Pulsed Electron Paramagnetic Resonance Methods Research Articles

Electronic Structure of a Diiron Complex: A Multitechnique Experimental Study of [(dppf)Fe(CO) 3]+/0.

Here we explore the electronic structure of the diiron complex [(dppf)Fe(CO)3]0/+ [10/+; dppf = 1,1'-bis(diphenylphosphino)ferrocene] in two oxidation states by an advanced multitechnique experimental approach. A combination of magnetic circular dichroism, X-ray absorption and emission, high-frequency electron paramagnetic resonance (EPR), and Mössbauer spectroscopies is used to establish that oxidation of 10 occurs on the carbonyl iron ion, resulting in a low-spin iron(I) ion. It is shown that an unequivocal result is obtained by combining several methods. Compound 1+ displays slow spin dynamics, which is used here to study its geometric structure by means of pulsed EPR methods. Surprisingly, these data show an association of the tetrakis[3,5-bis(trifluoromethylphenyl)]borate counterion with 1+.

Epr Spectroscopy of Adsorbed Spin-Labeled Peptides: Immobilization of Trichogin on the Titanium Oxide

The TOAC-spin-labeled peptide Trichogin GA IV adsorbed on the TiO2 surface is studied. It is shown that the continuous wave (CW) electron paramagnetic resonance (EPR) spectrum does not depend on temperature in a wide range of 77–300 K. A pulsed EPR method of electron spin echo (ESE) utilizing a two-pulse sequence (π/2-τ-π) is used to study temperature dependence of the phase relaxation time, TF. The TF values are found to change from 750 ns to 100 ns in the interval of 77–300 K. The pulsed electronelectron double resonance (PELDOR) measurements utilizing the pulse sequence((π/2)A,-T-πB,-(τ-T)-πA) show that the space distribution of spin labels on the surface remains uniform irrespective of the temperature, and provide the fractal dimension of the surface of 2.7±0.1. The obtained results testify that EPR pulse experiments can be used to study adsorbed spin-labeled molecules at room temperatures, i.e. not only at cryogenic temperatures.

EPR Spectroscopy of Impurity Thulium Ions in Yttrium Orthosilicate Single Crystals

The frequency-field and orientation dependences of the electron paramagnetic resonance (EPR) spectra are measured for impurity Tm3+ ions in yttrium orthosilicate (Y2SiO5) single crystals by stationary EPR spectroscopy in the frequency range of 50–100 GHz at 4.2 K. The position of the impurity ion in the crystal lattice and its magnetic characteristics are determined. The temperature dependences of the spin–lattice and phase relaxation times are measured by pulse EPR methods in the temperature range of 5–15 K and the high efficiency of the direct single-phonon mechanism of spin–lattice relaxation is established. This greatly shortens the spin–lattice relaxation time at low temperatures and makes impurity Tm3+ ions in Y2SiO5 a promising basis for the implementation of high-speed quantum memory based on rare-earth ions in dielectric crystals.

Localization of Guest Molecules in Nanopores by Pulsed EPR Spectroscopy

The localization of guest molecules at the molecular scale in mesoporous host materials is crucial for applications in heterogeneous catalysis, chromatography, drug delivery, and in different biomedical applications. Here, we present for the first time the precise localization of different guest molecules inside the mesoporous organosilica material UKON2a with a pore size of 6 nm. We exploited paramagnetic probe molecules 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (4-amino-TEMPO) and 4-carboxy-TEMPO in combination with a deuteration strategy. Applying a complementary set of different pulsed electron paramagnetic resonance methods, we obtained information about the dimensionality of the spatial distribution and local concentration via double electron–electron resonance experiments, orientation of the guest molecules with respect to the pore walls via electron spin echo envelope modulation spectroscopy, and about the distance between guest molecules and pore walls via electron nuclear double resonance spe...

EPR Study of Sc2SiO5:Nd143 Isotopically Pure Impurity Crystals

The Sc2SiO5 single crystals doped with 0.001 at.% of the 143Nd3+ ion were studied by continuous-wave and pulse electron paramagnetic resonance methods. The g-tensors and hyperfine structure tensors for two magnetically non-equivalent Nd ions were obtained. The spin–spin and spin–lattice relaxation times were measured at 9.82 GHz in the temperature range from 4 to 10 K. It was established that three relaxation processes contribute to the spin–lattice relaxation processes. There are one-phonon spin–phonon interaction, two-phonon Raman interaction and two-phonon Orbach–Aminov relaxation processes. It was established that spin–spin relaxation time is of the same magnitude for neodymium ion doped in Sc2SiO5 and in Y2SiO5.

Using Hyperfine Electron Paramagnetic Resonance Spectroscopy to Define the Proton-Coupled Electron Transfer Reaction at Fe-S Cluster N2 in Respiratory Complex I.

Energy-transducing respiratory complex I (NADH:ubiquinone oxidoreductase) is one of the largest and most complicated enzymes in mammalian cells. Here, we used hyperfine electron paramagnetic resonance (EPR) spectroscopic methods, combined with site-directed mutagenesis, to determine the mechanism of a single proton-coupled electron transfer reaction at one of eight iron-sulfur clusters in complex I, [4Fe-4S] cluster N2. N2 is the terminal cluster of the enzyme's intramolecular electron-transfer chain and the electron donor to ubiquinone. Because of its position and pH-dependent reduction potential, N2 has long been considered a candidate for the elusive "energy-coupling" site in complex I at which energy generated by the redox reaction is used to initiate proton translocation. Here, we used hyperfine sublevel correlation (HYSCORE) spectroscopy, including relaxation-filtered hyperfine and single-matched resonance transfer (SMART) HYSCORE, to detect two weakly coupled exchangeable protons near N2. We assign the larger coupling with A(1H) = [-3.0, -3.0, 8.7] MHz to the exchangeable proton of a conserved histidine and conclude that the histidine is hydrogen-bonded to N2, tuning its reduction potential. The histidine protonation state responds to the cluster oxidation state, but the two are not coupled sufficiently strongly to catalyze a stoichiometric and efficient energy transduction reaction. We thus exclude cluster N2, despite its proton-coupled electron transfer chemistry, as the energy-coupling site in complex I. Our work demonstrates the capability of pulse EPR methods for providing detailed information on the properties of individual protons in even the most challenging of energy-converting enzymes.

Vanadyl as a Stable Structural Mimic of Reactive Ferryl Intermediates in Mononuclear Nonheme-Iron Enzymes

The iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases catalyze an array of challenging transformations via a common iron(IV)-oxo (ferryl) intermediate, which in most cases abstracts hydrogen (H•) from an aliphatic carbon of the substrate. Although it has been shown that the relative disposition of the Fe-O and C-H bonds can control the rate of H• abstraction and fate of the resultant substrate radical, there remains a paucity of structural information on the actual ferryl states, owing to their high reactivity. We demonstrate here that the stable vanadyl ion [(VIV-oxo)2+] binds along with 2OG or its decarboxylation product, succinate, in the active site of two different Fe/2OG enzymes to faithfully mimic their transient ferryl states. Both ferryl and vanadyl complexes of the Fe/2OG halogenase, SyrB2, remain stably bound to its carrier protein substrate (l-aminoacyl-S-SyrB1), whereas the corresponding complexes harboring transition metals (Fe, Mn) in lower oxidation states dissociate. In the well-studied taurine:2OG dioxygenase (TauD), the disposition of the substrate C-H bond relative to the vanadyl ion defined by pulse electron paramagnetic resonance methods is consistent with the crystal structure of the reactant complex and computational models of the ferryl state. Vanadyl substitution may thus afford access to structural details of the key ferryl intermediates in this important enzyme class.

Photoexcited Triplet State Kinetics Studied by Electron Paramagnetic Resonance Spectroscopy

Following the first evidence of the triplet character of the optically excited phosphorescent state of naphthalene by Hutchison and Mangum in 1958, electron paramagnetic resonance (EPR) spectroscopy has become widely used to study and understand the properties and kinetic characteristics of excited triplet states. This minireview gives an overview over EPR techniques based on continuous microwave methods using lock-in or direct detection as well as pulsed EPR methods with respect to their suitability for kinetic studies of excited triplet states. A short historical overview of the experimental and theoretical developments in this field of research as well as of the triplet systems studied, with a final focus on fullerenes, is given. This may help newcomers to the field as a guide to the relevant literature.

Triarylmethyl Radicals: EPR Study of 13C Hyperfine Coupling Constants.

Triarylmethyl (TAM) radicals are widely used in Electron Paramagnetic Resonance (EPR) spectroscopy as spin labels and in EPR imaging as spin probes for in vivo oxymetry. One of the key advantages of TAMs is extremely narrow EPR line, especially in case of deuterated analogues (~5 μT). Another advantage is their slow spin relaxation even at physiological temperatures allowing, in particular, application of pulsed dipolar EPR methods for distance measurements in biomolecules. In this paper a large series of TAM radicals and their deuterated analogues is synthesized, and corresponding spectroscopic parameters including 13C hyperfine constants are obtained for the first time. The negligible dependence of 13C hyperfine constants on solvent, as well as on structure and number of substituents at para-C atoms of aromatic rings, has been found. In addition, we have demonstrated that 13C signals at natural abundance can be employed for successful room-temperature distance measurements using Pulsed Electron Double Resonance (PELDOR or DEER).

Water accessibility in a membrane-inserting peptide comparing Overhauser DNP and pulse EPR methods.

Water accessibility is a key parameter for the understanding of the structure of biomolecules, especially membrane proteins. Several experimental techniques based on the combination of electron paramagnetic resonance (EPR) spectroscopy with site-directed spin labeling are currently available. Among those, we compare relaxation time measurements and electron spin echo envelope modulation (ESEEM) experiments using pulse EPR with Overhauser dynamic nuclear polarization (DNP) at X-band frequency and a magnetic field of 0.33 T. Overhauser DNP transfers the electron spin polarization to nuclear spins via cross-relaxation. The change in the intensity of the (1)H NMR spectrum of H2O at a Larmor frequency of 14 MHz under a continuous-wave microwave irradiation of the nitroxide spin label contains information on the water accessibility of the labeled site. As a model system for a membrane protein, we use the hydrophobic α-helical peptide WALP23 in unilamellar liposomes of DOPC. Water accessibility measurements with all techniques are conducted for eight peptides with different spin label positions and low radical concentrations (10-20 μM). Consistently in all experiments, the water accessibility appears to be very low, even for labels positioned near the end of the helix. The best profile is obtained by Overhauser DNP, which is the only technique that succeeds in discriminating neighboring positions in WALP23. Since the concentration of the spin-labeled peptides varied, we normalized the DNP parameter ϵ, being the relative change of the NMR intensity, by the electron spin concentration, which was determined from a continuous-wave EPR spectrum.

Study of the effects of hydroxyapatite nanocrystal codoping by pulsed electron paramagnetic resonance methods

The effect of codoping of hydroxyapatite (HAP) nanocrystals with average sizes of 35 ± 15 nm during “wet” synthesis by CO 3 2− carbonate anions and Mn2+ cations on relaxation characteristics (for the times of electron spin–spin relaxation) of the NO 3 2− nitrate radical anion has been studied. By the example of HAP, it has been demonstrated that the electron paramagnetic resonance (EPR) is an efficient method for studying anion–cation (co)doping of nanoscale particles. It has been shown experimentally and by quantummechanical calculations that simultaneous introduction of several ions can be energetically more favorable than their separate inclusion. Possible codoping models have been proposed, and their energy parameters have been calculated.

Mechanism of the CuII-catalyzed benzylic oxygenation of (aryl)(heteroaryl)methanes with oxygen.

A mechanistic study of the copper-catalyzed oxidation of the methylene group of aryl(di)azinylmethanes was performed. Initial reaction rates were measured making use of in situ IR reaction monitoring and a kinetic analysis of the reaction was executed. The reaction proved to be first order in oxygen concentration. For substrate and acid concentration, saturation kinetics due to O2 mass transfer limitation were observed. The occurrence of mass transfer limitation was further confirmed by examining the effect of the stirring rate on the initial reaction rate. Interestingly, the effect of the concentration of the catalyst on the rate shows that higher loadings result in a maximal initial rate, followed initially by a steady decrease and subsequently a rate plateau when the concentration is increased further. Mass transfer limitation and increased concentration of dinuclear catalytically active species rationalizes this hitherto unprecedented rate behavior. Continuous-wave and pulsed electron paramagnetic resonance methods were used to characterize the catalytic species present in the solution during the reaction and confirmed the presence of both mono- and dinuclear copper species. Analysis of a diverse substrate scope points towards imine-enamine tautomerization as a crucial process in the oxidation reaction. DFT calculations of these equilibrium constants (pKeq) provided us with a qualitative tool to predict whether or not a substrate is viable for oxidation under the reaction conditions developed.

Characterization of a Radical Intermediate in Lipoyl Cofactor Biosynthesis.

Lipoyl synthase (LipA) catalyzes the final step in the biosynthesis of the lipoyl cofactor, the insertion of two sulfur atoms at C6 and C8 of an n-octanoyl chain. LipA is a member of the radical S-adenosylmethionine (SAM) superfamily of enzymes and uses two [4Fe-4S] clusters to catalyze its transformation. One cluster binds in contact with SAM and donates the requisite electron for the reductive cleavage of SAM to generate two 5'-deoxyadenosyl 5'-radicals, which abstract hydrogen atoms from C6 and C8 of the substrate. By contrast, the second, auxiliary [4Fe-4S] cluster, has been hypothesized to serve as the sulfur donor in the reaction. Such a sacrificial role for an iron-sulfur cluster during catalysis has not been universally accepted. Use of a conjugated 2,4-hexadienoyl-containing substrate analogue has allowed the substrate radical to be trapped and characterized by continuous-wave and pulsed electron paramagnetic resonance methods. Here we report the observation of a (57)Fe hyperfine coupling interaction with the paramagnetic signal, which indicates that the iron-sulfur cluster of LipA and its substrate are within bonding distance.

Addition Rate Constants of Phosphorus- and Carbon-Centered Radicals to Double Bond of Monomers as Studied by a Pulsed Electron Paramagnetic Resonance Method.

Addition reaction of a radical to the double bond of a monomer, which is important at early stage of photopolymerization, has been studied by time-resolved (TR-) and pulsed electron paramagnetic resonance (EPR) spectroscopic methods. Reactions of one phosphorus-centered and three carbon-centered radicals attacking to several monomers have been employed. Intermediate radicals were identified by analyzing the recorded TR-EPR spectra, and the reaction rate constants were determined by the electron spin-echo detection method proposed by Weber and Turro [J. Phys. Chem. A, 2003, 107 (18), 3326 - 3334]. The quantum chemical calculation shows that the rate constants for the addition reactions are well-explained by introducing two factors of "enthalpy effect" and "polar effect" to control the activation barrier height. It was observed that the rate constants of the phosphorus-centered radical were larger than those of carbon-centered radicals for some monomers. The difference in the rate constants was argued on the basis of frequency factor and activation barrier both of which are influenced by an atom of radical center.

Double Electron-Electron Spin Resonance Tracks Flavodoxin Folding.

Protein folding is one of the important challenges in biochemistry. Understanding the folding process requires mapping of protein structure as it folds. Here we test the potential of distance determination between paramagnetic spin-labels by a pulsed electron paramagnetic resonance method. We use double electron-electron spin resonance (DEER) to study the denaturant-dependent equilibrium folding of flavodoxin. This flavoprotein is spin-labeled with MTSL ((1-oxy-,2,2,5,5-tetramethyl-d-pyrroline-3-methyl)-methanethiosulfonate) at positions 69 and 131. We find that nativelike spin-label separation dominates the distance distributions up to 0.8 M guanidine hydrochloride. At 2.3 M denaturant, the distance distributions show an additional component, which we attribute to a folding intermediate. Upon further increase of denaturant concentration, the protein expands and evidence for a larger number of conformations than in the native state is found. We thus demonstrate that DEER is a versatile technique to expand the arsenal of methods for investigating how proteins fold.

HYSCORE Analysis of the Effects of Substrates on Coordination of Water to the Active Site Iron in Tyrosine Hydroxylase.

Tyrosine hydroxylase is a mononuclear non-heme iron monooxygenase found in the central nervous system that catalyzes the hydroxylation of tyrosine to yield L-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. Catalysis requires the binding of tyrosine, a tetrahydropterin, and O₂ at an active site that consists of a ferrous ion coordinated facially by the side chains of two histidines and a glutamate. We used nitric oxide as a surrogate for O₂ to poise the active site iron in an S = ³/₂ {FeNO}⁷ form that is amenable to electron paramagnetic resonance (EPR) spectroscopy. The pulsed EPR method of hyperfine sublevel correlation (HYSCORE) spectroscopy was then used to probe the ligands at the remaining labile coordination sites on iron. For the complex formed by the addition of tyrosine and nitric oxide, TyrH/NO/Tyr, orientation-selective HYSCORE studies provided evidence of the coordination of one H₂O molecule characterized by proton isotropic hyperfine couplings (A(iso) = 0.0 ± 0.3 MHz) and dipolar couplings (T = 4.4 and 4.5 ± 0.2 MHz). These data show complex HYSCORE cross peak contours that required the addition of a third coupled proton, characterized by an A(iso) of 2.0 MHz and a T of 3.8 MHz, to the analysis. This proton hyperfine coupling differed from those measured previously for H₂O bound to {FeNO}⁷ model complexes and was assigned to a hydroxide ligand. For the complex formed by the addition of tyrosine, 6-methyltetrahydropterin, and NO, TyrH/NO/Tyr/6-MPH₄, the HYSCORE cross peaks attributed to H₂O and OH⁻ for the TyrH/NO/Tyr complex were replaced by a cross peak due to a single proton characterized by an A(iso) of 0.0 MHz and a dipolar coupling (T = 3.8 MHz). This interaction was assigned to the N₅ proton of the reduced pterin.

Mapping the Structure of Metalloproteins with RIDME.

Distance measurements in biological macromolecules represent a very active field of application of pulsed electron paramagnetic resonance (EPR) spectroscopy. The relatively recently introduced pulsed EPR method of relaxation-induced dipolar modulation enhancement (RIDME) is conceptually similar to the popular double electron-electron resonance (DEER), but is much more suitable for studying the structures of metalloproteins while using their native paramagnetic metal centers as structural reference points. In particular, RIDME can largely alleviate the sensitivity and orientational selectivity problems that limit the application of DEER to such systems. In this contribution, the theoretical principles, implementation, optimization, and available experimental examples of RIDME are described with the purpose of enhancing the familiarity with this technique and promoting its application.

Probing the Redox Chemistry of Titanium Silicalite‐1: Formation of Tetrahedral Ti3+ Centers by Reaction with Triethylaluminum

Transition-metal ions with open-shell configurations hold promise in the development of novel coordination chemistry and potentially unprecedented redox catalysis. Framework-substituted Ti(3+) ions with tetrahedral coordination are generated by reductive activation of titanium silicalite-1 with triethylaluminum, an indispensable co-catalyst for heterogeneous Ziegler-Natta polymerization catalysts. Continuous-wave and pulse electron paramagnetic resonance methods are applied to unravel details on the local environment of the reduced transition metal-ions, which are shown to be part of the silica framework by detection of (29)Si hyperfine interactions. The chemical accessibility of the reduced sites is probed using ammonia as probe molecule. Evidence is found for the coordination of a single ammonia molecule. Comparison to similar systems, such as TiAlPO-5, reveals clear differences in the coordination chemistry of the reduced Ti sites in the two solids, which may be understood considering the different electronic properties of the solid frameworks.

Exploring the Structure of the 100 Amino-Acid Residue Long N-Terminus of the Plant Antenna Protein CP29

The structure of the unusually long (∼100 amino-acid residues) N-terminal domain of the light-harvesting protein CP29 of plants is not defined in the crystal structure of this membrane protein. We studied the N-terminus using two electron paramagnetic resonance (EPR) approaches: the rotational diffusion of spin labels at 55 residues with continuous-wave EPR, and three sets of distances with a pulsed EPR method. The N-terminus is relatively structured. Five regions that differ considerably in their dynamics are identified. Two regions have low rotational diffusion, one of which shows α-helical character suggesting contact with the protein surface. This immobile part is flanked by two highly dynamic, unstructured regions (loops) that cover residues 10–22 and 82–91. These loops may be important for the interaction with other light-harvesting proteins. The region around residue 4 also has low rotational diffusion, presumably because it attaches noncovalently to the protein. This section is close to a phosphorylation site (Thr-6) in related proteins, such as those encoded by the Lhcb4.2 gene. Phosphorylation might influence the interaction with other antenna complexes, thereby regulating the supramolecular organization in the thylakoid membrane.

Refined Distances Between Paramagnetic Centers of a Multi‐Copper Nitrite Reductase Determined by Pulsed EPR (iDEER) Spectroscopy

Refined Distances Between Paramagnetic Centers of a Multi‐Copper Nitrite Reductase Determined by Pulsed EPR (<i>i</i>DEER) Spectroscopy