Resolving the EPR Spectra in the Cytochrome bc 1 Complex from Saccharomyces cerevisiae

Quinone molecules are ubiquitous in living organisms. They are found either within the lipid phase of the biological membrane (quinone pool) or are bound in specific binding sites within membrane-bound protein complexes. The biological function of such bound quinones is determined by their ability to be reduced and/or oxidized in two successive one-electron steps. As a result, quinones are involved as one- or two-electron donors or acceptors in a large number of biological electron-transfer steps occurring during respiratory or photosynthetic processes. The intermediate formed by a one-electron reduction step is a semiquinone, which is paramagnetic and can be studied by electron paramagnetic resonance (EPR) spectroscopy. Detailed studies of such states can provide important structural information on these intermediates in such electron-transfer processes. In this study, we focus on the redox-active ubiquinone-6 of the yeast cytochrome bc ₁ complex (QCR, ubiquinol: cytochrome c oxidoreductase) from Saccharomyces cerevisiae at the so-called Q_i site. Although the location of the Q_i binding pocket is quite well known, details about its exact binding are less clear. Currently, three different X-ray crystallographic studies suggest three different binding geometries for Q_i. Recent studies in the bacterial system (Rhodobacter sphaeroides) have suggested a direct coordination to histidine as proposed in the chicken heart crystal structure model. Using the yeast system we apply EPR and especially relaxation filtered hyperfine (REFINE) spectroscopy to study the Q_i binding site. ¹⁴N-electron spin-echo envelope modulation spectroscopy together with an inversion-recovery filter (REFINE) is applied to resolve the question of whether ¹⁴N modulations arise from interactions to Q _i ^·? or to the Rieske iron–sulphur center. These results are discussed with regard to the location and potential function of Q_i in the enzyme.     

在体电子自旋共振(EPR)波谱和成像技术在生物医学中的应用

活性氧和氮自由基(ROS/RNS)在一系列的人类疾病中扮演着双重角色. 它们可以是氧化剂, 诱导氧化状态, 导致组织损伤. 它们又可以是信号传导因子, 诱发保护性反应, 使得被调节的组织器官经受得起更强的损伤. 鉴于它们在生物医学中的重要作用, 检测它们产生和分布的技术的研究因而变得必要和紧迫. 在体电子自旋共振(EPR)波谱和成像技术渐已被应用于活体生物体系中用以表针和显像ROS/RNS. EPR 波谱特性(包括线宽、强度和寿命)以及空间分布信息已为动物甚至人体病理模型中氧化还原状态和氧分布的检测提供不可缺少的依据. 该文将简单描述和讨论一系列在体EPR 波谱和成像技术在器官和组织中的应用, 其中包括活体组织氧化还原状态, 活体组织氧分布和时间演化, 自由基空间以及谱-空间成像等.     

Preparation of allicin-whey protein isolate conjugates: Allicin extraction by water,conjugates’ ultrasound-assisted binding and its stability,solubility and emulsibility analysis

The instability of allicin makes it easily decomposed into various organic sulfur compounds, resulting in significant decrease in biological activity. In this study, allicin was firstly extracted with water, then bound with whey protein isolates (WPI) which were pretreated by ultrasound to form conjugates, and the stability, water solubility and emulsibility of conjugates were as well investigated. The research results showed that there were no significant differences in the extraction yields of allicin from water, 40% and 80% ethanol. Appropriate frequency (20/40 kHz), power (50 W/L) and time (20 min) of ultrasonic pretreatments significantly increased (P < 0.05) the sulfhydryl groups content of WPI by 35.05% over control, causing improvement in binding ability of protein to allicin. The binding process of allicin-WPI displayed good fit with Elovich kinetic model (R² = 0.9781). The mass retention rate of the conjugates (in 60% combination rate) with ultrasonic pretreating kept at 95.97% after 14 days of storage at 25 °C, whereas allicin’s mass retention rate was only 61.79% at same storage condition. The water solubility of the prepared conjugates was significantly higher than allicin. And with optimal condition ultrasonic pretreatment of WPI, the conjugates showed the highest emulsifying capacity and emulsion stability (49.56 m²/g, 10.06 min). In conclusion, the ultrasonically pretreated allicin-WPI conjugates exhibited better stability, water solubility and emulsifying properties compared to allicin, this expands the application field of allicin.     

Noninvasive mapping of the redox status in septic mouse by in vivo electron paramagnetic resonance imaging

Increased reactive oxygen species (ROS) contribute to numerous brain disorders, and ROS generation has been examined in diverse experimental models of lipopolysaccharide (LPS)-induced inflammation. The in vivo electron paramagnetic resonance (EPR)/nitroxide spin probe method has been used to analyze the redox status in animal models modulated by ROS generation. In this study, a blood–brain barrier (BBB)-permeable nitroxide spin probe, 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (HMP), was used as a redox-sensitive nitroxide probe. Magnetic resonance images of mouse head after the injection of HMP showed that HMP was distributed throughout all regions of the mouse head including the brain, suggesting that HMP can reveal redox information in all regions of the mouse head. After the injection of HMP through the mouse tail vein 6 h after the injection of LPS, three-dimensional (3D) EPR images were obtained each minute under a field scanning of 0.3 s and with 81 projections. The reduction reaction of HMP in septic mouse heads was remarkably accelerated compared to that in control mice, and this accelerated reaction was inhibited by aminoguanidine and allopurinol, which inhibit enzymatic activities of induced nitric oxide synthase and xanthine oxidase, respectively. Based on the pharmacokinetics of HMP in mouse heads, the half-life mapping of HMP was performed in LPS-treated mouse head. Half-life maps clearly show a difference in the redox status induced by ROS generation in the presence or absence of inhibitors of ROS-generating enzymes. The present results suggest that a 3D in vivo EPR imaging system combined with BBB-permeable HMP is a useful noninvasive tool for assessing changes in the redox status in rodent models of brain disease under oxidative stress.     

Assessment of Nitrones as In Vivo Redox Sensors

Spin traps such as 5,5-dimethyl-1-pyrroline N-oxide, α-phenyl-tert-butyl nitrone, α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone and newer generation 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide analogs have been known for years. What are the desired properties of good probes for measuring in vivo redox reactions in biological systems? These are specificity, sensitivity, rapid, high yield kinetics, low toxicity, high stability and easy to administer and target. Unfortunately, the nitrones perform poorly in almost all of these categories. Typical in vivo concentrations of spin trap approach 100 mM (assuming solubility and toxicity are not an issue), frequently yield 1% nitroxide or less stoichiometry, are typically unstable with time and frequently lack specificity. In vivo electron paramagnetic resonance (EPR) experiments need to have strong signals that correlate with redox chemistry. The resultant signal should be stable and not rapidly interconvert to other diamagnetic species. Fortunately, some newer probes of in vivo redox reactions in biological systems have come upon the horizon. In fact some have been around for a long time, but their virtues are becoming increasingly appreciated. This paper summarizes the disadvantages of nitrones versus the clear advantages of other probes of free radicals, redox state and the like by EPR. It also expands on the properties of nitroxides and nitrones as therapeutics.     

Effects of resveratrol and its analogs on scavenging hydroxyl radicals: Evaluation by EPR spin trapping method

Resveratrol (3,4′,5-trihydroxy-trans-stilbene) and six analogs, polyhydroxystilbenes, were synthesized. Their effects on scavenging hydroxyl radicals were studied by electron paramagnetic resonance (EPR) spin trapping method. The EPR signal intensity of the spin adduct of hydroxyl radical to 5,5-dimethyl-1-pyrroline N-oxide was detected and used as a standard for the evaluation of the effect of the seven compounds on scavenging hydroxyl radicals. While all seven compounds exhibited hydroxyl radical-scavenging activity, some of them proved to be more effective than resveratrol in this model. Another stable but low-intensity spin adduct was also observed by EPR. A possible assignment is proposed.     

Magnetic nanoparticles: reactive oxygen species generation and potential therapeutic applications

Magnetic nanoparticles have been demonstrated to produce reactive oxygen species (ROS), which play a major role in various cellular pathways, via Fenton and Haber-Weiss reaction. ROS act as a double-edged sword inside the body. At normal conditions, the generation of ROS is in balance with their elimination by scavenger systems, and they can promote cell proliferation as well as differentiation. However, at an increased level, they can cause damages to protein, lead to cellular apoptosis, and contribute to many diseases including cancer. Many recent studies proposed a variety of strategies to either suppress toxicity of ROS generation or exploit the elevated ROS levels for cancer therapy.     

Time-resolved electron paramagnetic resonance of radical pair intermediates in cryptochromes

Electron transfer plays a key role in many biological systems, including core complexes of photosynthesis and respiration. As this involves unpaired electron spins, electron paramagnetic resonance (EPR) is the method of choice to investigate such processes. Systems that show photo-induced charge separation and electron transfer are of particular interest, as here the processes can easily be synchronised to the experiment and therefore followed directly over its time course. One particular class of proteins, the cryptochromes, showing charge separation and in turn spin-correlated radical pairs upon excitation with blue light, have been investigated by time-resolved EPR spectroscopy in great detail and the results obtained so far are summarised in this contribution. Highlights include the first observation of spin-correlated radical pairs in these proteins, a fact with great impact on the proposed role as key part of a magnetic compass of migratory birds, as well as the assignment of the radical-pair partners and the unravelling of alternative and unexpected electron transfer pathways in these proteins, giving new insights into aspects of biological electron transfer itself.     

9,10-Diphenylanthracene as a matrix for MALDI-MS electron transfer secondary reactions

The most common secondary-ionization mechanism in positive ion matrix-assisted laser desorption/ionization (MALDI) involves a proton transfer reaction to ionize the analyte. Peptides and proteins are molecules that have basic (and acidic) sites that make them susceptible to proton transfer. However, non-polar, aprotic compounds that lack basic sites are more difficult to protonate, and creating charged forms of this type of analyte can pose a problem when conventional MALDI matrices are employed. In this case, forming a radical molecular ion through electron transfer is a viable alternative, and certain matrices may facilitate the process. In this work, we investigate the performance of a newly developed electron-transfer secondary reaction matrix: 9,10-diphenylanthracene (9,10-DPA). The use of 9,10-DPA as matrix for MALDI analysis has been tested using several model compounds. It appears to promote ionization through electron transfer in a highly efficient manner as compared to other potential matrices. Thermodynamic aspects of the observed electron transfers in secondary-ionization reactions were also considered, as was the possibility for kinetically controlled/endothermic, electron-transfer reactions in the MALDI plume. Copyright ? 2012 John Wiley & Sons, Ltd.     

EPR Study of Ligand Effects in Spin Triads of Bis(o-Semiquinonato)Copper(II) Complexes

We report the X-band (9 GHz) electron paramagnetic resonance (EPR) study of series of bis(o-semiquinonato)copper(II) complexes with different ligands. It was found previously, that exchange interactions in spin triads of these compounds are very sensitive to the structure of the ligand coordinated to the central copper(II) ion. Ligand moderates the copper–radical and radical–radical exchange interactions and strongly changes the magnetic properties of the compound. Depending on a ligand, ferromagnetic or antiferromagnetic character of exchange dominates in the system. The EPR study of these complexes allowed us to obtain information on zero-field splitting parameters and their distributions in the studied compounds. The EPR results compliment previously obtained spectroscopic data on these compounds and suggest the pronounced plasticity of the clusters manifested in the broad distributions of their rhombicity parameters.     

Antisite defects in plastically-deformed GaAs: An alternative analysis

A revision is presented of the accepted view that the observed increease in electron paramagnetic resonance (EPR) with plastic deformation in GaAs is due to the generation of As antisite defects. It is proposed instead that only compensating deep acceptor defects are generated. The increase of the EPR signal from As antisites can then be attributed to the compensation of the neutral As antisites that were already present in the as-grown GaAs. The nonquenching extrinsic absorption can be attributed to the acceptors. The same analysis suggests that in the case of electron and neutron irradiation, both acceptors and As antisites are generated. These proposals succeed in eliminating some recently imposed complexities in the relationship between As antisites and EL2.     

High-Pressure EPR Spectroscopy Studies of the <Emphasis Type="Italic">E. coli</Emphasis> Lipopolysaccharide Transport Proteins LptA and LptC

The use of pressure is an advantageous approach to the study of protein structure and dynamics, because it can shift the equilibrium populations of protein conformations toward higher energy states that are not of sufficient population to be observable at atmospheric pressure. Recently, the Hubbell group at the University of California, Los Angeles, reintroduced the application of high pressure to the study of proteins by electron paramagnetic resonance (EPR) spectroscopy. This methodology is possible using X-band EPR spectroscopy due to advances in pressure intensifiers, sample cells, and resonators. In addition to the commercial availability of the pressure generation and sample cells by Pressure Biosciences Inc., a five-loop–four-gap resonator required for the initial high-pressure EPR spectroscopy experiments by the Hubbell group, and those reported here, was designed by James S. Hyde and built and modified at the National Biomedical EPR Center. With these technological advances, we determined the effect of pressure on the essential periplasmic lipopolysaccharide (LPS) transport protein from Escherichia coli, LptA, and one of its binding partners, LptC. LptA unfolds from the N-terminus to the C-terminus, binding of LPS does not appreciably stabilize the protein under pressure, and monomeric LptA unfolds somewhat more readily than oligomeric LptA upon pressurization to 2 kbar. LptC exhibits a fold and relative lack of stability upon LPS binding similar to LptA, yet adopts an altered, likely monomeric, folded conformation under pressure with only its C-terminus unraveling. The pressure-induced changes likely correlate with functional changes associated with binding and transport of LPS.     

A Multifrequency (X-, Q-, and W-band) EPR and DFT Study of a Photopolymerizable Dental Resin

The free radicals generated during the polymerization process of Z100 (3 M ESPE) dental resin were examined by electron paramagnetic resonance (EPR) in X-, Q- and W-bands. Experimental generation and spectra simulations were associated with density functional theory (DFT) calculations to determine the molecular structure and explain the EPR spectrum formation. It was assumed that the EPR spectrum was formed by the sum of two different types of radicals: “propagating” and allylic. The spectra simulations and DFT calculations showed good agreement, indicating that the proposed model fully explained the nine lines of the EPR spectrum in X-band and showed that the spectrum formation is the sum of “9 + 5” lines, rather than the “5 + 4” lines predicted early. Simulations in Q- and W-bands showed very close correlation and were essential to support the proposed model.     

新型X波段多功能EPR谱仪的设计与性能

本文设计和研制了一款新型X波段多功能电子顺磁共振（electron paramagnetic resonance,EPR）谱仪,并为其开发一款新的控制和读出系统（control and readout system,CRS）来操控微波脉冲的产生和信号的采集,提高了系统的集成度和可扩展性. 该谱仪可实现常规的连续波EPR（continuous-wave EPR,cw-EPR）、脉冲EPR（pulsed EPR）和瞬态EPR（transient EPR,trEPR）实验,并装配了6~300 K的无液氦变温装置,以及兼具平行模式与垂直模式的新型双模连续波谐振腔和用于脉冲EPR及trEPR的介质腔. 针对新型EPR谱仪和新谐振腔,本文利用双模连续波、脉冲和瞬态三个不同方式的EPR实验,对其功能进行了验证.     

EPR of Spin Transitions in Complexes of Cu(hfac)2 with tert-Butylpyrazolylnitroxides

Polymer chain complexes [Cu(hfac)₂L^R] _n exhibit thermally and light-induced magnetic anomalies in many aspects similar to a spin crossover. These compounds attracted significant attention in the field of molecular magnetism and have been extensively studied by electron paramagnetic resonance (EPR) during the last several years. All compounds studied so far were based on copper(II) ions bridged by pyrazolyl-substituted nitronylnitroxides. The present work reports the first EPR study of complexes of Cu(hfac)₂ with tert-butylpyrazolylnitroxides—a new type of nitroxide ligand expected to modify exchange interaction pathways and physical properties of the crystals. The Q-band EPR spectra of three representative novel compounds are principally different from those studied previously, supporting the assumption that the magnetic motif of the compound has changed. Dominant intercluster exchange interactions are now found along the structural polymer chains. This complicates the EPR detection of phase transitions to some extent; however, theoretical modeling of the observed spectral changes allows for unambiguous assignment of different spin states and transitions between them. The magnitudes of intercluster exchange interaction were estimated to be ca. 0.1–1.5 cm^?1 for the studied compounds.     

In Vivo EPR Imaging of a Perfluoroalkyl Radical in Mouse Abdomen

Although it is thought that perfluoro-2,4-dimethyl-3-isopropyl-3-pentyl (PFR-2) is a candidate for electron paramagnetic resonance (EPR) imaging agents because of its high stability, no study has been made yet on the EPR imaging of PFR-2. In this study, EPR imaging of a phantom including PFR-2 and mice that had received PFR-2 was performed by an in vivo EPR imaging system operating at an EPR frequency of 700 MHz equipped with a bridged loop-gap resonator (inner diameter, 41 mm; axial length, 10 mm). Because PFR-2 is insoluble in water, it was dissolved in perfluorocarbon. The PFR-2 solution was put in cylindrical sample tubes with various inner diameters, and these sample tubes were placed together in a larger cylindrical sample tube filled with a physiological saline solution, which was used as a phantom. The spatial resolution was estimated to be about 3 mm on the basis of EPR imaging of the phantom. EPR images of mice that had received a PFR-2 injection via the intraperitoneal route indicated that PFR-2 remained in the peritoneal cavity even 2 days after the injection. This finding suggests that it is possible to perform EPR imaging of experimental animals using PFR-2 as an imaging agent which persists in a biological system. Authors' address: Hidekatsu Yokoyama, National Institute of Advanced Industrial Science and Technology, 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya, Aichi 463-8560, Japan     

EPR and ENDOR Investigation of Rhodosemiquinone in Bacterial Reaction Centers Formed by B-Branch Electron Transfer

In photosynthetic bacteria, light-induced electron transfer takes place in a protein called the reaction center (RC) leading to the reduction of a bound ubiquinone molecule, Q_B, coupled with proton binding from solution. We used electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) to study the magnetic properties of the protonated semiquinone, an intermediate proposed to play a role in proton coupled electron transfer to Q_B. To stabilize the protonated semiquinone state, we used a ubiquinone derivative, rhodoquinone, which as a semiquinone is more easily protonated than ubisemiquinone. To reduce this low-potential quinone we used mutant RCs modified to directly reduce the quinone in the Q_B site via B-branch electron transfer (Paddock et al. in Biochemistry 44:6920–6928, 2005). EPR and ENDOR signals were observed upon illumination of mutant RCs in the presence of rhodoquinone. The EPR signals had g values characteristic of rhodosemiquinone (g _x  = 2.0057, g _y  = 2.0048, g _z  ~ 2.0018) at pH 9.5 and were changed at pH 4.5. The ENDOR spectrum showed couplings due to solvent exchangeable protons typical of hydrogen bonds similar to, but different from, those found for ubisemiquinone. This approach should be useful in future magnetic resonance studies of the protonated semiquinone.     

Apoptosis induced by sonodynamic treatment by protoporphyrin IX on MDA-MB-231 cells

Sonodynamic therapy (SDT) is a promising modality for cancer treatment, involving the synergistic interaction of ultrasound and some chemical compounds termed as sono-sensitizers. It has been found that SDT can lead to apoptotic cell death because of the induction of direct sonochemical and subsequent redox reactions. However, the detailed mechanisms are not clear. This study was to identify the cytotoxic effects of ultrasound-activated protoporphyrin IX (PpIX) on MDA-MB-231 cells. The fluorescence microscope was used to detect the sub-cellular localization of PpIX. Several distinct sonochemical effects were found after SDT treatment, including the decrease of cell viability, generation of intracellular ROS, the loss of mitochondrial membrane potential. The activation of some special apoptosis-associated proteins [Caspase-9, Caspase-3 and polypeptide poly (ADP-robose) polymerase] was evaluated by western blotting. The results show that PpIX mediated SDT (PpIX-SDT) treatment could obviously inhibit the proliferation of MDA-MB-231 cells, and which was significantly reduced by the pan-Caspase inhibitor z-VAD-fmk and the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC). Further, SDT induced a conspicuous loss of mitochondrial membrane potential (MMP) and a mass of ROS accumulation in MDA-MB-231 cells at 1 h post-treatment and the SDT-treated cells showed obvious Caspase-3 and Caspase-9 activation, and PARP cleavage at 6 h after treatment. And, the general apoptosis marker-Caspase-3 activation-was also greatly relieved by NAC. These findings primarily indicate a Caspase-depended apoptosis could be induced by PpIX-SDT in MDA-MB-231 cells, and the intracellular ROS was involved during the apoptotic process.     

Ultra-stable temperature control in EPR experiments: thermodynamics of gel-to-liquid phase transition in spin-labeled phospholipid bilayers and bilayer perturbations by spin labels

An ultra-stable variable temperature accessory for EPR experiments with biological samples has been designed and tested. The accessory is comprised from a digitally controlled circulator bath that pumps fluid through high-efficiency aluminum radiators attached to an EPR resonator of a commercial X-band EPR spectrometer. Temperature stability of this new accessory after a 15 min re-equilibration is at least +/-0.007 K. For a standard 1-cm-long capillary sample arranged inside an EPR tube filled with silicon oil, the temperature variations do not exceed +/-0.033 K over the sample temperature range from 283 to 333 K. This new accessory has been tested by carrying out a comparative spin-labeling EPR and differential scanning calorimetry (DSC) study of the gel-to-liquid phase transition in multilamellar vesicles (MLV) composed of a synthetic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC). We demonstrate that the gel-to-liquid phase transition temperatures of MLV DMPC measured by EPR and DSC agree within +/-0.02 K experimental error even though the sample for EPR study was labeled with 1 mol% of 5PC (1-palmitoyl-2-stearoyl-(5-doxyl)-sn-glycero-3 phosphocholine). Cooperative unit number measured by EPR, N=676+/-36, was almost 50% higher than that obtained from DSC (N=458+/-18). These high values of N indicate that (i) the lipid domains should include at least several spin-labeled lipid molecules and (ii) the spin-probe 5PC molecules are not excluded into domains that are different from the bulk lipid phase as was speculated earlier. Overall, our data provide DSC and EPR evidence that in studies of the gel-to-liquid phase transition, the effect of bilayer perturbation by spin-labeled lipids is negligible and therefore thermodynamic parameters of the phase transition can be accurately measured by spin-labeling EPR. This might serve as an indication when spin-labeled molecules with structures similar to those of lipids are introduced at low concentrations, they are easily accommodated by fluid phospholipid bilayers without significant losses of the lipid cooperativity.     

基于EPR方法的天然产物抗氧化性能研究(英文)

自旋捕捉EPR、用于动物模型的低温EPR以及自旋标记EPR等多种EPR方法已经用于天然产物的抗氧化活性研究中.虽然对于抗氧化剂而言其必备条件之一就是具有体外清除自由基的能力,但是外源性抗氧化剂在生物体内抗氧化剂抗氧化能力评价的方法更有实际意义.通过对超过200种中药和一些传统中药处方的研究,已经建立并详细描述了用于评价天然产物抗氧化能力的EPR筛选方法.通过EPR方法可以获得天然产物在分子水平、细胞水平以及组织水平抗氧化能力的比较全面的评价.