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The first consistent series of mononuclear 17‐electron complexes of three Group 7 elements has been isolated in crystalline form and studied by X‐ray diffraction and spectroscopic methods. The paramagnetic compounds have a composition of [M0(CO)(CNp‐F‐ArDArF2)4] (M=Mn, Tc, Re; ArDArF2=2,6‐(3,5‐(CF3)2C6H3)2C6H2F) and are stabilized by four sterically encumbering isocyanides, which prevent the metalloradicals from dimerization. They have a square pyramidal structure with the carbonyl ligands as apexes. The frozen‐solution EPR spectra of the rhenium and technetium compounds are clearly anisotropic with large 99Tc and 185,187Re hyperfine interactions for one component. High‐field EPR (Q band and W band) has been applied for the elucidation of the EPR parameters of the manganese(0) complex. [ABSTRACT FROM AUTHOR]

In this review on advanced biomolecular EPR spectroscopy, which addresses both the EPR and NMR communities, considerable emphasis is put on delineating the complementarity of NMR and EPR regarding the measurement of interactions and dynamics of large molecules embedded in fluid-solution or solid-state environments. Our focus is on the characterization of protein structure, dynamics and interactions, using sophisticated EPR spectroscopy methods. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultrafast electronics for signal data handling and processing have pushed the limits of EPR spectroscopy to new horizons reaching millimeter and sub-millimeter wavelengths and 15 T Zeeman fields. Expanding traditional applications to paramagnetic systems, spin-labeling of biomolecules has become a mainstream multifrequency approach in EPR spectroscopy. In the high-frequency/high-field EPR region, sub-micromolar concentrations of nitroxide spin-labeled molecules are now sufficient to characterize reaction intermediates of complex biomolecular processes. This offers promising analytical applications in biochemistry and molecular biology where sample material is often difficult to prepare in sufficient concentration for NMR characterization. For multifrequency EPR experiments on frozen solutions typical sample volumes are of the order of 250 L (S-band), 150 L (X-band), 10 L (Q-band) and 1 L (W-band). These are orders of magnitude smaller than the sample volumes required for modern liquid- or solid-state NMR spectroscopy. An important additional advantage of EPR over NMR is the ability to detect and characterize even short-lived paramagnetic reaction intermediates (down to a lifetime of a few ns). Electron-nuclear and electron-electron double-resonance techniques such as electron-nuclear double resonance (ENDOR), ELDOR-detected NMR, PELDOR (DEER) further improve the spectroscopic selectivity for the various magnetic interactions and their evolution in the frequency and time domains. PELDOR techniques applied to frozen-solution samples of doubly spin-labeled proteins allow for molecular distance measurements ranging up to about 100 Å. For disordered frozen-solution samples high-field EPR spectroscopy allows greatly improved orientational selection of the molecules within the laboratory axes reference system by means of the anisotropic electron Zeeman interaction. Single-crystal resolution is approached at the canonical g-tensor orientations--even for molecules with very small g-anisotropies. Unique structural, functional, and dynamic information about molecular systems is thus revealed that can hardly be obtained by other analytical techniques. On the other hand, the limitation to systems with unpaired electrons means that EPR is less widely used than NMR. However, this limitation also means that EPR offers greater specificity, since ordinary chemical solvents and matrices do not give rise to EPR in contrast to NMR spectra. Thus, multifrequency EPR spectroscopy plays an important role in better understanding paramagnetic species such as organic and inorganic radicals, transition metal complexes as found in many catalysts or metalloenzymes, transient species such as light-generated spin-correlated radical pairs and triplets occurring in protein complexes of photosynthetic reaction centers, electron-transfer relays, etc. Special attention is drawn to high-field EPR experiments on photosynthetic reaction centers embedded in specific sugar matrices that enable organisms to survive extreme dryness and heat stress by adopting an anhydrobiotic state. After a more general overview on methods and applications of advanced multifrequency EPR spectroscopy, a few representative examples are reviewed to some detail in two Case Studies: (I) High-field ELDOR-detected NMR (EDNMR) as a general method for electron-nuclear hyperfine spectroscopy of nitroxide radical and transition metal containing systems; (II) High-field ENDOR and EDNMR studies of the Oxygen Evolving Complex (OEC) in Photosystem II, which performs water oxidation in photosynthesis, i.e., the light-driven splitting of water into its elemental constituents, which is one of the most important chemical reactions on Earth. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • High-Q resonator for millimeter wave EPR has been designed, modelled, and tested. • The resonator is based on forming a detect in 1D photonic band gap structure. • Nanoporous ceramics is used for flat aqueous sample holders with tunable properties. • The resonator provides a tenfold gain in concentration sensitivity vs. cylindrical cavities. Abstract High-field EPR provides significant advantages for studying structure and dynamics of molecular systems possessing an unpaired electronic spin. However, routine use of high-field EPR in biophysical research, especially for aqueous biological samples, is still facing substantial technical difficulties stemming from high dielectric millimeter wave (mmW) losses associated with non-resonant absorption by water and other polar molecules. The strong absorbance of mmW's by water also limits the penetration depth to just fractions of mm or even less, thus making fabrication of suitable sample containers rather challenging. Here we describe a radically new line of high Q-factor mmW resonators that are based on forming lattice defects in one-dimensional photonic band-gap (PBG) structures composed of low-loss ceramic discs of λ/4 in thickness and having alternating dielectric constants. A sample (either liquid or solid) is placed within the E = 0 node of the standing mm wave confined within the defect. A resonator prototype has been built and tested at 94.3 GHz. The resonator performance is enhanced by employing ceramic nanoporous membranes as flat sample holders of controllable thickness and tunable effective dielectric constant. The experimental Q-factor of an empty resonator was ≈ 420. The Q-factor decreased slightly to ≈ 370 when loaded with a water-containing nanoporous disc of 50 μm in thickness. The resonator has been tested with a number of liquid biological samples and demonstrated about tenfold gain in concentration sensitivity vs. a high-Q cylindrical TE 012 -type cavity. Detailed HFSS Ansys simulations have shown that the resonator structure could be further optimized by properly choosing the thickness of the aqueous sample and employing metallized surfaces. The PBG resonator design is readily scalable to higher mmW frequencies and is capable of accommodating significantly larger sample volumes than previously achieved with either Fabry-Perot or cylindrical resonators. [ABSTRACT FROM AUTHOR]

The g factor of paramagnetic defects in commercial high performance carbon fibers was determined by a double resonance experiment based on the Overhauser shift due to hyperfine coupled protons. Our carbon fibers exhibit a single, narrow and perfectly Lorentzian shaped ESR line and a g factor slightly higher than g free with g = 2.002644 = g free · ( 1 + 162 ppm ) with a relative uncertainty of 15 ppm . This precisely known g factor and their inertness qualify them as a high precision g factor standard for general purposes. The double resonance experiment for calibration is applicable to other potential standards with a hyperfine interaction averaged by a process with very short correlation time. [ABSTRACT FROM AUTHOR]

[Display omitted] • High-field (36 T) EPR spectrometer. • High-resolution (<1 mT) THz EPR spectroscopy. • Resolved BDPA g-tensor. • Gd spin label g-anisotropy. Electron Paramagnetic Resonance (EPR) is a powerful technique to study materials and biological samples on an atomic scale. High-field EPR in particular enables extracting very small g -anisotropies in organic radicals and half-filled 3d and 4f metal ions such as MnII (3d5) or GdIII (4f7), and resolving EPR signals from unpaired spins with very close g -values, both of which provide high-resolution details of the local atomic environment. Before the recent commissioning of the high-homogeneity Series Connected Hybrid magnet (SCH, superconducting + resistive) at the National High Magnetic Field Laboratory (NHMFL), the highest-field, high-resolution EPR spectrometer available was limited to 25 T using a purely resistive "Keck" magnet at the NHMFL. Herein, we report the first EPR experiments performed using the SCH magnet capable of reaching the field of 36 T, corresponding to an EPR frequency of 1 THz for g = 2. The magnet's intrinsic homogeneity (25 ppm, that is 0.9 mT at 36 T over 1 cm diameter, 1 cm length cylinder) was previously established by NMR. We characterized the magnet's temporal stability (5 ppm, which is 0.2 mT at 36 T over one-minute, the typical acquisition time) using 2,2-diphenyl-1-picrylhydrazyl (DPPH). This high resolution enables resolving the weak g -anisotropy of 1,3-bis(diphenylene)-2-phenylallyl (BDPA), Δ g = 2.5 × 10–4 obtained from measurements at 932 GHz and 33 T. Subsequently, we recorded EPR spectra at multiple frequencies for two GdIII complexes with potential applications as spin labels. We demonstrated a significant reduction in line broadening in Gd[DTPA], attributed to second order zero field splitting, and a resolution enhancement of g -tensor anisotropy for Gd[sTPATCN]-SL. [ABSTRACT FROM AUTHOR]

Known since the ancient times, cotton continues to be one of the essential materials for the human civilization. Cotton fibers are almost pure cellulose and contain both crystalline and amorphous nanodomains with different physicochemical properties. While understanding of interactions between the individual cellulose chains within the crystalline phase is important from a perspective of mechanical properties, studies of the amorphous phase lead to characterization of the essential transport parameters, such as solvent diffusion, dyeing, drug release, and toxin absorption, as well as more complex processes of enzymatic degradation. Here, we describe the use of spin probe electron paramagnetic resonance methods to study local polarity and heterogeneous viscosity of two types of unprocessed cotton fibers, G. hirsutum and G. barbadense, harvested in the State of North Carolina, USA. These fibers were loaded with two small molecule nitroxide probes that differ in polarity-Tempo and its more hydrophilic derivative Tempol-using a series of polar and non-polar solvents. The electron paramagnetic resonance spectra of the nitroxide-loaded cotton fibers were analyzed both semi-empirically and by least-squares simulations using a rigorous stochastic theory of electron paramagnetic resonance spectra developed by Freed and coworkers. A software package and least-squares fitting protocols were developed to carry out automatic simulations of multi-component electron paramagnetic resonance spectra in both first-derivative and the absorption forms at multiple resonance frequencies such as X-band (9.5 GHz) and W-band (94.3 GHz). The results are compared with the preceding electron paramagnetic resonance spin probe studies of a commercial bleached cotton sheeting carried out by Batchelor and coworkers. One of the results of this study is a demonstration of a co-existence of cellulose nanodomains with different physicochemical properties such as polarity and microviscosity that are affected by solvents and temperature. Spin labeling studies also revealed a macroscopic heterogeneity in the domain distribution along the cotton fibers and a critical role the cuticular layer is playing as a barrier for spin probe penetration. Finally but not lastly, the simultaneous multi-component least-squares simulation method of electron paramagnetic resonance spectra acquired at different resonant frequencies and the display forms (e.g., absorption and first-derivative displays) and the strategy of spectral parameter sharing could be potentially applicable to other heterogeneous biological systems in addition to the cotton fibers studies here. [ABSTRACT FROM AUTHOR]

In this review on advanced biomolecular EPR spectroscopy, which addresses both the EPR and NMR communities, considerable emphasis is put on delineating the complementarity of NMR and EPR regarding the measurement of interactions and dynamics of large molecules embedded in fluid-solution or solid-state environments. Our focus is on the characterization of protein structure, dynamics and interactions, using sophisticated EPR spectroscopy methods. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultrafast electronics for signal data handling and processing have pushed the limits of EPR spectroscopy to new horizons reaching millimeter and sub-millimeter wavelengths and 15 T Zeeman fields. Expanding traditional applications to paramagnetic systems, spin-labeling of biomolecules has become a mainstream multifrequency approach in EPR spectroscopy. In the high-frequency/high-field EPR region, sub-micromolar concentrations of nitroxide spin-labeled molecules are now sufficient to characterize reaction intermediates of complex biomolecular processes. This offers promising analytical applications in biochemistry and molecular biology where sample material is often difficult to prepare in sufficient concentration for NMR characterization. For multifrequency EPR experiments on frozen solutions typical sample volumes are of the order of 250 μL (S-band), 150 μL (X-band), 10 μL (Q-band) and 1 μL (W-band). These are orders of magnitude smaller than the sample volumes required for modern liquid- or solid-state NMR spectroscopy. An important additional advantage of EPR over NMR is the ability to detect and characterize even short-lived paramagnetic reaction intermediates (down to a lifetime of a few ns). Electron⁻nuclear and electron⁻electron double-resonance techniques such as electron⁻nuclear double resonance (ENDOR), ELDOR-detected NMR, PELDOR (DEER) further improve the spectroscopic selectivity for the various magnetic interactions and their evolution in the frequency and time domains. PELDOR techniques applied to frozen-solution samples of doubly spin-labeled proteins allow for molecular distance measurements ranging up to about 100 Å. For disordered frozen-solution samples high-field EPR spectroscopy allows greatly improved orientational selection of the molecules within the laboratory axes reference system by means of the anisotropic electron Zeeman interaction. Single-crystal resolution is approached at the canonical g-tensor orientations—even for molecules with very small g-anisotropies. Unique structural, functional, and dynamic information about molecular systems is thus revealed that can hardly be obtained by other analytical techniques. On the other hand, the limitation to systems with unpaired electrons means that EPR is less widely used than NMR. However, this limitation also means that EPR offers greater specificity, since ordinary chemical solvents and matrices do not give rise to EPR in contrast to NMR spectra. Thus, multifrequency EPR spectroscopy plays an important role in better understanding paramagnetic species such as organic and inorganic radicals, transition metal complexes as found in many catalysts or metalloenzymes, transient species such as light-generated spin-correlated radical pairs and triplets occurring in protein complexes of photosynthetic reaction centers, electron-transfer relays, etc. Special attention is drawn to high-field EPR experiments on photosynthetic reaction centers embedded in specific sugar matrices that enable organisms to survive extreme dryness and heat stress by adopting an anhydrobiotic state. After a more general overview on methods and applications of advanced multifrequency EPR spectroscopy, a few representative examples are reviewed to some detail in two Case Studies: (I) High-field ELDOR-detected NMR (EDNMR) as a general method for electron⁻nuclear hyperfine spectroscopy of nitroxide radical and transition metal containing systems; (II) High-field ENDOR and EDNMR studies of the Oxygen Evolving Complex (OEC) in Photosystem II, which performs water oxidation in photosynthesis, i.e., the light-driven splitting of water into its elemental constituents, which is one of the most important chemical reactions on Earth.

Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for characterizing conformational sampling and dynamics in biological macromolecules. Here we demonstrate that nitroxide spectra collected at frequencies higher than X-band (~9.5 GHz) have sensitivity to the timescale of motion sampled by highly dynamic intrinsically disordered proteins (IDPs). The 68 amino acid protein IA3, was spin-labeled at two distinct sites and a comparison of X-band, Q-band (35 GHz) and W-band (95 GHz) spectra are shown for this protein as it undergoes the helical transition chemically induced by tri-fluoroethanol. Experimental spectra at W-band showed pronounced line shape dispersion corresponding to a change in correlation time from ~0.3 ns (unstructured) to ~0.6 ns (α-helical) as indicated by comparison with simulations. Experimental and simulated spectra at X- and Q-bands showed minimal dispersion over this range, illustrating the utility of SDSL EPR at higher frequencies for characterizing structural transitions and dynamics in IDPs. [ABSTRACT FROM AUTHOR]

Highlights: [•] We have applied W-band ELDOR-detected NMR to a nitroxide radical. [•] EDNMR method is shown to be capable of characterizing heterogeneous environments. [•] Superior sensitivity of EDNMR as compared to ENDOR method is demonstrated. [Copyright &y& Elsevier]

Highlights: [•] Analysis of ASR contributions in glassy solutions for DNP-enhanced solid-state NMR. [•] High-field CW EPR measurements at the same magnetic field as MAS-DNP experiments. [•] Impact of glass rigidity on DNP efficiencies. [ABSTRACT FROM AUTHOR]

Abstract: The complex [Fe(H2O)6]SiF6 is one of the most stable and best characterized high-spin Fe(II) salts and as such, is a paradigm for the study of this important transition metal ion. We describe high-frequency and -field electron paramagnetic resonance studies of both pure [Fe(H2O)6]SiF6 and [Zn(H2O)6]SiF6 doped with 8% of Fe(II). In addition, frequency domain magnetic resonance spectroscopy was applied to these samples. High signal-to-noise, high resolution spectra were recorded which allowed an accurate determination of spin Hamiltonian parameters for Fe(II) in each of these two, related, environments. For pure [Fe(H2O)6]SiF6, the following parameters were obtained: D =+11.95(1)cm−1, E =0.658(4)cm−1, g =[2.099(4),2.151(5),1.997(3)], along with fourth-order zero-field splitting parameters: and , which are rarely obtainable by any technique. For the doped complex, D =+13.42(1)cm−1, E =0.05(1)cm−1, g =[2.25(1),2.22(1),2.23(1)]. These parameters are in good agreement with those obtained using other techniques. Ligand-field theory was used to analyze the electronic absorption data for [Fe(H2O)6]SiF6 and suggests that the ground state is 5A1, which allows successful use of a spin Hamiltonian model. Density functional theory and unrestricted Hartree–Fock calculations were performed which, in the case of latter, reproduced the spin Hamiltonian parameters very well for the doped complex. [Copyright &y& Elsevier]

Abstract: Cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2 involves reaction of a peroxide-induced Tyr385 radical with arachidonic acid (AA) to form an AA radical that reacts with O2. The potential for isomeric AA radicals and formation of an alternate tyrosyl radical at Tyr504 complicate analysis of radical intermediates. We compared the EPR spectra of PGHS-1 and -2 reacted with peroxide and AA or specifically deuterated AA in anaerobic, single-turnover experiments. With peroxide-treated PGHS-2, the carbon-centered radical observed after AA addition was consistently a pentadienyl radical; a variable wide-singlet (WS) contribution from mixture of Tyr385 and Tyr504 radicals was also present. Analogous reactions with PGHS-1 produced EPR signals consistent with varying proportions of pentadienyl and tyrosyl radicals, and two additional EPR signals. One, insensitive to oxygen exposure, is the narrow singlet tyrosyl radical with clear hyperfine features found previously in inhibitor-pretreated PGHS-1. The second type of EPR signal is a narrow singlet lacking detailed hyperfine features that disappeared upon oxygen exposure. This signal was previously ascribed to an allyl radical, but high field EPR analysis indicated that ~90% of the signal originates from a novel tyrosyl radical, with a small contribution from a carbon-centered species. The radical kinetics could be resolved by global analysis of EPR spectra of samples trapped at various times during anaerobic reaction of PGHS-1 with a mixture of peroxide and AA. The improved understanding of the dynamics of AA and tyrosyl radicals in PGHS-1 and -2 will be useful for elucidating details of the cyclooxygenase mechanism, particularly the H-transfer between tyrosyl radical and AA. [Copyright &y& Elsevier]

Abstract: We introduce atomic hydrogen trapped in an octaisobutylsilsesquioxane nanocage (H@iBuT8) as a new molecular high-precision magnetic field standard for high-field EPR spectroscopy of organic radicals and other systems with signals around g =2. Its solid-state EPR spectrum consists of two 0.2mT wide lines separated by about 51mT and centered at g ≈2. The isotropic g factor is 2.00294(3) and essentially temperature independent. The isotropic 1H hyperfine coupling constant is 1416.8(2)MHz below 70K and decreases slightly with increasing temperature to 1413.7(1)MHz at room temperature. The spectrum of the standard does not overlap with those of most organic radicals, and it can be easily prepared and is stable at room temperature. [ABSTRACT FROM AUTHOR]

Abstract: High-field electron paramagnetic resonance (HF EPR) has been employed to investigate the primary electron donor electronic structure of Blastochloris viridis heterodimer mutant reaction centers (RCs). In these mutants the amino acid substitution His(M200)Leu or His(L173)Leu eliminates a ligand to the primary electron donor, resulting in the loss of a magnesium in one of the constituent bacteriochlorophylls (BChl). Thus, the native BChl/BChl homodimer primary donor is converted into a BChl/bacteriopheophytin (BPhe) heterodimer. The heterodimer primary donor radical in chemically oxidized RCs exhibits a broadened EPR line indicating a highly asymmetric distribution of the unpaired electron over both dimer constituents. Observed triplet state EPR signals confirm localization of the excitation on the BChl half of the heterodimer primary donor. Theoretical simulation of the triplet EPR lineshapes clearly shows that, in the case of mutants, triplet states are formed by an intersystem crossing mechanism in contrast to the radical pair mechanism in wild type RCs. Photooxidation of the mutant RCs results in formation of a BPhe anion radical within the heterodimer pair. The accumulation of an intradimer BPhe anion is caused by the substantial loss of interaction between constituents of the heterodimer primary donor along with an increase in the reduction potential of the heterodimer primary donor D/D+ couple. This allows oxidation of the cytochrome even at cryogenic temperatures and reduction of each constituent of the heterodimer primary donor individually. Despite a low yield of primary donor radicals, the enhancement of the semiquinone–iron pair EPR signals in these mutants indicates the presence of kinetically viable electron donors. [Copyright &y& Elsevier]

Abstract: The reaction intermediates of reduced bovine Cytochrome c Oxidase (CcO) were trapped following its reaction with oxygen at 50μs–6ms by innovative freeze-quenching methods and studied by EPR. When the enzyme was reduced with either ascorbate or dithionite, distinct radicals were generated; X-band (9GHz) and D-band (130GHz) CW-EPR measurements support the assignments of these radicals to ascorbyl and sulfur dioxide anion radical (), respectively. The X-band spectra show a linewidth of 12G for the ascorbyl radical and 11G for the radical and an isotropic g-value of 2.005 for both species. The D-band spectra reveal clear distinctions in the g-tensors and powder patterns of the two species. The ascorbyl radical spectrum displays approximate axial symmetry with g-values of gx =2.0068, gy =2.0066, and gz =2.0023. The radical has rhombic symmetry with g-values of gx =2.0089, gy =2.0052, and gz =2.0017. When the contributions from the ascorbyl and radicals were removed, no protein-based radical on CcO could be identified in the EPR spectra. [Copyright &y& Elsevier]

Abstract: One of the most puzzling questions of manganese and iron superoxide dismutases (SODs) is what is the basis for their metal-specificity. This review summarizes our findings on the Mn(II) electronic structure of SODs and related synthetic models using high-field high-frequency electron paramagnetic resonance (HFEPR), a technique that is able to achieve a very detailed and quantitative information about the electronic structure of the Mn(II) ions. We have used HFEPR to compare eight different SODs, including iron, manganese and cambialistic proteins. This comparative approach has shown that in spite of their high structural homology each of these groups have specific spectroscopic and biochemical characteristics. This has allowed us to develop a model about how protein and metal interactions influence protein pK, inhibitor binding and the electronic structure of the manganese center. To better appreciate the thermodynamic prerequisites required for metal discriminatory SOD activity and their relationship to HFEPR spectroscopy, we review the work on synthetic model systems that functionally mimic Mn-and FeSOD. Using a single ligand framework, it was possible to obtain metal-discriminatory “activity” as well as variations in the HFEPR spectra that parallel those found in the proteins. Our results give new insights into protein-metal interactions from the perspective of the Mn(II) and new steps towards solving the puzzle of metal-specificity in SODs. [Copyright &y& Elsevier]

Abstract: The five-coordinate mono-halide mononuclear Zn(II) complexes [Zn(tpa)X]+ (tpa=tris(2-pyridylmethyl)amine; X=I ([Zn(tpa)I]I; 1a), Br ([Zn(tpa)Br](ZnBr4)0.5; 2a) and Cl ([Zn(tpa)Cl](ZnCl4)0.5; 3a)) and the six-coordinate mononuclear complex [Zn(tpa)(NCS)2] (4a) have been synthesized and characterized by X-ray crystallography. The [Zn(tpa)X]+ complexes doped with the corresponding [Mn(tpa)X2] complexes (X=I (1b), Br (2b) and Cl (3b)) have been synthesized and their electronic properties investigated by multifrequency high field EPR (HF-EPR) (95–285GHz). The magnetically diluted conditions allow the determination of the hyperfine coupling constant A (∣A∣=68.10−4 cm−1 for 1b–3b). The zero-field splitting parameters (D and E) found for 1b–3b are comparable to those found for neat samples of the [Mn(tpa)X2] complexes (1b: ∣D∣=0.635cm−1, ∣E/D∣=0.189; 2b: ∣D∣=0.360cm−1, ∣E/D∣=0.192; 3b: ∣D∣=0.115cm−1, ∣E/D∣=0.200). The efficacy of using multifrequency EPR under dilute conditions to precisely determine spin Hamiltonian parameters is discussed. [Copyright &y& Elsevier]

Abstract: Orientation selection on two nitroxide-labelled shape-persistent molecules is demonstrated by high-field pulsed electron–electron double resonance experiments at a frequency of 95GHz with a commercial spectrometer. The experiments are performed with fixed observer and pump frequencies by variation of the magnetic field, so that the variation of both the dipolar frequencies and the modulation depths can be analyzed. By applying the deadtime-free four-pulse double electron–electron resonance (DEER) sequence, the lineshapes of the dipolar spectra are obtained. In the investigated linear biradical and equilateral triradical the nitroxide labels undergo restricted dynamics, so that their relative orientations are not fixed, but are correlated to some extent. In this situation, the general dependence of the dipolar spectra on the observer field can be satisfyingly modelled by simple geometrical models that involve only one rotational degree of freedom for the biradical and two rotational degrees of freedom for the triradical. A somewhat better agreement of the dipolar lineshapes for the biradical is obtained by simulations based on a molecular dynamics trajectory. For the triradical, small but significant deviations of the lineshape are observed with both models, indicating that the technique can reveal deficiencies in modelling of the conformational ensemble of a macromolecule. [Copyright &y& Elsevier]

Abstract: We describe a field-lock/field-sweep system for the use in superconducting magnets. The system is based on a commercially available field mapping unit and a custom designed broad-band 1H NMR probe. The NMR signal of a small water sample is used in a feedback loop to set and control the magnetic field to high accuracy. The current instrumental configuration allows field sweeps of ±0.4T and a resolution of up to 10−5 T (0.1G) and the performance of the system is demonstrated in a high-field electron paramagnetic resonance (EPR) application. The system should also be of utility in other experiments requiring precise and reproducible sweeps of the magnetic field such as DNP, ENDOR or PELDOR. [Copyright &y& Elsevier]

Abstract: The polarity of protein surfaces is one of the factors driving protein–protein interactions. High-field, spin-label EPR at 95GHz, i.e., 10 times higher than conventional EPR, is an upcoming technique to determine polarity parameters of the inside of proteins. Here we show that by 275GHz EPR even the small polarity differences of sites at the protein surface can be discriminated. To do so, four single cysteine mutations were introduced at surface sites (positions 12, 27, 42, and 118) of azurin and spin labeled. By 275GHz EPR in frozen solution, polarity/proticity differences between all four sites can be resolved, which is impossible by 95GHz EPR. In addition, by 275GHz EPR, two spectral components are observed for all mutants. The difference between them corresponds to one additional hydrogen bond. [Copyright &y& Elsevier]

Abstract: The reactive intermediates formed in the catalase-peroxidase from Synechocystis PCC6803 upon reaction with peroxyacetic acid, and in the absence of peroxidase substrates, are the oxoferryl-porphyrin radical and two subsequent protein-based radicals that we have previously assigned to a tyrosyl (Tyr ) and tryptophanyl (Trp ) radicals by using multifrequency Electron Paramagnetic Resonance (EPR) spectroscopy combined with deuterium labeling and site-directed mutagenesis. In this work, we have further investigated the Trp in order to identify the site for the tryptophanyl radical formation, among the 26 Trp residues of the enzyme and to possibly understand the protein constraints that determine the selective formation of this radical. Based on our previous findings about the absence of the Trp intermediate in four of the Synechocystis catalase-peroxidase variants on the heme distal side (W122F, W106A, H123Q, and R119A) we constructed new variants on Trp122 and Trp106 positions. Trp122 is very close to the iron on the heme distal side while Trp106 belongs to a short stretch (11 amino acid residues on the enzyme surface) that is highly conserved in catalase-peroxidases. We have used EPR spectroscopy to characterize the changes on the heme microenvironment induced by these mutations as well as the chemical nature of the radicals formed in each variant. Our findings identify Trp106 as the tryptophanyl radical site in Synechocystis catalase-peroxidase. The W122H and W106Y variants were specially designed to mimic the hydrogen-bond interactions of the naturally occurring Trp residues. These variants clearly demonstrated the important role of the extensive hydrogen-bonding network of the heme distal side, in the formation of the tryptophanyl radical. Moreover, the fact that W106Y is the only Synechocystis catalase-peroxidase variant of the distal heme side that recovers a catalase activity comparable to the WT enzyme, strongly indicates that the integrity of the extensive hydrogen-bonding network is also essential for the catalatic activity of the enzyme. [Copyright &y& Elsevier]

This short review compiles high-field electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies on different intermediate amino acid radicals, which emerge in wild-type and mutant class I ribonucleotide reductase (RNR) both in the reaction of protein subunit R2 with molecular oxygen, which generates the essential tyrosyl radical, and in the catalytic reaction, which involves a radical transfer between subunits R2 and R1. Recent examples are presented, how different amino acid radicals (tyrosyl, tryptophan, and different cysteine-based radicals) were identified, assigned to a specific residue, and their interactions, in particular hydrogen bonding, were investigated using high-field EPR and ENDOR spectroscopy. Thereby, unexpected diiron-radical centers, which emerge in mutants of R2 with changed iron coordination, and an important catalytic cysteine-based intermediate in the substrate turnover reaction in R1 were identified and characterized. Experiments on the essential tyrosyl radical in R2 single crystals revealed the so far unknown conformational changes induced by formation of the radical. Interesting structural differences between the tyrosyl radicals of class Ia and Ib enzymes were revealed. Recently accurate distances between the tyrosyl radicals in the protein dimer R2 could be determined using pulsed electron–electron double resonance (PELDOR), providing a new tool for docking studies of protein subunits. These studies show that high-field EPR and ENDOR are important tools for the identification and investigation of radical intermediates, which contributed significantly to the current understanding of the reaction mechanism of class I RNR. [Copyright &y& Elsevier]

Two current frontiers in EPR research are high-field (v0 70 GHz, B0 > 2:5 T) electron paramagnetic resonance (EPR) and high-field electron-nuclear double resonance (ENDOR). This review focuses on recent advances in high-field ENDOR and its applications to the study of proteins containing native paramagnetic sites. It concentrates on two aspects; the first concerns the determination of the location of protons and is related to the site geometry, and the second focuses on the spin density distribution within the site, which is inherent to the electronic structure. Both spin density and proton locations can be derived from ligand hyperfine couplings determined by ENDOR measurements. A brief description of the experimental methods is presented along with a discussion of the advantages and disadvantages of high-field ENDOR compared with conventional X-band (∼ 9:5 GHz) experiments. Specific examples of both protein single crystals and frozen solutions are then presented. These include the determination of the coordinates of water ligand protons in the Mn(II) site of concanavalin A, the detection of hydrogen bonds in a quinone radical in the bacterial photosynthetic reaction center as well as in the tyrosyl radical in ribonuclease reductase, and the study of the spin distribution in copper proteins. The copper proteins discussed are the type I copper of azurin and the binuclear CuA center in a number of proteins. The last part of the review presents a brief discussion of the interpretation of hyperfine couplings using quantum chemical calculations, primarily density functional theory (DFT) methods. Such methods are becoming an integral part of the data analysis tools, as they can facilitate signal assignment and provide the ultimate relation between the experimental hyperfine couplings and the electronic wave function. [ABSTRACT FROM AUTHOR]

In this communication we present a complimentary Mössbauer- and EPR-study of the time dependance of the reaction of substrate free P450cam with peracetic acid within a time region ranging from 8 ms up to 5 min. An Fe(IV) species as well as a tyrosyl radical residing on the amino acid residue Tyr96 have been identified as reaction intermediates. These species possibly are formed by the reduction of compound I by means of transferring an electron from Tyr 96 to the heme moiety. [ABSTRACT FROM AUTHOR]

The frozen solution electron paramagnetic resonance spectrum of the primary donor cation P+⋅ in reaction centers of site-directed mutants of Rhodobacter (Rb.) sphaeroides has been obtained at a microwave frequency ν=360 GHz and a magnetic field B0=12.8 T. Due to the high Zeeman resolution of the powder pattern, all three principal components of the rhombic g-tensors at T=160 K could be determined with high accuracy. We compare spectra of the site-directed mutants, in which the axial ligand histidine M202 of the primary donor is replaced by glutamic acid (HE(M202)) or leucine (HL(M202)), with those of the strain R26, whose primary donor is similar to that of the wild type and only lacks the carotenoid. For HE(M202), this is the first determination of its g-tensor with the principal components gxx=2.00335(3), gyy=2.00236(2) and gzz=2.00191(2). While in R26 the primary donor is a bacteriochlorophyll a dimer, the HL(M202) and HE(M202) mutants have previously been shown to be bacteriochlorophyll:bacteriopheophytin heterodimers. Their g-tensor anisotropy Δg=gxx−gzz shows significant variations in opposite directions when compared with R26, with an increased anisotropy for HE(M202) and a decreased one for HL(M202). Calculations employing Density Functional Theory suggest that the observed shifts originate in different torsional angles of the acetyl group attached to the spin-carrying bacteriochlorophyll half L of the dimer. [Copyright &y& Elsevier]

Recent high-field EPR studies have revealed important differences between the g-matrices of the photoexcited triplet and the cation radical states of the primary donor P in bacterial photosynthetic reaction centers. One of the more unusual differences is the temperature dependence in the g-tensor of the triplet state 3P, which contrasts with the temperature independence that has been observed in the g-tensor of the cation state P+. This difference leads us to suggest that intersystem (singlet–triplet) spin–orbit coupling could play a significant role in determining the electronic structure of the triplet state, 3P. Such coupling may also be important for explaining other poorly understood characteristics of 3P, including the temperature dependence of its zero-field splitting D, and the reduction of D relative to the triplet state in monomeric bacteriochlorophyll. We present a survey of ZFS data from a wide variety of bacteriochlorophyll species that supports this idea. Implications of this model for the electronic structure of the primary donor are discussed. [Copyright &y& Elsevier]

The g-matrix of photosynthetic pigments has been studied in the last decade due to the advent of high-field EPR techniques. It can be observed when the spectral splitting of the principal g-factor components is larger than the linewidth due to unresolved hyperfine splitting and if there is no g-strain obscuring it. For large organic molecules such as the primary electron donor in photosynthetic reaction centers (RC) this usually requires fields above 11 T, or, for fields between 3 and 11 T, full deuteration and/or single crystal work. Here we present for the first time the fully resolved rhombic EPR spectrum of the primary donor of Blastochloris viridis (formerly called Rhodopseudomonas viridis), a purple photosynthetic bacterium containing bacteriochlorophyll b. As was the case for Rhodobacter sphaeroides, g-strain is negligible for this radical up to a field of 24 T. The temperature dependence of the g-anisotropy is presented and compared with that of the bacteriochlorophyll a-containing Rb. sphaeroides and plant photosystem I. A slight shift in the principal components of the g-matrix is observed at temperatures below 70 K, where it becomes more axial. The experimental work is complemented with theoretical calculations for g using the semi-empirical INDO/S method as implemented in the program ZINDO. The theoretical results generally agree well with the experiment. This indicates that a satisfactory description of the anisotropic g-tensor for radical cations of large planar molecules like the chlorophylls as well as their aggregates, e.g., reaction center primary donor special pairs, is possible with this relatively cheap semi-empirical approach. [Copyright &y& Elsevier]

Low-temperature EPR spectroscopy with frequencies between 95 and 345 GHz and magnetic fields up to 12 T has been used to study metal sites in proteins or inorganic complexes and free radicals. The high-field EPR method was used to resolve g-value anisotropy by separating it from overlapping hyperfine couplings. The presence of hydrogen bonding interactions to the tyrosyl radical oxygens in ribonucleotide reductases were detected. At 285 GHz the g-value anisotropy from the rhombic type 2 Cu(II) signal in the enzyme laccase has its g-value anisotropy clearly resolved from slightly different overlapping axial species. Simple metal site systems with S>½ undergo a zero-field splitting, which can be described by the spin Hamiltonian Hs = βSgB+D[Sz2 - S(S +1)/3+(E/D)(Sx2-Sy2]. From high-frequency EPR, the D values that are small compared to the frequency (high-field limit) can be determined directly by measuring the distance of the outermost signal to the center of the spectrum, which corresponds to (2S-)∗ &verbar;D&verbar;. For example, D values of 0.8 and 0.3 cm-1 are observed for S=5/2 Fe(III)-EDTA and transferrin, respectively. When D values are larger compared to the frequency and in the case of half-integer spin systems, they can be obtained from the frequency dependence of the shifts of geff, as observed for myoglobin in the presence (D=5 cm-1) or absence (D=9.5 cm-1) of fluoride. The 285 and 345 GHz spectra of the Fe(II)-NO-EDTA complex show that it is best described as a S=3/2 system with D=11.5 cm)1, E=0.1 cm-1, and gx=gy=gz=2.0. Finally, the effects of HF-EPR on X-band EPR silent states and weak magnetic interactions are demonstrated. [ABSTRACT FROM AUTHOR]

A three-pulse stimulated ESE at 130 GHz (D-band) of nitroxide biradical in a molecular glass shows envelope modulation (ESEEM) when the time separation between the first and the second pulses is varied. This ESEEM originates from relaxation-induced flips of the spin partner during the mixing period between the second and third pulses. These flips alternates the local dipolar field in which the resonant spins precess before second and after third pulses. The ESEEM is different for different spectral positions, due to orientation selectivity. For toluene glassy solution at 35 K Fourier transform shows pronounced peak reflecting singularity of the Pake resonance pattern. Increasing temperature up to 80 K results in appearance of a strong additional peak ascribed to alteration of the resonance field induced by methyl group reorientation. [Copyright &y& Elsevier]

Chemical oxidation of the chlorosomes from Chloroflexus aurantiacus and Chlorobium tepidum green bacteria produces bacteriochlorophyll radicals, which are characterized by an anomalously narrow EPR signal compared to in vitro monomeric BChl c.+ [Van Noort PI, Zhu Y, LoBrutto R and Blankenship RE (1997) Biophys J 72: 316–325]. We have performed oxidant concentration and temperature-dependent X-band EPR measurements in order to elucidate the line narrowing mechanism. The linewidth decreases as the oxidant concentration is increased only for Chloroflexus indicating that for this system Heisenberg spin exchange is at least partially responsible for the EPR spectra narrowing. For both species the linewidth is decreasing on increasing the temperature. This indicates that temperature-activated electron transfer is the main narrowing mechanism for BChl radicals in chlorosomes. The extent of the electron transfer process among different BChl molecules has been evaluated and a comparison between the two species representative of the two green bacteria families has been made. In parallel, high frequency EPR experiments have been performed on the oxidized chlorosomes of Chloroflexus and Chlorobium at 110 and 330 GHz in the full temperature range investigated at X-band. The g-tensor components obtained from the simulation of the 330 GHz EPR spectrum from Chlorobium show the same anisotropy as those of monomeric Chl a.+ [Bratt PJ, Poluektov OG, Thurnauer MC, Krzystek J, Brunel LC, Schrier J, Hsiao YW, Zerner M and Angerhofer A (2000) J Phys Chem B 104: 6973–6977]. The spectrum of Chloroflexus has a nearly axial g-tensor with reduced anisotropy compared to Chlorobium and monomeric Chl a in vitro. g-tensor values and temperature dependence of the linewidth have been discussed in terms of the differences in the local structure of the chlorosomes of the two families. [ABSTRACT FROM AUTHOR]

[Display omitted] • Resolution of 19F chemical shift anisotropies (CSA) in ENDOR spectra at 9.4 T. • CSA reveals the orientation of 19F moieties and rotation of CF 3 groups. • CSA determined from analysis of ENDOR spectra. • Code of simulation routine is made available. Pulsed 19F ENDOR spectroscopy provides a selective method for measuring angstrom to nanometer distances in structural biology. Here, the performance of 19F ENDOR at fields of 3.4 T and 9.4 T is compared using model compounds containing one to three 19F atoms. CF 3 groups are included in two compounds, for which the possible occurrence of uniaxial rotation might affect the distance distribution. At 9.4 T, pronounced asymmetric features are observed in many of the presented 19F ENDOR spectra. Data analysis by spectral simulations shows that these features arise from the chemical shift anisotropy (CSA) of the 19F nuclei. This asymmetry is also observed at 3.4 T, albeit to a much smaller extent, confirming the physical origin of the effect. The CSA parameters are well consistent with DFT predicted values and can be extracted from simulation of the experimental data in favourable cases, thereby providing additional information about the geometrical and electronic structure of the spin system. The feasibility of resolving the CSA at 9.4 T provides important information for the interpretation of line broadening in ENDOR spectra also at lower fields, which is relevant for developing methods to extract distance distributions from 19F ENDOR spectra. [ABSTRACT FROM AUTHOR]

• A new dinuclear and five new mononuclear carboxylate complexes with metronidazole have been synthesized. • All compounds were structurally determined by single crystal X-ray diffraction. • Some of the complexes were examined with High Field EPR. • The Hirshfeld surface analysis showed the intermolecular interactions in all the complexes. • The antimicrobial activity studies of the synthesized complexes were examined. A dinuclear paddle-wheel complex [Cu 2 (PTA) 4 (mnz) 2 ] (1) and mononuclear [M(BBA) 2 (mnz) 2 (H 2 O) 2 ] complexes, where PTA = p -methylbenzoate, BBA = 2-bromobenzoate, mnz = metronidazole and M = Cu(II) (2), Co(II) (3), Ni(II) (4), Zn(II) (5), Cd(II) (6) were prepared by adding metronidazole to the mixture of divalent metal nitrate salts and arylcarboxylate acid in alkaline aqueous solution under ambient conditions. The complexes were characterized by elemental analysis, spectroscopy (FTIR, UV–visible and NIR-Vis-UV), single crystal X-ray diffraction (SCXRD), Hirshfeld surface analysis and High-Field EPR. The asymmetry of the d − d bands observed in the electronic diffuse − reflectance spectra of cobalt(II) and nickel(II) complexes is due to the symmetry lowering from O h to elongated D 4h in trans − [MN 2 O 2 O 2 ′] chromophores. Large zero-field splitting was observed in the HF EPR spectra of the Ni(II) and Co(II) complexes which was modeled by using the CASSCF method. The antimicrobial activity was studied using the agar well diffusion method. The antimicrobial tests of these new compounds were carried out on Candida albicans and five bacterial strains (three Gram-negative and two Gram-positive), and were compared to their respective parent ligands. A broad antimicrobial spectrum was observed in some of the complexes. [ABSTRACT FROM AUTHOR]

• DEER theory is elaborated using spherical harmonic expansions. • DEER spectra consist of individual modified Pake pattern components. • Conformational distribution suppresses higher degree components. • Theory enables model-based simulations of DEER data with orientation selection. • Theory enables model-free analysis of DEER data with orientation selection. Double electron–electron resonance (DEER) and other pulse electron paramagnetic resonance (EPR) techniques are valuable tools for determining distances between paramagnetic centres. DEER theory is well developed for a scenario where relative orientations of paramagnetic centres do not affect the DEER data. In particular, such theory enables a number of approaches for extracting distance distributions. However, in a more general case, when orientation selection effects become substantial, the analytical theory of DEER is less well developed, therefore quite commonly researchers rely on a comparison of some model-based simulations with experimental data. This work elaborates the theory of DEER with orientation selection effects, focusing on a scenario of a moderate conformational disorder, leading to an orientation distribution in a pair of paramagnetic centres. The analytical treatment based on expansions into spherical harmonics, provides important insights into the structure of DEER data. As follows from this treatment, DEER spectra with orientation selection can be represented as a linear combination of modified Pake pattern (MPP) components. The conformational disorder has a filtering effect on the weights of MPP components, specifically by significantly suppressing MPP components of higher degrees. The developed theory provides a pathway for model-based simulations of DEER data where orientation distribution is defined by analytical functions with parameters. The theory based on spherical harmonics expansions was also applied to develop an iterative processing algorithm based on Tikhonov regularization, which disentangles the distance and orientation information in a model-free manner. As an input, this procedure takes several DEER datasets measured at various positions of an EPR line, and outputs a distance distribution and orientation distribution information encoded in a set of coefficients related to the weights of MPP components. The model-based and model-free approaches based on the developed theory were validated for a nitroxide biradical and a spin-labelled protein. [ABSTRACT FROM AUTHOR]

We present a novel approach to study transient protein-protein complexes with standard, 9 GHz, and high-field, 95 GHz, electron paramagnetic resonance (EPR) and paramagnetic NMR at ambient temperatures and in solution. We apply it to the complex of yeast mitochondrial iso-1-cytochrome c (Cc) with cytochrome c peroxidase (CcP) with the spin label [1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate] attached at position 81 of Cc (SL-Cc). A dissociation constant K D of 20±4×10 -6  M (EPR and NMR) and an equal amount of stereo-specific and encounter complex (NMR) are found. The EPR spectrum of the fully bound complex reveals that the encounter complex has a significant population (60 %) that shares important features, such as the Cc-interaction surface, with the stereo-specific complex., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Applications of distance measurements by pulse dipolar electron-paramagnetic resonance (PD-EPR) spectroscopy to structural biology are based on introducing spin labels (SLs) at well-defined locations in the biomacromolecule. The most commonly used SLs are nitroxyl radicals, but recently SLs based on high-spin Gd(3+) (S=7/2) complexes have been shown to be an attractive alternative for PD-EPR, particularly double electron-electron resonance (DEER), at spectrometer frequencies higher than 30 GHz. In this chapter, we describe the advantage of using this new family of SLs in terms of sensitivity, stability, and chemical diversity. We present current labeling strategies for proteins, discuss the approximations under which DEER data analysis of a pair of Gd(3+) SLs (GdSLs) is equivalent to that of a pair of S=1/2 SLs, and discuss the reduction in multispin effects in a cluster of GdSLs, as opposed to a cluster of nitroxide labels, which can be found in oligomeric systems. In addition, we provide a brief overview of the current, rather limited, knowledge of Gd(3+) phase relaxation behavior and describe experimental strategies in terms of optimizing sensitivity. The possibility of using several types of SLs in a system allows one to isolate effects due to the chemical nature of the SL itself; several such examples are presented, focusing on comparing nitroxide and GdSLs. Finally, we will discuss the initial results on in-cell DEER with GdSLs., (© 2015 Elsevier Inc. All rights reserved.)