Continuous-wave electron paramagnetic resonance experiments are conducted to examine the performance of the microwave source.The outstanding performance shows a prospect of wide applications of the microwave source in numerous fields of science.

Temperature-Dependent Formation of Redox Sites in Molybdenum Trioxide Studied by Electron Paramagnetic Resonance Spectroscopy

The formation and qualification of redox sites in transition metal oxides are always the active fields related to electronics, catalysis, sensors, and energy-storage units. In the present study, the temperature dependence of thermal reduction of MoO3 was surveyed at the range of 350℃ to 750℃. Upon reduction, the formed redox species characterized by EPR spectroscopy are the MoVion and superoxide anion radical (O2-) when the reduction was induced at the optimal temperature of 300-350℃. When heating-up from 350℃, the EPR signals started to decline in amplitude. The signals in the range of 400-450℃ decreased to half of that at 350℃, and then to zero at ~600℃. Further treatment at even higher temperature or prolonged heating time at 500℃ caused more reduction and more free electrons were released to the MoO3 bulk, which results in a delocalized means similar to the antiferromagnetic coupling. These data herein are helpful to prepare and study the metal-oxide catalysts.

Identification of Superoxide O2^- during KNO3-NaNO2-NaNO3 Salt by Electron UV-Vis Absorption Spectroscopy Thermal Decomposition of Molten Paramagnetic Resonance and

On account of excellent thermal physical properties, molten nitrates/nitrites salt has been widely employed in heat transfer and thermal storage industry, especially in concentrated solar power system. The thermal stability study of molten nitrate/nitrite salt is of great importance for this system, and the decomposition mechanism is the most complicated part of it. The oxide species O2^2- and O2^- were considered as intermediates in molten KNO3-NaNO3 while hard to been detected in high temperature molten salt due to their trace concentration and low stability. In this work, the homemade in situ high temperature UV- Vis instrument and a commercial electron paramagnetic resonance were utilized to supply evidence for the formation of superoxide during a slow decomposition process of heat transfer salt （HTS, 53 wt% KNO3/40 wt% NaNO2/7 wt% NaNO3）. It is found that the superoxide is more easily generated from molten NaNO2 compared to NaNO3, and it has an absorption band at 420-440 nm in HTS which red shifts as temperature increases. The band is assigned to charge-transfer transition in NaO2 or KO2, responsible for the yellow color of the molten nitrate/nitrite salt. Furthermore, the UV absorption bands of molten NaNO2 and NANO3 are also obtained and compared with that of HTS.

Electron paramagnetic resonance study of amphiphiles partitioning behavior in desiccation-tolerant moss during dehydration

Desiccation tolerance is a crucial characteristic for desert moss surviving in arid regions. Desiccation procedure always induces amphiphiles transferring from the polar cytoplasm into lipid bodies. The behavior of amphiphiles transferring can contribute to the enhancement of desiccation tolerance and the reduction of plasma membrane integrity simultaneously. The effects of amphiphiles partitioning into the lipid phase during water loss has been studied for pollen and seeds using electron paramagnetic resonance （EPR） spectroscopy. However, desiccation-tolerant high plants occur among mosses, several angiosperms and higher plants seeds or pollens. They have different strategies for survival in dehydration and rehydration. A desiccation-tolerant moss Tortula desertorurn was used to investigate the behaviors of amphiphilic molecules during drying by spin label technology. There are small amount of amphiphilic probes partitioning into membrane during moss leaves dehydration, comparing with that in higher plants. Cytoplasm viscosity changed from 1.14 into glass state only dehydration less than 60 min. Moss leaves lost plasma membrane integrity slightly,from 0.115 to 0.237, occurred simultaneously with amphiphiles partition. The results showed the more advantages of mosses than higher plants in adapting fast dehydration. We propose that EPR spin label is feasible for studying the amphiphiles partitioning mechanisms in membrane protection and damage for desiccation-tolerant mosses.

Electron paramagnetic resonance studies of the chelate-based ionic liquid in different solvents

The electron paramagnetic resonance spectra of the chelate-based ionic liquid[C_(10)mim][Cu(F_6-acac)_3]in different solvents have been obtained at 120 K.It was found that the values of the^(63)Cu hyperfine coupling constants(A_(IL))of[C_(10)mim][Cu(F_6-acac)_3]in molecular solvents were from 116 to 180 Gauss.Moreover,the A_(IL)values in general ionic liquids are more complicated,and two sets of peaks can often be observed in their electron paramagnetic resonance spectra.Based on the Kamlet-Taft parameters,relative permittivity,the experimental results were discussed in terms of solvation effect and coordination of the solvents.

Electron paramagnetic resonance characterization of aluminum ion implantation-induced defects in 4H-SiC

Deep-level defects in silicon carbide(SiC)are critical to the control of the performance of SiC electron devices.In this paper,deep-level defects in aluminumion-implanted 4H-SiC after high-temperature annealingwere studied using electron paramagnetic resonance(EPR)spectroscopy at temperatures of 77 K and 123 K under different illumination conditions.Results showed that the main defect in aluminum ion-implanted 4H-SiC was the positively charged carbon vacancy(VC+),and the higher the doping concentration was,the higher was the concentration of VC+.Itwas found that the type of material defectwas independent of the doping concentration,although more VC+defects were detected during photoexcitation and at lower temperatures.These results should be helpful in the fundamental research of p-type 4H-SiC fabrication in accordance with functional device development.

X-Ray Diffraction, Electron Paramagnetic Resonance and Optical Absorption Study of Bauxite

The bauxite mineral obtained from Araku, Vishakapatnam district of Andhra Pradesh, India is used in the present work. Structural characterization was performed by X-ray diffraction (XRD). The mineral was found to be gibbsite in phase. The transitional metal ions present were investigated using electron paramagnetic resonance (EPR) and optical absorption spectra. The EPR results suggest that Fe3+ has replaced Al3+ in the unit cell of bauxite. The optical absorption spectrum is due to Fe3+ which indicates that it is in distorted octahedral environment. The near-infrared (NIR) spectrum is due to water fundamentals and combination overtones, which confirm the formula of the compound. The impurities in the mineral are identified using spectroscopic techniques.

Electron Paramagnetic Resonance and Optical Absorption Studies on Copper Ions in Mixed Alkali Cadmium Phosphate Glasses

Electron paramagnetic resonance （EPR） and optical absorption studies were carried out at room temperature on copper doped mixed alkali cadmium phosphate （LiNaCdP） glasses to understand the nature and symmetry of dopant. Three samples with varying concentrations of alkali ions have been prepared. The spin Harniltonian parameters obtained from room temperature EPR spectra are： gli=2.437, 9⊥=2.096, A‖=117×10-4 cm-1, A⊥=26×10-4 cm-1 for LiNaCdP1, g‖=2.441, g⊥=2.088, A‖=121×10-4 cm-1, A⊥=25×10-4 cm-1 for LiNaCdP2and g‖=2.433,g⊥=2.096, A‖=125×10 4cm-1, A⊥=32×10-4cm-1 for LiNaCdP3. TheseEPR results indicate that the dopant Cu2＋ ion enters the glass matrix into a tetragonally elongated octahedral site. The bonding parameters evaluated by correlating optical and EPR data suggest that bonding between the central metal ion and ligands is partially covalent. The mixed alkali effect in cadmium phosphate glasses was reported.

Effect of local structure on electron paramagnetic resonance spectra for trigonal [Cr(H_2O)_6]^(3+) coordination complex in the sulfate alums series:a ligand field theory study

A simple theoretical method is introduced for studying the interrelation between electronic and molecular structures.By diagonalizing the 120 × 120 complete energy matrices,the relationships between zero-field splitting （ZFS） parameter D and local distortion parameter △θ for Cr^3＋ ions doped,separately,in α- and β- alums are investigated.Our results indicate that there exists an approximately linear relationship between D and △θ in a temperature range 4.2-297 K and the signs of D and △θ are opposite to each other.Moreover,in order to understand the contribution of spin-orbit coupling coefficient ζ to ZFS parameter D,the relation between D and ζ is also discussed.

Theoretical Studies of Electron Paramagnetic Resonance Parameters for Cr^4＋ Ions in Ca2GeO4 Crystals

The electron paramagnetic resonance (EPR) parameters (zero-Geld splitting Dand g factors g_‖, g_⊥) of Cr~(4+) ions in Ca_2 GeO_4 crystals have been calculated from thecomplete high-order perturbation formulas of EPR parameters for a 3d~2 ion in trigonal MX_4clusters. In these formulas, in addition to the contributions to EPR parameters from the widely usedcrystal-field (CF) mechanism, the contributions from the charge-transfer (CT) mechanism (which areoften neglected) are included. From the calculations, it is found that for the high valence state3d~n ions in crystals, the reasonable explanation of EPR parameters (in particular, the g factors)should take both the CF and CT mechanisms into account.

Monitoring the autoproteolysis of hiv-1 protease by site-directed spin-labeling and electron paramagnetic resonance spectroscopy

Site-directed spin-labeling with continuous wave electron paramagnetic resonance spectroscopy was used to monitor autoproteolysis of HIV-1 protease, an enzyme essential for viral maturation. Two protein constructs were examined, namely subtype F and the circulating recombinant form CRF01_A/E. As the protease undergoes self-cleavage, protein unfolds and small peptide fragments containing the spin label are generated, which collectively give rise to a sharp spectral component that is easily discernable in the high-field resonance line in the EPR spectrum. By monitoring the intensity of this spectral component over time, the autoproteolytic stability of each construct was characterized under various conditions. Data were collected for samples stored at 4 °C, 25 °C, and 37 °C, and on a subtype F HIV-1 protease sample stored at 25 °C and containing the FDA-approved protease inhibitor Tipranavir. As expected, the rate of autoproteolysis decreased as the storage temperature was lowered. Minimal autoproteolysis was seen for the sample that contained Tipranavir, providing direction for future spectroscopic studies of active protease samples. When compared to standard methods of monitoring protein degradation such as gel electrophoresis or chromatographic analyses, spin-labeling with CW EPR offers a facile, real-time, non-consuming way to monitor autoproteolysis or protein degradation. Additionally, mass spectrometry studies revealed that the N-termini of both constructs are sensitive to degradation and that the sites of specific autoproteolysis vary.

Elucidating solvent effects on the stability of phenoxyl radicals in monohydric alcohols via electron paramagnetic resonance

Research on solvent effects is an important and long-standing topic,but there still is some room,especially for the special solvent effect of fluoroalcohols.In this work,we investigated the stability of phenoxyl radical in monohydric alcohol solvents through in-situ electron paramagnetic resonance detections.The decay behavior of phenoxyl radical showed a reasonable relationship with the mesoscopic structure of alcohols,characterized by smalland wide-angle X-ray scattering.Moreover,the distinct solvent effects of fluoroalcohols were emphasized,and the significant influence of van der Waals distance in the solvents was suggested.Overall,the stability of phenoxyl radical in alcohols was quantified and correlated with the solvent structures.We believe that the established method for stability study on radicals will encourage solvent effect studies on various organic reactions,and the proposed solvent effects in fluoroalcohols may inspire the development of green solvents in both industrial conversions and organic synthesis.

Advancements and Prospects in Continuous Wave Time-resolved Electron Paramagnetic Resonance

This brief review highlights the techniques and diverse applications of time-resolved electron paramagnetic resonance(TREPR)spectroscopy,underscoring its essential role in elucidating the structures,spin dynamics,and reactivities of open-shell systems.Furthermore,we discuss the limitations of traditional TREPR methodologies,particularly their challenges in directly observing reactive radical intermediates under real-world reaction conditions.Lastly,we present the latest advancements in TREPR technology developed in our laboratory,specifically ultrawide single-sideband phase-sensitive detection(U-PSD)TREPR,highlighting its significant impact and tremendous potential in advancing free radical chemistry research.We envision promising future applications of TREPR and its pivotal role in enhancing our understanding of mechanisms involved in complex radical processes and photocatalysis.

高频高场下EPR谱仪的研究进展

电子顺磁共振(EPR)是一种研究磁性材料微观信息的测量手段.由于早期的EPR研究受到磁场强度和微波频率的限制,一些材料相关的微观信息并不能得到清晰地显示.近些年来,随着强磁场技术和微波技术的发展,连续波电子顺磁共振(cw-EPR)谱仪和脉冲电子顺磁共振(pulsed EPR)谱仪在高频高场下得到了充分的应用,同时谱仪的灵敏度和分辨率等技术指标也得到了比较好的提升.本文主要介绍了在高频高场下EPR谱仪的原理和构造,国内外发展历程和研究现状,以及在相关领域的最新应用.

EPR谱图模拟软件EasySpin的图形用户界面设计

EasySpin是一款较为流行的电子顺磁共振(EPR)谱图模拟和拟合软件,LabVIEW是一种图形化编程语言开发环境.本文介绍了一款使用LabVIEW为EasySpin设计的图形用户界面LV-EasySpin.LV-EasySpin提供了一种直观的操作方法来实现连续波EPR多组分波谱的可视化模拟和拟合.本文辅以各种模式下的实例进行说明,阐述了LV-EasySpin的设计思路与实现方案,最终希望具有简洁、易操作界面的LV-EasySpin可以降低用户使用EasySpin分析EPR谱图的难度.

In Situ Electron Paramagnetic Resonance Reveals Fading Mechanism of Mn-Based Prussian Blue Analogue:Accelerated Mn Dissolution Due to Charge Delocalization

Contrasting with Fe-based Prussian blue analogues(PBAs),Mn-based PBAs with higher energy density are more promising cathode materials for Na-ion batteries.However,fast capacity fading has severely impeded its practical use,which is still not well understood.To elucidate the fading mechanism,in situ and ex situ electron paramagnetic resonance are employed here.The results first demonstrate the charge delocalization of Mn2+and Mn dissolution during cycles,which are further proved to be highly related.Our work reveals the inherent shortcoming of Mn-based PBA cathodes in liquid electrolyte.

晶体(NH_4)_2C_4H_4O_6∶VO^(2+)的EPR参量及局域结构的研究

采用叠加模型和双旋-轨耦合参量模型,建立了结构参数与EPR参量之间的定量关系;较好地解释了[VO(H2O)5]2+络离子的局域结构和EPR参量;研究结果发现,(NH4)2C4H4O6:VO2+晶体中络离子[VO(H2O)5]2+的键长为R//≈0.130nm,R⊥≈0.195nm;在(NH4)2C4H4O6:VO2+晶体中,局域结构沿C4轴方向呈压缩的八面体结构;所得EPR参量的理论计算与实验测量数据符合很好。

电子顺磁共振技术在化工基础研究中的应用进展

电子顺磁共振(electron paramagnetic resonance,EPR)技术能够检测自由基、过渡金属离子及缺陷等顺磁性物质,谱图具有高特异性,背景信号少,既可以检测溶液样品,也可以检测固态样品,且检测限低。在化工基础研究中,尤其是自由基相关过程研究中,EPR技术的优势是其他谱学无可比拟的。但是化工领域的科学家们使用EPR技术并不广泛。综述了EPR在化工基础相关研究中应用的一些例子,包括催化材料的表征、活性中间体的表征、溶剂性质表征及材料性能表征四个方面,希望能让更多化工领域工作者了解EPR技术,并使用EPR技术解决化工问题。